甘蓝型油菜显性光叶突变体BnaA.GL基因定位和表皮蜡质分析
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摘要
植物地上部分覆盖着一层蜡质作为表皮的保护层,角质层蜡质决定的功能对于植物十分重要。蜡质具有防止紫外线辐射损伤,减少植物表面水分过度蒸发和抵抗病虫害侵入等功能,在植物对外界环境适应,减少生物和非生物胁迫方面起重要作用。
一般来说,甘蓝型油菜和茎表皮均有蜡粉覆盖,而光叶突变体则表皮蜡减少或缺失,颜色亮绿有光泽。目前在芸薹属植物无蜡(光叶亮绿)性状的遗传规律方面有一定的研究,但该性状在不同材料之间存在着多样性。尽管在模式植物拟南芥中已经鉴定和克隆了很多蜡质合成转运及调控等相关基因,但是芸薹属作物的相关研究还非常少,关于甘蓝型油菜无蜡光叶性状定位方面的研究,目前尚未见报道。本研究是首次对一个显性光叶突变体进行表型描述和定位研究。光叶突变体GL展示出了表皮蜡质的急剧减少和表皮渗透性的增强,生化分析表明光叶突变体蜡质总量降低以及成分发生了改变。通过遗传分析和定位,将光叶基因BnaA.GL定位在A9连锁群末端,为了比较野生型和光叶突变体在表达水平上的差异,进行了cDNA芯片的比较分析,结果显示一组与脂肪酸合成及蜡质合成代谢相关的基因受到抑制,其表达量下降。这些信息为我们进一步精细定位和克隆该基因,探索蜡质调控的机理奠定了基础。主要研究结果如下：
1.表皮形态学观察
扫描电镜显示正常材料野生型WT和光叶突变体GL叶片及茎表皮蜡质差异很大,突变体表皮蜡质晶体明显减少,尤其是叶片。透射电镜显示,光叶突变体GL叶片和茎表皮层亲锇性弱,电子密度显著减少,但是比WT要厚。光叶突变体GL表皮蜡质减少和表皮结构的变化,导致其渗透性增加。TB测试显示光叶突变体叶表皮更容易被染色,叶绿素浸提速率和失水率均高于WT。
2.遗传分析和基因定位
野生型WT和突变体GL正反交F1都表现为光叶,在F2群体中,光叶与正常叶比例符合3：1,BC1群体中,光叶和正常叶比例符合1：1。遗传分析表明,光叶性状是受单个显性基因控制。
基因初步定位显示,光叶性状为形态标记Y被定位于N9连锁群末端。在此基础上,BSA法结合AFLP技术,以及联合多种方式开发SSR标记、IP标记,搜索该区段已经公布信息以及利用SNP芯片结合BSA法等,最终在一个BC3F2的1200单株的群体中,通过标记分析,将光叶基因BnaA.GL定位到A9连锁群末端,开发的分子标记距离目标基因0.67-1.33cM,遗憾的是标记均位于目标基因一侧。根据网站提供的白菜序列信息比对分析(http://brassicadb.org/brad/),与目标基因最近的分子标记距离白菜R9连锁群末端最后一个注释基因的距离250kb。该区段包括有白菜46个注释基因,与拟南芥基因组同源线性比对分析显示,共线性较好,对应拟南芥AT1G01190到AT1G02205区段。候选区段的最后一个白菜基因Bra032670是AT1G02205(CER1)的同源基因。CER1是与蜡质合成有关的酶,其T-DNA插入突变体表型是光杆不育的。因此我们认为该基因为可能的候选基因。
3.叶脂质分析和蜡质分析
通过气相色谱-质谱联用(GC-MS)的分析方法,对WT,突变体GL, DH系中的正常系DH line2和光叶系DH line7四个样本进行了脂质分析和蜡质分析。结果表明,突变体GL和光叶系DH line7显示出脂肪酸C16：0和C18：3的增加,而缺乏C22：0。超长链脂肪酸(VLCFAs),总含量显著减少。
蜡质分析中,薄板层析(TLC)分析结果显示,突变体GL和光叶系DH line7萌发后四周叶片提取的蜡质中缺乏烷烃,酮以及二级醇,而醛大量积累。GC-MS分析表明,突变体GL和光叶系DH line7显示除醛增加以外,蜡酯,初级醇和脂肪酸略有减少,而蜡质总含量显著降低。
4. cDNA芯片分析
由于定位方面进展缓慢,因此采用cDNA芯片分析的方法,试图进行差异表达基因分析,为定位提供一定指导,同时初步分析蜡质合成代谢途径的网络,解析蜡质合成受阻的原因。
以WT,突变体GL, DH系中的正常系混合成的正常RNA池DHNB和l光叶系混合成的光叶RNA池DHGB四个RNA为样本,利用加拿大PBI研发的甘蓝型油菜90KcDNA芯片进行了芯片分析。芯片结果显示,差异表达基因可以被分成很多的大类,包括：光合作用和碳代谢、脂和蜡质代谢、DNA的转录调控、生物和非生物逆境响应等,此外,还包括与与转录相关、发育相关以及未知的功能等类别。
在分析了脂肪酸代谢和蜡质代谢途径差异表达基因之后,发现在光叶材料中脂肪酸从头合成到蜡质合成整个代谢途径在光叶材料中都受到抑制,尤其是脱羰基途径中的CER1基因,下降倍数最多。藉此,对挑选出来与脂代谢等相关代谢途径的部分基因进行了Real-Time PCR验证,证实其中大部分基因表达情况与芯片结果一致。根据验证结果,初步绘制了蜡质代谢途径的简图。
5.候选基因分析
CER1被认为可能是脱羰基途径上编码催化醛脱羰反应的酶,拟南芥突变体cerl-1具有光杆表型,生化特征与甘蓝型油菜光叶突变体GL相似,芯片分析及Real-Time PCR验证了该基因严重受到抑制,且该基因的同源基因位于候选区段,因此将其作为候选基因,进行序列分析和基因转化实验。
序列分析表明,在甘蓝型油菜WT的cDNA中扩增出4种CDS拷贝,但在光叶突变体cDNA中未能扩增出CDS序列。在甘蓝型油菜WT的DNA中除扩增出含有内含子的拷贝外还扩增出不带有内含子的拷贝,可能为假基因。通过TAIL技术得到了启动子序列。经过比较测序,仅发现一个在WT和GL有差异的拷贝,且SNP差异位点位于内含子。
从光叶突变体基因组DNA中分离出这个含有SNP差异位点的拷贝,并构建了遗传转化载体,但遗憾的是没有得到预期的表型。
构建了过表达载体,所用启动子分别为CaMV35s启动子和油菜自身的启动子。两种载体在花絮浸染法转化拟南芥cerl-1突变体(SALK_008544C)时,未能获得阳性植株。在转化野生型拟南芥时,BnCER1在CaMV35s启动子驱动下的载体转化后没有得到表型,而BnCER1在自身启动子驱动下的载体转化后得到的转化阳性植株展示出了光杆不育的表型。经扫描电镜观察,其茎表皮蜡质急剧减少,花粉形态不正常。通过分析转基因植株与WT外源及内源CER1基因表达水平,初步推断可能是发生了共抑制现象。
6.甘蓝型油菜正常蜡质WT和光叶突变体GL抗逆性差异
用WT和和光叶突变体GL进行抗旱抗逆相关实验。通过设置包括对照,干旱处理,盐处理以及ABA处理三种处理。与对照相比,在不同处理下,蜡质积累均有增加,失水率,叶绿素浸提率和相对含水率均有下降,但变化幅度不同。与蜡质合成相关基因BnCER1在WT盐处理之后,基因表达量上升,在干旱和ABA处理之后均下降。
The aerial parts of land plants are covered with cuticular waxes. The wax was important and play role in limit non-stomatal water loss and gaseous exchange and protect plants from ultraviolet radiation and pathogen attack.
Generally, the leaf and stem are covered with cuticular waxes, but the glossy mutants are shiny green without the waxes. There were extensively studies on the inheritance of glossy waxless character in Brassica, it demonstrated diversity among different species.
A group of cuticular wax related genes were identified and cloned in Arabidopisis thaliana, but there were few researches on the cuticular wax in Brassica, especially there have been no reports of the molecular marker study on the glossy waxless character in Brassica napus. This study presented the first report on the characterization and genetic mapping of a novel dominant glossy mutant (BnaA.GL) in Brassica napus. The glossy mutant (GL mutant) exhibited abnormal cuticular wax deposition and increased water permeability. Biochemical analysis showed a decreased total cuticular waxes and alteration of wax composition. We did genetic analysis and mapped the BnaA.GL gene close to the end of chromosome A9. In search of clues underlying the molecular basis of the glossy phenotype, we conducted a microarray analysis and performed a comparison of the global transcript level between the wide type and the mutants. It was revealed that the wax biosynthetic genes were down-regulated in the glossy mutant. Data presented in the study on genetic mapping of the BnaA.GL gene and transcriptome alterations in the mutant pave the road for future efforts in cloning this gene and understanding the regulations of wax biosynthesis pathways in plants. The main points in this study as follow:
1. The morphological observation about the glossy mutant
Scanning electron microscopy corroborated the reduction of wax on the leaf and stem surface in GL mutan, especially the leaf. Transmission electron microscopy revealed that the GL mutant epidermis had a cuticle membrane that was less osmiophilic, as indicated by the reduced electron density, but more than twice as thick compared with that of the wild type. Toluidine blue test showed that the GL mutant was stained more easily, in addition, the GL mutant rapidly lost chlorophyll content and showed a significantly higher water loss rate when compared with WT, which indicated the mutants were compromised in the strength of water permeability barrier.
2. Genetica analysis and mapping of the BnaA.GL gene
The F1(or RF1) plants of reciprocal crosses between WT and the GL mutant were all glossy, indicating that the glossy trait was dominant. The BC1progeny developed from the crosses between the F1plant and WT displayed a ratio of glossy to normal plants at a1:1ratio. Moreover, the glossy to normal phenotype ratio in the F2population was approximately3:1, indicating that this was a case in which one Mendelian locus controlled the glossy trait. Our initial mapping using the glossy leaf phenotype as a morphological marker (named Y) mapped the genetic lesion to linkage group A9from an F2population derived from WT x the glossy mutant. We then used an AFLP assay in combination with BSA and developed SSR markers, IP markers, in addition, an SNP Chip assay in combination with BSA was used. Finally, based on300normal individuals from a BC3F2population (1200), we were able to map these markers to a region of0.67-1.33cM in the flanking region of the BnaA.GL gene. All markers linked to the gene were used to compare the micro-colinearity of the regions flanking the genes with B. rapa and Arabidopsis. By comparative mapping with B. rapa using http://brassicadb.org/brad/database, the distance from the closest marker and the last annotated gene on R9was calculated as only250kb, there were46genes in the region, the homologous region of BnaA.GL in Arabidopsis was between AT1G01190and AT1G02205. Interestingly, a homolog of Arabidopsis gene CER1(AT1G02205), Bra032670was located in the mapped region of the BnaA. GL. CER1encodes aldehyde decarbonylase, the stem was glossy and the pollen was sterility in the mutant cerl-1. So we thought it was the candidate gene.
3. Fatty acid composition of leaf and cuticular wax analysis
GC-MS analysis of the leaf fatty acid composition indicated that the C16:0and C18:3were increased, but no C22:0was detected in GL mutant and DH line7, the total VLCFAs were decreased.
Thin-layer chromatography (TLC) analysis revealed alkanes, ketones and secondary alcohols were not detected, but aldehydes were increased in the GL mutant. GC-MS analysis indicated that the primary alcohols, wax esters and fatty acids were decreased, and total waxes were decreased.
4cDNA microarray assay
In search of clues underlying the molecular basis of the glossy phenotype and providing more information for mapping. We then conducted a microarray analysis using RNA from leaf tissue of similar developmental stages and performed a comparison of the global transcript level between WT vs. the GL mutant and double haploid normal line bulk (DHNB) segregates vs. DH glossy line bulk (DHGB) segregates. These genes fell into several categories, including fatty acids biosynthesis, DNA-depend transcription, the responses to stress and to abiotic or biotic stimuli and photosynthesi, in addition, including genes related to protein biosynthesis, transcription, development and so on. A major functional category of the down-regulated genes emerged from the DHNB vs. DHGB microarray analysis included those encoding a subset of genes related to the fatty acid biosynthesis and wax biosynthesis pathway. It was revealed that wax biosynthetic genes were down-regulated, especially the decarbonylation pathway of wax biosynthesis was compromised and BnCER1was one of those most severely down-regulated in the glossy mutant. The different expression patterns of these genes in the GL mutants were verifiable by quantitative real-time PCR (qRT-PCR). Based on those, we made a simplified cuticular wax biosynthetic pathway.
5. Candidate gene analysis
CER1was the aldehyde decarbonylase. The phenotype and biochemic character of Arabidopisis cerl-1mutant was similar with the GL mutant. In light of the fact that Bra032670at the end of the chromosome was the homologous to CER1(AT1G02205), the genetic lesion responsible for the glossy phenotype in Arabidopsis.We thought it was the candidate gene.
Sequence analysis indicated that there were4copies of CDS in WT, but we didn't amplify any copies from the cDNA of GL mutant. Except the copies with introns, we amplified copies without introns. Promoter sequence was obtained by TAIL-PCR. However, there was no apparent sequence alteration between WT and the GL mutant except in the fifth intron where three SNPs were identified.
The copy with SNPs differences obtainted from the GL mutant was transformed to the WT in Brassica napus, but we didn't get the expected phenotype.
The vectors of over expression BnCER1were construeted under the CaMV35S promoter and its own promoter in B. napus. Both of them were transformed in Arabidopisis cerl mutant (SALK_008544C), but no positive transformed plants were obtained.
The vector of over expression BnCER1under the CaMV35S promoter was transformed in WT of Arabidopisis, but no significance differences were detected compared with WT. The vector of over expression BnCER1under its own promoter was transformed to WT in Arabidopisis, T1plants originating from independent TO kanamycin-resistant transformants exhibited glossy stem and reduced fertility. Scanning electron microscopy showed the reduction of wax on the stem surface in transformants, the pollen was abnormal and infertile. In transgenic plants, the expression level of CER1was lower than the WT, transgenes may be silenced together with the homologous endogenous genes in some cases. This phenomenon is called cosuppression.
6. The difference of stress resistance between WT and GL muatant
Four-weeks plants of WT and GL muatant were used for all stress treatments. NaCl stress was imposed by subirrigation with NaCl solution, for water deficit treatments, pots were deprived of water, ABA treatment consisted of spraying with ABA. Compared with the control plants, plants respond to all treatment by increasing the deposition of leaf cuticular waxes, meanwhile the chlorophyll-extraction rates and water loss rate were decreased, but the changing extent varied with different treatments. Results here also revealed that salt stress-induced accumulation of waxes involves accumulation of wax related gene CER1, but reduction of CER1under drought and ABA treatments.
一般来说,甘蓝型油菜和茎表皮均有蜡粉覆盖,而光叶突变体则表皮蜡减少或缺失,颜色亮绿有光泽。目前在芸薹属植物无蜡(光叶亮绿)性状的遗传规律方面有一定的研究,但该性状在不同材料之间存在着多样性。尽管在模式植物拟南芥中已经鉴定和克隆了很多蜡质合成转运及调控等相关基因,但是芸薹属作物的相关研究还非常少,关于甘蓝型油菜无蜡光叶性状定位方面的研究,目前尚未见报道。本研究是首次对一个显性光叶突变体进行表型描述和定位研究。光叶突变体GL展示出了表皮蜡质的急剧减少和表皮渗透性的增强,生化分析表明光叶突变体蜡质总量降低以及成分发生了改变。通过遗传分析和定位,将光叶基因BnaA.GL定位在A9连锁群末端,为了比较野生型和光叶突变体在表达水平上的差异,进行了cDNA芯片的比较分析,结果显示一组与脂肪酸合成及蜡质合成代谢相关的基因受到抑制,其表达量下降。这些信息为我们进一步精细定位和克隆该基因,探索蜡质调控的机理奠定了基础。主要研究结果如下：
1.表皮形态学观察
扫描电镜显示正常材料野生型WT和光叶突变体GL叶片及茎表皮蜡质差异很大,突变体表皮蜡质晶体明显减少,尤其是叶片。透射电镜显示,光叶突变体GL叶片和茎表皮层亲锇性弱,电子密度显著减少,但是比WT要厚。光叶突变体GL表皮蜡质减少和表皮结构的变化,导致其渗透性增加。TB测试显示光叶突变体叶表皮更容易被染色,叶绿素浸提速率和失水率均高于WT。
2.遗传分析和基因定位
野生型WT和突变体GL正反交F1都表现为光叶,在F2群体中,光叶与正常叶比例符合3：1,BC1群体中,光叶和正常叶比例符合1：1。遗传分析表明,光叶性状是受单个显性基因控制。
基因初步定位显示,光叶性状为形态标记Y被定位于N9连锁群末端。在此基础上,BSA法结合AFLP技术,以及联合多种方式开发SSR标记、IP标记,搜索该区段已经公布信息以及利用SNP芯片结合BSA法等,最终在一个BC3F2的1200单株的群体中,通过标记分析,将光叶基因BnaA.GL定位到A9连锁群末端,开发的分子标记距离目标基因0.67-1.33cM,遗憾的是标记均位于目标基因一侧。根据网站提供的白菜序列信息比对分析(http://brassicadb.org/brad/),与目标基因最近的分子标记距离白菜R9连锁群末端最后一个注释基因的距离250kb。该区段包括有白菜46个注释基因,与拟南芥基因组同源线性比对分析显示,共线性较好,对应拟南芥AT1G01190到AT1G02205区段。候选区段的最后一个白菜基因Bra032670是AT1G02205(CER1)的同源基因。CER1是与蜡质合成有关的酶,其T-DNA插入突变体表型是光杆不育的。因此我们认为该基因为可能的候选基因。
3.叶脂质分析和蜡质分析
通过气相色谱-质谱联用(GC-MS)的分析方法,对WT,突变体GL, DH系中的正常系DH line2和光叶系DH line7四个样本进行了脂质分析和蜡质分析。结果表明,突变体GL和光叶系DH line7显示出脂肪酸C16：0和C18：3的增加,而缺乏C22：0。超长链脂肪酸(VLCFAs),总含量显著减少。
蜡质分析中,薄板层析(TLC)分析结果显示,突变体GL和光叶系DH line7萌发后四周叶片提取的蜡质中缺乏烷烃,酮以及二级醇,而醛大量积累。GC-MS分析表明,突变体GL和光叶系DH line7显示除醛增加以外,蜡酯,初级醇和脂肪酸略有减少,而蜡质总含量显著降低。
4. cDNA芯片分析
由于定位方面进展缓慢,因此采用cDNA芯片分析的方法,试图进行差异表达基因分析,为定位提供一定指导,同时初步分析蜡质合成代谢途径的网络,解析蜡质合成受阻的原因。
以WT,突变体GL, DH系中的正常系混合成的正常RNA池DHNB和l光叶系混合成的光叶RNA池DHGB四个RNA为样本,利用加拿大PBI研发的甘蓝型油菜90KcDNA芯片进行了芯片分析。芯片结果显示,差异表达基因可以被分成很多的大类,包括：光合作用和碳代谢、脂和蜡质代谢、DNA的转录调控、生物和非生物逆境响应等,此外,还包括与与转录相关、发育相关以及未知的功能等类别。
在分析了脂肪酸代谢和蜡质代谢途径差异表达基因之后,发现在光叶材料中脂肪酸从头合成到蜡质合成整个代谢途径在光叶材料中都受到抑制,尤其是脱羰基途径中的CER1基因,下降倍数最多。藉此,对挑选出来与脂代谢等相关代谢途径的部分基因进行了Real-Time PCR验证,证实其中大部分基因表达情况与芯片结果一致。根据验证结果,初步绘制了蜡质代谢途径的简图。
5.候选基因分析
CER1被认为可能是脱羰基途径上编码催化醛脱羰反应的酶,拟南芥突变体cerl-1具有光杆表型,生化特征与甘蓝型油菜光叶突变体GL相似,芯片分析及Real-Time PCR验证了该基因严重受到抑制,且该基因的同源基因位于候选区段,因此将其作为候选基因,进行序列分析和基因转化实验。
序列分析表明,在甘蓝型油菜WT的cDNA中扩增出4种CDS拷贝,但在光叶突变体cDNA中未能扩增出CDS序列。在甘蓝型油菜WT的DNA中除扩增出含有内含子的拷贝外还扩增出不带有内含子的拷贝,可能为假基因。通过TAIL技术得到了启动子序列。经过比较测序,仅发现一个在WT和GL有差异的拷贝,且SNP差异位点位于内含子。
从光叶突变体基因组DNA中分离出这个含有SNP差异位点的拷贝,并构建了遗传转化载体,但遗憾的是没有得到预期的表型。
构建了过表达载体,所用启动子分别为CaMV35s启动子和油菜自身的启动子。两种载体在花絮浸染法转化拟南芥cerl-1突变体(SALK_008544C)时,未能获得阳性植株。在转化野生型拟南芥时,BnCER1在CaMV35s启动子驱动下的载体转化后没有得到表型,而BnCER1在自身启动子驱动下的载体转化后得到的转化阳性植株展示出了光杆不育的表型。经扫描电镜观察,其茎表皮蜡质急剧减少,花粉形态不正常。通过分析转基因植株与WT外源及内源CER1基因表达水平,初步推断可能是发生了共抑制现象。
6.甘蓝型油菜正常蜡质WT和光叶突变体GL抗逆性差异
用WT和和光叶突变体GL进行抗旱抗逆相关实验。通过设置包括对照,干旱处理,盐处理以及ABA处理三种处理。与对照相比,在不同处理下,蜡质积累均有增加,失水率,叶绿素浸提率和相对含水率均有下降,但变化幅度不同。与蜡质合成相关基因BnCER1在WT盐处理之后,基因表达量上升,在干旱和ABA处理之后均下降。
The aerial parts of land plants are covered with cuticular waxes. The wax was important and play role in limit non-stomatal water loss and gaseous exchange and protect plants from ultraviolet radiation and pathogen attack.
Generally, the leaf and stem are covered with cuticular waxes, but the glossy mutants are shiny green without the waxes. There were extensively studies on the inheritance of glossy waxless character in Brassica, it demonstrated diversity among different species.
A group of cuticular wax related genes were identified and cloned in Arabidopisis thaliana, but there were few researches on the cuticular wax in Brassica, especially there have been no reports of the molecular marker study on the glossy waxless character in Brassica napus. This study presented the first report on the characterization and genetic mapping of a novel dominant glossy mutant (BnaA.GL) in Brassica napus. The glossy mutant (GL mutant) exhibited abnormal cuticular wax deposition and increased water permeability. Biochemical analysis showed a decreased total cuticular waxes and alteration of wax composition. We did genetic analysis and mapped the BnaA.GL gene close to the end of chromosome A9. In search of clues underlying the molecular basis of the glossy phenotype, we conducted a microarray analysis and performed a comparison of the global transcript level between the wide type and the mutants. It was revealed that the wax biosynthetic genes were down-regulated in the glossy mutant. Data presented in the study on genetic mapping of the BnaA.GL gene and transcriptome alterations in the mutant pave the road for future efforts in cloning this gene and understanding the regulations of wax biosynthesis pathways in plants. The main points in this study as follow:
1. The morphological observation about the glossy mutant
Scanning electron microscopy corroborated the reduction of wax on the leaf and stem surface in GL mutan, especially the leaf. Transmission electron microscopy revealed that the GL mutant epidermis had a cuticle membrane that was less osmiophilic, as indicated by the reduced electron density, but more than twice as thick compared with that of the wild type. Toluidine blue test showed that the GL mutant was stained more easily, in addition, the GL mutant rapidly lost chlorophyll content and showed a significantly higher water loss rate when compared with WT, which indicated the mutants were compromised in the strength of water permeability barrier.
2. Genetica analysis and mapping of the BnaA.GL gene
The F1(or RF1) plants of reciprocal crosses between WT and the GL mutant were all glossy, indicating that the glossy trait was dominant. The BC1progeny developed from the crosses between the F1plant and WT displayed a ratio of glossy to normal plants at a1:1ratio. Moreover, the glossy to normal phenotype ratio in the F2population was approximately3:1, indicating that this was a case in which one Mendelian locus controlled the glossy trait. Our initial mapping using the glossy leaf phenotype as a morphological marker (named Y) mapped the genetic lesion to linkage group A9from an F2population derived from WT x the glossy mutant. We then used an AFLP assay in combination with BSA and developed SSR markers, IP markers, in addition, an SNP Chip assay in combination with BSA was used. Finally, based on300normal individuals from a BC3F2population (1200), we were able to map these markers to a region of0.67-1.33cM in the flanking region of the BnaA.GL gene. All markers linked to the gene were used to compare the micro-colinearity of the regions flanking the genes with B. rapa and Arabidopsis. By comparative mapping with B. rapa using http://brassicadb.org/brad/database, the distance from the closest marker and the last annotated gene on R9was calculated as only250kb, there were46genes in the region, the homologous region of BnaA.GL in Arabidopsis was between AT1G01190and AT1G02205. Interestingly, a homolog of Arabidopsis gene CER1(AT1G02205), Bra032670was located in the mapped region of the BnaA. GL. CER1encodes aldehyde decarbonylase, the stem was glossy and the pollen was sterility in the mutant cerl-1. So we thought it was the candidate gene.
3. Fatty acid composition of leaf and cuticular wax analysis
GC-MS analysis of the leaf fatty acid composition indicated that the C16:0and C18:3were increased, but no C22:0was detected in GL mutant and DH line7, the total VLCFAs were decreased.
Thin-layer chromatography (TLC) analysis revealed alkanes, ketones and secondary alcohols were not detected, but aldehydes were increased in the GL mutant. GC-MS analysis indicated that the primary alcohols, wax esters and fatty acids were decreased, and total waxes were decreased.
4cDNA microarray assay
In search of clues underlying the molecular basis of the glossy phenotype and providing more information for mapping. We then conducted a microarray analysis using RNA from leaf tissue of similar developmental stages and performed a comparison of the global transcript level between WT vs. the GL mutant and double haploid normal line bulk (DHNB) segregates vs. DH glossy line bulk (DHGB) segregates. These genes fell into several categories, including fatty acids biosynthesis, DNA-depend transcription, the responses to stress and to abiotic or biotic stimuli and photosynthesi, in addition, including genes related to protein biosynthesis, transcription, development and so on. A major functional category of the down-regulated genes emerged from the DHNB vs. DHGB microarray analysis included those encoding a subset of genes related to the fatty acid biosynthesis and wax biosynthesis pathway. It was revealed that wax biosynthetic genes were down-regulated, especially the decarbonylation pathway of wax biosynthesis was compromised and BnCER1was one of those most severely down-regulated in the glossy mutant. The different expression patterns of these genes in the GL mutants were verifiable by quantitative real-time PCR (qRT-PCR). Based on those, we made a simplified cuticular wax biosynthetic pathway.
5. Candidate gene analysis
CER1was the aldehyde decarbonylase. The phenotype and biochemic character of Arabidopisis cerl-1mutant was similar with the GL mutant. In light of the fact that Bra032670at the end of the chromosome was the homologous to CER1(AT1G02205), the genetic lesion responsible for the glossy phenotype in Arabidopsis.We thought it was the candidate gene.
Sequence analysis indicated that there were4copies of CDS in WT, but we didn't amplify any copies from the cDNA of GL mutant. Except the copies with introns, we amplified copies without introns. Promoter sequence was obtained by TAIL-PCR. However, there was no apparent sequence alteration between WT and the GL mutant except in the fifth intron where three SNPs were identified.
The copy with SNPs differences obtainted from the GL mutant was transformed to the WT in Brassica napus, but we didn't get the expected phenotype.
The vectors of over expression BnCER1were construeted under the CaMV35S promoter and its own promoter in B. napus. Both of them were transformed in Arabidopisis cerl mutant (SALK_008544C), but no positive transformed plants were obtained.
The vector of over expression BnCER1under the CaMV35S promoter was transformed in WT of Arabidopisis, but no significance differences were detected compared with WT. The vector of over expression BnCER1under its own promoter was transformed to WT in Arabidopisis, T1plants originating from independent TO kanamycin-resistant transformants exhibited glossy stem and reduced fertility. Scanning electron microscopy showed the reduction of wax on the stem surface in transformants, the pollen was abnormal and infertile. In transgenic plants, the expression level of CER1was lower than the WT, transgenes may be silenced together with the homologous endogenous genes in some cases. This phenomenon is called cosuppression.
6. The difference of stress resistance between WT and GL muatant
Four-weeks plants of WT and GL muatant were used for all stress treatments. NaCl stress was imposed by subirrigation with NaCl solution, for water deficit treatments, pots were deprived of water, ABA treatment consisted of spraying with ABA. Compared with the control plants, plants respond to all treatment by increasing the deposition of leaf cuticular waxes, meanwhile the chlorophyll-extraction rates and water loss rate were decreased, but the changing extent varied with different treatments. Results here also revealed that salt stress-induced accumulation of waxes involves accumulation of wax related gene CER1, but reduction of CER1under drought and ABA treatments.
引文
1.曹阳,沈向群,李峰.小白菜花茎无蜡粉性状遗传分析.中国蔬菜,2008,3：20-22
2.陈志谊,王玉环,殷尚智.水稻纹枯病抗性机制的研究.中国农业科学,1992,25(4)：41-46
3.冯辉,杨晓飞,辛彬,赵鹏涛,王玉刚.无蜡粉型青梗菜核基因雄性不育系转育研究.沈阳农业大学学报,2010,41(2)：142-146
4.黄镇.分子标记辅助选择培育新型甘蓝型油菜隐性细胞核雄性不育系.[博士学位论文].武汉：华中农业大学图书馆,2008
5.康立功,齐凤坤,许向阳,李景富.番茄叶片蜡质和角质层与芝麻斑病菌侵染的关系.中国蔬菜,2010,(18)：47-50
6.李海英,刘亚光,杨庆凯.大豆叶片结构与灰斑病抗性的研究Ⅱ.大豆叶片组织结构与灰斑病抗性的关系.中国油料作物学报,2012,24(2)：74-76
7.李景涛.结球甘蓝无蜡粉亮绿性状遗传分析及分子标记研究.[硕士学位论文].北京：中国农业科学院,2012
8.李灵芝.3个水稻WAX2同源基因的蜡质合成及抗逆性功能分析.[硕士学位论文].湖南长沙：湖南农业大学,2012
9.李艳,惠有为,张仲凯,黄兴奇,李毅.转查耳酮合酶基因矮牵牛共抑制的研究.中国科学,2001,31(5)：401-407
10.刘仁虎,孟金陵.MapDraw,在Excel中绘制遗传连锁图的宏.遗传,2003,25(3)：317-321
11.刘忠松,官春云,陈社员,莫鉴国,Osborn T甘蓝型油菜显性无蜡粉基因的染色体组定位.湖南农业大学学报(自然科学版),2000,26(3)：182-184
12.卢艳丽.不同类型玉米种质分子特征分析及耐旱相关性状的连锁—连锁不平衡联合作图.[博士学位论文].四川雅安：四川农业大学,2011
13.卢泳全,汪旭升,黄伟素,肖天霞,郑燕,吴为人.基于水稻内含子长度多态性开发禾本科扩增共有序列遗传标记.中国农业科学,2006,39(3)：433-439
14.陆光远,杨光圣,傅廷栋.一个简便的适合于分析油菜中SSR位点的检测体系.中国油料作物学报,2003,25：79-81
15.马永彪.甘蓝型油菜与白菜比较作图和比较QTL研究.[硕士学位论文].武汉：华中农业大学图书馆,2009
16.莫鉴国,李万渠,彭云强等.甘蓝型油菜无蜡粉种植材料的改良以及在杂交育种上的应用.种子,1999,105(5)：18-20
17.牟香丽.结球甘蓝“无蜡粉亮叶”突变体性状分析.[硕士学位论文].哈尔滨：东北农业大学,2013
18.倪郁,郭彦军.植物超长链脂肪酸及角质层蜡质生物合成相关酶基因研究现状.遗传,2008,15(05)：561-567
19.任菲,张荣佳,陈强,白艳波,黄菲,李雪梅.ABA和SA对于提高植物抗旱及抗盐性的研究进展.生物技术通报,2012,6(03)：17-21
20.孙玉燕.利用拟南芥BOUNTIFUL和HARDY基因以及表皮蜡质合成基因提高番茄耐旱和耐盐性.中国农业科学院,2012
21.宋超.菌核病菌、UV-B低温胁迫条件下拟南芥表皮蜡质的响应机制研究.[硕士学位论文].重庆：西南大学,2013
22.王婧,刘泓利,宋超,倪郁.甘蓝型油菜叶表皮蜡质组分及结构与菌核病抗性关系.植物生理学报,2012,48：958-964
23.王美芳,陈巨莲,原国辉,雷振生,吴政卿,赵献林.植物表面蜡质对植食性昆虫的影响研究进展.生态环境学报,2009,18(3)：1155-1160
24.王玉锋,黄霁月,杨金水.基因工程培育可恢复的植物雄性不育系的研究进展.遗传,2011,15(01)：40-47
25.谢彦周.甘蓝型油菜隐性细胞核雄性不育上位抑制基因的精细定位.[博士学位论文].武汉：华中农业大学图书馆,2010
26.易斌.甘蓝型油菜隐性核不育基因Bnmsl的精细定位和克隆.[博士学位论文].武汉：华中农业大学图书馆,2007
27.张海禄,齐军仓.大麦叶片表皮蜡质含量与抗旱性的关系研究.新疆农业科学,2012,49(1)：22-27
28.张志飞,饶力群,向佐湘,胡晓敏,王晓杰.高羊茅叶片表皮蜡质含量与其抗旱性的关系.西北植物学报,2007,27(7)：1417-1421
29.赵建峰,沈向群,张海楼.菜薹无蜡粉性状遗传规律初探.园艺学报,2006,3(33)：538
30.周熙荣,李树林,顾龙弟.显性核不育油菜无蜡粉纯合两型系的选育.上海农业学报,1999,(4)：25-27
31.周熙荣,李树林,庄静.甘蓝型油菜无蜡粉隐性核不育两型系的选育.上海农业学报,2002,18(1)：20-24
32.曾芳琴.油菜S45AB隐性核不育分子机理与应用研究.[博士学位论文].武汉：华中农业大学图书馆,2010
33. Aarts MGM, Keijzer CJ, Stiekema WJ, Pereira A. Molecular characterization of the CERlgene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell,1995,7:2115-2127
34. Adamski NM, Bush MS, Simmonds J, Turner AS, Mugford SG, Jones A, Findlay K, Pedentchouk N, von Wettstein-Knowles P, Uauy C. The inhibitor of wax 1 locus (Iw1) prevents formation of P-and OH-β-diketones in wheat cuticular waxes and maps to a sub-cM interval on chromosome arm 2BS. Plant Journal,2013,74(6):989-1002
35. Aharoni A, Dixit S, Jetter R, Thoenes E, vanArkel G, PereiraA. The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance when Overexpressed in Arabidopsis. Plant Cell,2004,16:2463-2480
36. Angadi SP, Singh JP, Anand IJ. Inheritance on non-waxiness and tolerance to aphids in Indian mustard. Journal of Oilseeds Research,1987,4(2):265-267
37. Anstey TH, Moore JF. Inheritance of glossy foliage and cream petal. Heredity,1954, 45:39-41.
38. Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR. Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science,1992,258:1353-1355
39. Ayele M, Haas BJ, Kumar N, Wu H, Xiao YL, Van Aken S, Utterback TR, Wortman JR, White OR, Town CD. Whole genome shotgun sequencing of Brassica oleracea and its application to gene discovery and annotation in Arabidopsis. Genome Research,2005,15:487-495
40. Babula D, Kaczmarek M, Barakat A, Delseny M, Quiros CF, Sadowski J. Chromosomal mapping of Brassica oleracea based on ESTs from Arabidopsis thaliana:complexity of the comparative map. Mol Genet Genomics,2003,268:656-665
41. Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da Costa M, Boutin JP, Miquel M, Tellier F, Domergue F, Markham JE, Beaudoin F, Napier JA, Faure JD. The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development. P Natl Acad Sci Usa,2008,105(38): 14727-14731
42. Baker E. Chemistry and morphology of plant epicuticular waxes. In:Cutler D F, Alvin K L, Price C E eds., The Plant Culture. New York:Academic Press,1982. 139-165
43. Barrs HD, Weatherley PE. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust JBiol Sci,1962,15:413-428
44. Beaudoin F, Gable K, Sayanova O, Dunn T, Napier JA. A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-ketoreductase. JBiol Chem,2002,277(13):11481-11488
45. Beaudoin F, Wu X, Li F, Haslam RP, Markham JE, Zheng H, Napier JA, Kunst L Functional characterization of the Arabidopsis thaliana b-ketoacyl-CoA reductase candidates of the fatty acid elongase. Plant Physiol,2009,150(3):1174-1191
46. Bernard A, Domergue F, Pascal S, Jetter R, Renne C, Faure JD, Haslam RP, Napier JA, Lessire R, Joubes J. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell,2012,24(7): 3106-3118
47. Bernard A, Joubes J. Arabidopsis cuticular waxes:Advances in synthesis, export and regulation. Progress in Lipid Research,2013,52(1):110-129
48. Bessire M, Chassot C, Jacquat AC, Humphry M, Borel S, Petetot J, Metraux JP, Nawrath C. A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea. Embo J,2007,26(8):2158-2168
49. Bianchi, G. Murelli, C. and Ottaviano, E. Maize pollen lipids. Phytochemistry.1990, 29(3):739-744
50. Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X W, Yephremov A, Samuels L. Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J,2007,52(3):485-498
51. Bourdenx B, Bernard A, Domergue F, Pascal S, Leger A, Roby D, Pervent M, Vile D, Haslam RP, Napier J A, Lessire R, Joubes J. Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiol,2011,156(1):29-45
52. Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB. Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell,2003,15:1020-33
53. Brown GG, Formanova N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung WY, Landry BS. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J.2003,35(2):262-72.
54. Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL. WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA. 2004,101(13):4706-4711
55. Cameron KD, Teece MA, Smart LB. Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco. Plant Physiol,2006,140(1):176-183
56. Cardoza v, Stewart CN. Inereased Agrobacterium-mediated transformation and rooting effieiencies in eanola (Brassica napus L.) from hypoeotyl segment explants. Plant Cell Rep,2003,21(6):599-604
57. Chen X, Goodwin SM, Boroff VL, Liu X, Jenks MA. Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell,2003,15(5):1170-1185
58. Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X. BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol,2011,11:136
59. Cheng F, Mandakova T, Wu J, Xie Q, Lysak MA, Wang XW. Deciphering the Diploid Ancestral Genome of the Mesohexaploid Brassica rapa. Plant Cell,2013, 25(5):1541-1554
60. Cheung F, Trick M, Drou N, Lim Y R, Park J Y, Kwon S J, Kim J A, Scott R, Pires JC, Paterson AH, Town C, Bancroft I. Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence. Plant Cell,2009,21(7):1912-1928
61. Cheung WY, Friesen L, Rakow GFW, Seguin-Swartz G, Landry BS. A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]. Theor Appl Genet, 1997,94(6):841-851
62. Clough SJ, Bent AE. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal,1998,16(6):735-743
63. Costaglioli P, Joubes J, Garcia C, Stef M, Arveiler B, Lessire R, Garbay B. Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray. analysis. Biochim Biophys Acta,2005,1734(3):247-258
64. DeBono A, Yeats TH, Rose J, Bird D, Jetter R, Kunst L, Samuelsa L. Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface. Plant Cell,2009,21(4):1230-1238
65. Decottignies A, Gotteau A. Complete inventory of the yeast ABC proteins. Nature Genet,1997,15(2):137-45
66. Dietrich CR, Perera MADN, Yandeau-Nelson MD, Meeley RB, Nikolau BJ, Schnable PS. Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development. Plant Journa,2005,1.42:844-61
67. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus,1990,12:13-15
68. Dun XL, Zhou ZF, Xia SQ, Wen J, Yi B, Shen JX, Ma CZ, Tu JX, Fu TD. BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus. Plant J,2011,68(3):532-545
69. Farnham MW. Glossy and nonglossy near-isogenic lines USVL115-GL, USVL115-NG, USVL188-GL, and USVL188-NG of broccoli. Hortscience,2010,45(4):660-662
70. Gable K, Garton S, Napier J A, Dunn TM. Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system. JExp Bot,2004,55(396):543-545
71. Gao M, Li G, Quiros CF. Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana. Theor Appl Genet,2005,111(5):949-955
72. Gao M, Li G, Potter D, McCombie WR, Quiros CF. Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana. Plant cell reports,2006,25(6):592-598
73. Gonzalez A, Ayerbe L. Effect of terminal water stress on leaf epicuticular wax load, residual transpiration and grain yield in barley. Euphytica,2010,172 (3):341-349
74. Greer S, Wen M, Bird D, Wu X, Samuels L, Kunst L, Jetter R. The cytochrome P450 enzyme CYP96A15 is the mid-chain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis thaliana. Plant Physiol,2007,145(3):653-67
75. Haslam TM, Manas-Fernandez A, Zhao LF, Kunst L. Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths. Plant Physiol 2012,160(3):1164-1174
76. Haslam TM, Kunst L. Extending the story of very-long-chain fatty acid elongation. Plant Science 2013,210:93-107
77. Heyn FW. Marker genes in Brassica napus. Eucarpia Cruciferae Newsletter,1977,2: 9
78. Hong CP, Kwon SJ, Kim JS, Yang TJ, Park BS, Lim YP. Progress in Understanding and Sequencing the Genome of Brassica rapa. Int J Plant Genomics,2008,58:28-37
79. Hooker TS, Lam P, Zheng H, Kunst L. A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis. Plant Cell,2007,19(3):904-913
80. Hu TT, Pattyn P, Bakker EG, Cao J, Cheng JF, Clark RM, Fahlgren N, Fawcett JA, Grimwood J, Gundlach H, Haberer G, Hollister JD, Ossowski S, Ottilar RP, Salamov AA, Schneeberger K, Spannagl M, Wang X, Yang L, Nasrallah ME, et al. The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nature Genetics,2011,43(5):476-81
81. Hwang KT, Weller CL, Cuppett SL, Hanna MA. Changes in composition and thermal transition temperatures of grain sorghum wax during storage. Industrial Crops Products,2004,19(2):125-132
82. Hatakeyama K, Suwabe K, Tomita RN, Kato T, Nunome T, Fukuoka H, Matsumoto S. Identification and Characterization of Crrla, a Gene for Resistance to Clubroot Disease(Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS ONE,2013, 8(1):e54745.
83. Islam M A, Du H, Ning J, Ye H, Xiong. Characterization of Glossyl homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Molecular Biology,2009,70(4):443-456
84. Jambhulkar SJ, Raut RN. Inheritance of flower color and leaf waxiness in Brassica carinata A. Braun. Cruciferae Newsletter,1995,17:66-67
85. Jeffee CE. The fine structure of the plant cuticle. In:Riederer M, Muller C eds., Biology of the Plant Cuticle, Annual Plant Review. Blackwell Publishers,2006.11-110
86. Jenks MA, Tuttle H A, Eigenbrode SD, Feldmann KA. Leaf Epicuticular Waxes of the Eceriferum Mutants in Arabidopsis. Plant Physiol,1995,108(1):369-377
87. Karaba A. Improvement of water use efficiency in rice and tomato using Arabidopsis wax biosynthetic genes and transcription factors. [Ph. D. Dissertation]. The Netherlands:Wageningen University,2007
88. Kim H, Lee SB, Kim HJ, Min MK, Hwang I, Suh MC. Characterization of glycosylphosphatidylinositol-anchored lipid transfer protein 2 (LTPG2) and overlapping function between LTPG/LTPG1 and LTPG2 in cuticular wax export or accumulation in Arabidopsis thaliana. Plant Cell Physiol,2012,53(8):1391-1403
89. Kinnunen H, Huttunen S, Laakso K. UV-absorbing compounds and waxes of scots pine needles during a third growing season of supplemental UV-B. Environ Pollution, 2001,112 (2):215-220
90. Klein I, Salkadi B, Varadi A. An inventory of the human ABC proteins. Biochem Biophys Acta,1999,1461:237-262
91. Klypina N, Hanson SF. Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs. Plant Sci,2008,175(3):312-20
92. Koch k, Ensikat H J. The hydrophobic coatings of plant surfaces:Epicuticular wax crystals and their morphologies, crystallinity and molecular self-assembly. Micron, 2008,39:759-772.
93. Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol,2001,21(1):109-125
94. Kolattukudy P E. Enzymatic synthesis of fatty alcohols in Brassica oleracea. Arch.Biochem. Biophys,1971,142(2):701-709
95. Kosambi DD. The estimation of map distances from recombination values. Ann Eugen,1944,12(1):172-175
96. Kuhlmann F, Mueller C. UV-B impact on aphid performance mediated by plant quality and plant changes induced by aphids. Plant Biol,2010,12 (4):676-684
97. Kunst L, Samuels AL. Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res,2003,42(1):51-80
98. Kunst L, Samuels L. Plant cuticles shine:advances in wax biosynthesis and export. Curr Opin Plant Biol,2009,12(6):721-727
99. Kunst L, Taylor DC, Underhill E W. Fatty acid elongation in developing seed of Arabidopsis thaliana. Plant Physiol Biochem,1992,30(4):425-434
100. Ladaniya MS, Shyam S, Mahalle B. Suboptimum low temperature storage of 'Nagpur'mandarin as influenced by wax coating and intermittent warming. Indian J Horti,2005,62(1):1-7
101. Lagercrantz U, Lydiate D. Comparative genome analysis in Brassica. Genetics, 1996,144(4):1903-1909
102. Lam P, Zhao LF, McFarlane HE, Aiga M, Lam V, Hooker TS, Kunst L. RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis. Plant Physiol,2012,159(4):1385-1395
103. Lander E, Green P, Abrahamson J, Barlow A, Daley M, Lincoln S, Newburg L. MAPMAKER:an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics,1987,1(2):174-181
104.Landry BS, Hubert N, Etoh T, Harada JJ, Lincoln SE. A genetic map for Brassica napus based on restriction fragment length polymorphisms detected with expressed DNA sequences. Genome,1991,34(4):543-552.
105. Lee SB, Jung SJ, Go YS, Kim HU, Kim JK, Cho HJ, Park OK, Suh MC. Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress. Plant J,2009,60(3):462-475
106. Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L. Identification of the wax ester synthase/acyl-coenzyme A:Diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol,2008,148(1): 97-107
107. Li X, Chen L, Hong M, Zhang Y, Zu F, Wen J, Yi B, Ma CZ, Shen JX, Tu JX, Fu TD. A Large Insertion in bHLH Transcription Factor BrTT8 Resulting in Yellow Seed Coat in Brassica rapa. Plos One,2012,7(9):e44145
108. Li-Beisson Y., et al. Acyl-lipid metabolism. In:The Arabidopsis Book. American Society of Plant Biologists,2010.8:e0133
109. Lu SY, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA. Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J,2009, 59(4):553-564
110. Lukens L, Zou F, Lydiate D, Parkin I, Osbom T. Comparison of Brassica oleracea genetic map with the genome of Arabidopsis thaliana. Genetics,2003,164(1):359-372
111. Lundqvist U, von Wettstein-Knowles P. Dominant mutations at Cer-yy change barley spike wax into leaf blade wax. Carlsberg Res Communication,1982, 47(1):29-43
112.Mackova J. The role of plant cuticle in water loss protection. [Ph.D. Thesis]. University of South Bohemia,2010
113. Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L. CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell,1999,11:825-38
114. Millar AA, Kunst L. Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J,1997,12(1):121-131
115. Mo JG, Li WQ, Wang JS. Inheritance and Agronomic Performance of the Wax-less. Character in Brassica napus L. Plant Breeding,1992,108(3):256-259
116. Mo JG, Li WQ, Yu Q, Bodnaryk R P. Inheritance of the wax-less character of Brassica napus Nilla glossy. Canadian Journal of Plant Science,1995,75:893-894
117. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell,1990,2(4):279-289
118. North C, Priestley WG. A lossy-leaved mutant of Brussels sprout. Horticultural Research,1962,1:95-99
119.Oshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M. MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. Plant Cell,2013, 25(5):1609-24
120.Panjabi P, Jagannath A, Bisht NC, Padmaja KL, Sharma S, Gupta V, Pradhan AK, Pental D. Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers:homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes. BMC Genomics,2008,9:113
121. Park JJ, Jin P, Yoon J, Yang JI, Jeong HJ, Ranathunge K, Schreiber L, Franke R, Lee IJ, An G. Mutation in Wilted Dwarf and Lethal 1 (WDL1) causes abnormal cuticle formation and rapid water loss in rice. Plant Mol Biol,2010,74(1-2):91-103
122. Parkin IAP, Gulden SM, Sharpe AG, Lukens L, Trick M, Osbom TC, Lydiate D J. Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana. Genetics,2005,171:765-781
123. Parkin IAP, Sharpe AG, Lydiate DJ. Patterns of genome duplication within the Brassica napus genome. Genome,2003,46:291-303
124. Pascal S, Bernard A, Sorel M, Pervent M, Vile D, Haslam RP, Napier JA, Lessire R, Domergue F, Joubes J. The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process. Plant J, 2013,73(5):733-746
125. Pighin JA, Zheng HQ, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL. Plant cuticular lipid export requires an ABC transporter. Science,2004, 306(5696):702-704
126. Porra RJ, Thompson WA, Kriedemann PE. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents:verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta,1989,975(3): 384-394
127. Post-Beittenmiller D. Biochemistry and molecular biology of wax production in plants. Annu Rev Plant Physiol Plant Mol Biol,1996,47:405-430.
128. Priestley WG, Wills AB. Glossy-leaf in collard (Brassica oleracea L. var. acephala D. C.):An incomplete dominant. Euphytaca,1966,15:389-394
129. Priestly WG, Complementary'glossy' genes in Brussels sprout and their application to the production of F1 cultivars. Horticulture Research,1967,7:74-75
130. Pulsifer IP, Kluge S, Rowland O. Arabidopsis long-chain acyl-CoA synthetase 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiol Biochem,2012,51:31-39
131. Qaderi MM, Yeung EC, Reid DM. Growth and physiological responses of an invasive alien species, silene noctiflora, during two developmental stages to four levels of ultraviolet-B radiation. Ecoscience,2008,15 (2):150-159
132. Rana D, van den Boogaart T, O'Neill CM, Hynes L, Bent E, Macpherson L, Park J P, Kim YP, Bancroft I. Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives. Plant J,2004,40:725-733
133. Riederer M, Miiller C. Biology of the Plant Cuticle.2006,145-175
134.Rossak M, Smith M, Kunst L. Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana. Plant Mol Biol,2001,46(6): 717-725
135. Rowland O, Lee R, Franke R, Schreiber L, Kunst L. The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1. FEBS Lett,2007, 581:3538-44
136. Rowland O, Zheng HQ, Hepworth SR, Lam P, Jetter R, Kunst L. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol,2006,142(3):866-877
137.Roudier F, Gissot L, Beaudoin F, Haslam R, Michaelson L, Marion J, Molino D, Lima A, Bach L, Morin H, Tellier F, Palauqui JC, Bellec Y, Renne C, Miquel M, Dacosta M, Vignard J, Rochat C, Markham JE, Moreau P, Napier J, Faure JD. Very-long-chain fatty acids are involved in polar auxin transport and developmental patterning in Arabidopsis. Plant Cell,2010,22:364-375
138. Russin JS, Guo BZ, Tubajika KM, Brown RL, Cleveland TE, Wid strom NW. Comparison of Kernel Wax from Corn Genotypes Resistant or Susceptible to Aspergillus flavus. Biochem Cell Biol,1997,87 (5):529-533
139. Ryder CD, Smith LB, Teakle GR, King GJ. Contrasting genome organisation:two regions of the Brassica oleracea genome compared with collinear regions of the Arabidopsis thaliana genome. Genome,2001,44:808-817
140. Sakuradani E, Zhao L F, Haslam T M, Kunst L. The CER22 gene required for the synthesis of cuticular wax alkanes in Arabidopsis thalianais allelic to CER1. Planta, 2013,237(3):731-738
141. Samdur MY, Manivel P, Jain VB, Chikani BM, Gor HK, Desai S, Misra JB. Genotypic differences and water-deficit induced enhancement in epicuticular wax load in peanut. Crop Sci,2003,43(4):1294-1299
142. Samuels AL, Kunst L, Jetter R. Sealing plant surfaces:cuticular wax formation by epidermal cells. Annu Rev Plant Biol,2008,59:683-707
143. Sangtarash MH, Qaderi MM, Chinnappa CC, Reid DM. Differential sensitivity of canola(Brassica napus) seedlings to ultraviolet-B radiation, water stress and abscisic acid. Environ Exp Bot,2009,66 (2):212-219
144. Schnurr J, Shockey J, Browse J. The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell,2004,16:629-642
145. Schranz ME, Lysak MA, Mitchell-Olds T. The ABC's of comparative genomics in the Brassicaceae:building blocks of crucifer genomes. Trends Plant Sci,2006,11: 535-542
146. Shockey JM, Fulda MS, Browse JA. Arabidopsis contains nine long-chain acyl-Coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant Physiol,2002,129:1710-1722
147. Shen WY, Li JQ, Dauk M, Huang Y, Periappuram C, Wei YD, Zou JT. Metabolic and Transcriptional Responses of Glycerolipid Pathways to a Perturbation of Glycerol 3-Phosphate Metabolism in Arabidopsis.J BIOL CHEM,2010,285(30): 22957-22965
148. Sieber P, Schorderer M, Ryser U, Buchala A, Kolattukudy P, Metraux J P, Nawrath C. Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell,2000, 12:721-738
149. Singh D, Singh A B, and Singh S P. Note on inheritance of a new mutant with glaucous surface in Indian colza(Brassica campestris L. var. sarson Prain). Indian Journal of Agricultural Sciences,1969,39:1133-1134
150. Singh, D. Bloomless mutant in Brassica campestris var. sarson. Sci. Cult.1954,19: 454
151.Slocum MK, Figdore SS, Kennard WC, Suzuki JY, Osborn TC. Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea. Theor Appl Genet,1990,80(1):57-64
152. Snowdon RJ, Friedrich T, Friedt W, Kohler W. Identifying the chromosomes of theA-and C-genome diploid Brassica species B. rapa (syn. campestris) and oleracea in their amphidiploid B. napus. Theor Appl Genet,2002,104(4):533-538
153. Snowdon RJ, Kohler W, Friedt W, Kohler A. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor Appl Genet,1997,95(8): 1320-1324
154. Song KM, Suzuki JY, Slocum MK, Williams PM, Osborn TC. A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci. Theor Appl Genet,1991,82(3):296-304
155. Stringam GR. Genetics of two bloomless mutants in Brassica campestris. Canadian Journal of Genetic and Cytology,1976,18(2):225-230
156. Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F. Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol,2005,139(4):1649-1665
157. Tanaka T, Tanaka H, Machida C, Watanabe M, Machida Y. A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis. The Plant Journal,2004,37(1):139-146
158. Tatlioglu T. Inheritance of waxlessness in kohlrabi(Brassica oleracea L. var. gongylodes) and its utilization in hybrid seed production. Plant Breeding,1989,102: 215-221
159. Thampson KF. Cytoplasmic male sterility in oil-seed rape. Heredity,1972,29:253-257
160. Todd J, Post-Beittenmiller D, Jaworski JG. KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase. Plant J.1999,17(2):119-30
161.Truco M, Quiros C. Structure and organization of the B genome based on a linkage map in Brassica nigra. Theor Appl Genet,1994,89(5):590-598
162. van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell,1990,2(4):291-2899
163. Vioque J, Kolattukudy PE. Resolution and purification of an aldehyde-generating and an alcohol-generating fatty acyl-CoA reductase from pea leaves (Pisum sativum L).Arch Biochem Biophys,1997,340(1):64-72
164. Von WKP. New alleles of Cer-yy and cer-b. Barley Genet Newsl.1991,20:66-68
165. Wang H, Hao J, Chen X, Hao Z, Wang X, Lou Y, Peng Y, Guo Z. Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol Biol,2007,65(6):799-815
166. Wang J, Long Y, Wu B, Liu J, Jiang C, Shi L, Zhao J, King GJ, Meng J. The evolution of Brassica napus FLOWERING LOCUS T paralogues in the context of inverted chromosomal duplication blocks. BMC evolutionary biology,2009,9:271
167. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F, Huang S, Li X, Hua W, Wang J, Wang X, Freeling M, Pires JC, Paterson AH, Chalhoub B, Wang B, et al. The genome of the mesopolyploid crop species Brassica rapa. Nature genetics,2011,43(10):1035-1039
168. Wang YH, Wan LY, Zhang LX, Zhang ZJ, Zhang HW, Quan RD, Zhou SR, Huang RF. An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice. Plant Mol Biol,2012,78(3):275-288
169. Weng H, Molina I, Shockey J, Browse J. Organ fusion and defective cuticle function in alacsl lacs2 double mutant of Arabidopsis. Planta,2010,231:1089-1100
170. Wu R, Li S, He S, Wassmann F, Yu C, Qin G, Schreiber L, Qu L J, Gu H. CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis. Plant Cell,2011,23(9):3392-3411
171. Xia SQ, Cheng L, Zu F, Dun XL, Zhou ZF, Yi B, Wen J, Ma CZ, Shen JX, Tu JX, Fu TD. Mapping of BnMs4 and BnRf to a common microsyntenic region of Arabidopsis thaliana chromosome 3 using intron polymorphism markers. TheorAppl Genet,2012,124(7):1193-1200
172. Yadav AK, Singh H, Yadava TP. Inheritance of non-waxy trait in Indian mustard and its reaction to the aphids. Journal of Oilseeds Research,1985,2:120-123
173. Yan J, Yang X, Shah T, Sanchez-VH, Li JS, Warburton M, Zhou Y, Crouch J H, Xu Y B. High-throughput SNP genotyping with the Golden Gate assay in maize. Molecular Breeding,2009,25(3):441-451
174. Yang MG, Yang QY, Fu TD, Zhou YM. Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance. Plant Cell Rep,2011,30(3):373-388
175. Yang TJ, Kim JS, Lim KB, Kwon SJ, Kim JA, Jin M, Park JY, Lim MH, Kim HI, Kim SH, Lim YP, Park BS. The Korea Brassica Genome Project:A glimpse of the Brassica genome based on comparative genome analysis with Arabidopsis. Comparative and Functional Genomics,2005,6(3):138-146
176. Yeats TH, Rose JK. The formation and function of plant cuticles. Plant Physiol, 2013,163(1):5-20
177. Yi B, Zeng FQ, Lei SL, Chen YN, Yao XQ, Zhu Y, Wen J, Shen JX, Ma CZ, Tu JX, Fu TD. Two duplicate CYP704B1 homologous genes BnMsl and BnMs2 are required for pollen exine formation and tapetal development in Brassica napus. The Plant Journal,2010,63(6):925-938
178. Yu H, Xie W, Li J, Zhou F, Zhang Q. A whole-genome SNP array (RICE6K) for genomic breeding in rice. Plant Biotechnol J,2013, doi:10.1111/pbi.12113
179. Yu J, Zhao M, Wang X, Tong C, Huang S, Tehrim S, Liu Y, Hua W, Liu S. Bolbase: a comprehensive genomics database for Brassica oleracea. BMC Genomics,2013, 14(1):664
180. Zhang JY, Blancaflor EB, Sumner L W,Wang ZY. Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance. Plant Mol Biol,2007,64(3):265-278
181. Zhang JY, Broeckling CD, Blancaflor EB, Sledge M, Sumner LW, Wang ZY. Overexpression of WXP1, a putative Medicago truncatulaAP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa(Medicago sativa). Plant J,2005,42(5):689-707
182. Zhang X, Liu ZY, Wang P, Wang QS, Yang S, Feng H. Fine mapping of BrWaxl, a gene controlling cuticular wax biosynthesis in Chinese cabbage(Brassica rapa L. ssp. pekinensis). Mol Breeding,2013, DOI 10.1007/s11032-013-9914-0
183. Zhang J, Lu Y, Yuan Y, Zhang X, Geng J, Chen Y, Cloutier S, McVetty PB, Li G. Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa. Plant Mol Biol.2009,69(5):553-63
184. Zhao L, Katavic V, Li F, Haughn GW, Kunst L. Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis. Plant J,2010,64(6): 1048-1058
185. Zheng H, Rowland O, Kunst L. Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell,2005,17(5):1467-1481
186. Zinsou V, Wydra K, Ahohuendo B, Schreiber L. Leaf waxes of cassava(Manihot esculenta Crantz) in relation to ecozong and resistance to Xanthomonas blight. Euphytica,2006,149(1):189-198
2.陈志谊,王玉环,殷尚智.水稻纹枯病抗性机制的研究.中国农业科学,1992,25(4)：41-46
3.冯辉,杨晓飞,辛彬,赵鹏涛,王玉刚.无蜡粉型青梗菜核基因雄性不育系转育研究.沈阳农业大学学报,2010,41(2)：142-146
4.黄镇.分子标记辅助选择培育新型甘蓝型油菜隐性细胞核雄性不育系.[博士学位论文].武汉：华中农业大学图书馆,2008
5.康立功,齐凤坤,许向阳,李景富.番茄叶片蜡质和角质层与芝麻斑病菌侵染的关系.中国蔬菜,2010,(18)：47-50
6.李海英,刘亚光,杨庆凯.大豆叶片结构与灰斑病抗性的研究Ⅱ.大豆叶片组织结构与灰斑病抗性的关系.中国油料作物学报,2012,24(2)：74-76
7.李景涛.结球甘蓝无蜡粉亮绿性状遗传分析及分子标记研究.[硕士学位论文].北京：中国农业科学院,2012
8.李灵芝.3个水稻WAX2同源基因的蜡质合成及抗逆性功能分析.[硕士学位论文].湖南长沙：湖南农业大学,2012
9.李艳,惠有为,张仲凯,黄兴奇,李毅.转查耳酮合酶基因矮牵牛共抑制的研究.中国科学,2001,31(5)：401-407
10.刘仁虎,孟金陵.MapDraw,在Excel中绘制遗传连锁图的宏.遗传,2003,25(3)：317-321
11.刘忠松,官春云,陈社员,莫鉴国,Osborn T甘蓝型油菜显性无蜡粉基因的染色体组定位.湖南农业大学学报(自然科学版),2000,26(3)：182-184
12.卢艳丽.不同类型玉米种质分子特征分析及耐旱相关性状的连锁—连锁不平衡联合作图.[博士学位论文].四川雅安：四川农业大学,2011
13.卢泳全,汪旭升,黄伟素,肖天霞,郑燕,吴为人.基于水稻内含子长度多态性开发禾本科扩增共有序列遗传标记.中国农业科学,2006,39(3)：433-439
14.陆光远,杨光圣,傅廷栋.一个简便的适合于分析油菜中SSR位点的检测体系.中国油料作物学报,2003,25：79-81
15.马永彪.甘蓝型油菜与白菜比较作图和比较QTL研究.[硕士学位论文].武汉：华中农业大学图书馆,2009
16.莫鉴国,李万渠,彭云强等.甘蓝型油菜无蜡粉种植材料的改良以及在杂交育种上的应用.种子,1999,105(5)：18-20
17.牟香丽.结球甘蓝“无蜡粉亮叶”突变体性状分析.[硕士学位论文].哈尔滨：东北农业大学,2013
18.倪郁,郭彦军.植物超长链脂肪酸及角质层蜡质生物合成相关酶基因研究现状.遗传,2008,15(05)：561-567
19.任菲,张荣佳,陈强,白艳波,黄菲,李雪梅.ABA和SA对于提高植物抗旱及抗盐性的研究进展.生物技术通报,2012,6(03)：17-21
20.孙玉燕.利用拟南芥BOUNTIFUL和HARDY基因以及表皮蜡质合成基因提高番茄耐旱和耐盐性.中国农业科学院,2012
21.宋超.菌核病菌、UV-B低温胁迫条件下拟南芥表皮蜡质的响应机制研究.[硕士学位论文].重庆：西南大学,2013
22.王婧,刘泓利,宋超,倪郁.甘蓝型油菜叶表皮蜡质组分及结构与菌核病抗性关系.植物生理学报,2012,48：958-964
23.王美芳,陈巨莲,原国辉,雷振生,吴政卿,赵献林.植物表面蜡质对植食性昆虫的影响研究进展.生态环境学报,2009,18(3)：1155-1160
24.王玉锋,黄霁月,杨金水.基因工程培育可恢复的植物雄性不育系的研究进展.遗传,2011,15(01)：40-47
25.谢彦周.甘蓝型油菜隐性细胞核雄性不育上位抑制基因的精细定位.[博士学位论文].武汉：华中农业大学图书馆,2010
26.易斌.甘蓝型油菜隐性核不育基因Bnmsl的精细定位和克隆.[博士学位论文].武汉：华中农业大学图书馆,2007
27.张海禄,齐军仓.大麦叶片表皮蜡质含量与抗旱性的关系研究.新疆农业科学,2012,49(1)：22-27
28.张志飞,饶力群,向佐湘,胡晓敏,王晓杰.高羊茅叶片表皮蜡质含量与其抗旱性的关系.西北植物学报,2007,27(7)：1417-1421
29.赵建峰,沈向群,张海楼.菜薹无蜡粉性状遗传规律初探.园艺学报,2006,3(33)：538
30.周熙荣,李树林,顾龙弟.显性核不育油菜无蜡粉纯合两型系的选育.上海农业学报,1999,(4)：25-27
31.周熙荣,李树林,庄静.甘蓝型油菜无蜡粉隐性核不育两型系的选育.上海农业学报,2002,18(1)：20-24
32.曾芳琴.油菜S45AB隐性核不育分子机理与应用研究.[博士学位论文].武汉：华中农业大学图书馆,2010
33. Aarts MGM, Keijzer CJ, Stiekema WJ, Pereira A. Molecular characterization of the CERlgene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell,1995,7:2115-2127
34. Adamski NM, Bush MS, Simmonds J, Turner AS, Mugford SG, Jones A, Findlay K, Pedentchouk N, von Wettstein-Knowles P, Uauy C. The inhibitor of wax 1 locus (Iw1) prevents formation of P-and OH-β-diketones in wheat cuticular waxes and maps to a sub-cM interval on chromosome arm 2BS. Plant Journal,2013,74(6):989-1002
35. Aharoni A, Dixit S, Jetter R, Thoenes E, vanArkel G, PereiraA. The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance when Overexpressed in Arabidopsis. Plant Cell,2004,16:2463-2480
36. Angadi SP, Singh JP, Anand IJ. Inheritance on non-waxiness and tolerance to aphids in Indian mustard. Journal of Oilseeds Research,1987,4(2):265-267
37. Anstey TH, Moore JF. Inheritance of glossy foliage and cream petal. Heredity,1954, 45:39-41.
38. Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR. Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science,1992,258:1353-1355
39. Ayele M, Haas BJ, Kumar N, Wu H, Xiao YL, Van Aken S, Utterback TR, Wortman JR, White OR, Town CD. Whole genome shotgun sequencing of Brassica oleracea and its application to gene discovery and annotation in Arabidopsis. Genome Research,2005,15:487-495
40. Babula D, Kaczmarek M, Barakat A, Delseny M, Quiros CF, Sadowski J. Chromosomal mapping of Brassica oleracea based on ESTs from Arabidopsis thaliana:complexity of the comparative map. Mol Genet Genomics,2003,268:656-665
41. Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da Costa M, Boutin JP, Miquel M, Tellier F, Domergue F, Markham JE, Beaudoin F, Napier JA, Faure JD. The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development. P Natl Acad Sci Usa,2008,105(38): 14727-14731
42. Baker E. Chemistry and morphology of plant epicuticular waxes. In:Cutler D F, Alvin K L, Price C E eds., The Plant Culture. New York:Academic Press,1982. 139-165
43. Barrs HD, Weatherley PE. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust JBiol Sci,1962,15:413-428
44. Beaudoin F, Gable K, Sayanova O, Dunn T, Napier JA. A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-ketoreductase. JBiol Chem,2002,277(13):11481-11488
45. Beaudoin F, Wu X, Li F, Haslam RP, Markham JE, Zheng H, Napier JA, Kunst L Functional characterization of the Arabidopsis thaliana b-ketoacyl-CoA reductase candidates of the fatty acid elongase. Plant Physiol,2009,150(3):1174-1191
46. Bernard A, Domergue F, Pascal S, Jetter R, Renne C, Faure JD, Haslam RP, Napier JA, Lessire R, Joubes J. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell,2012,24(7): 3106-3118
47. Bernard A, Joubes J. Arabidopsis cuticular waxes:Advances in synthesis, export and regulation. Progress in Lipid Research,2013,52(1):110-129
48. Bessire M, Chassot C, Jacquat AC, Humphry M, Borel S, Petetot J, Metraux JP, Nawrath C. A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea. Embo J,2007,26(8):2158-2168
49. Bianchi, G. Murelli, C. and Ottaviano, E. Maize pollen lipids. Phytochemistry.1990, 29(3):739-744
50. Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X W, Yephremov A, Samuels L. Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J,2007,52(3):485-498
51. Bourdenx B, Bernard A, Domergue F, Pascal S, Leger A, Roby D, Pervent M, Vile D, Haslam RP, Napier J A, Lessire R, Joubes J. Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiol,2011,156(1):29-45
52. Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB. Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell,2003,15:1020-33
53. Brown GG, Formanova N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung WY, Landry BS. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J.2003,35(2):262-72.
54. Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL. WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA. 2004,101(13):4706-4711
55. Cameron KD, Teece MA, Smart LB. Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco. Plant Physiol,2006,140(1):176-183
56. Cardoza v, Stewart CN. Inereased Agrobacterium-mediated transformation and rooting effieiencies in eanola (Brassica napus L.) from hypoeotyl segment explants. Plant Cell Rep,2003,21(6):599-604
57. Chen X, Goodwin SM, Boroff VL, Liu X, Jenks MA. Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell,2003,15(5):1170-1185
58. Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X. BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol,2011,11:136
59. Cheng F, Mandakova T, Wu J, Xie Q, Lysak MA, Wang XW. Deciphering the Diploid Ancestral Genome of the Mesohexaploid Brassica rapa. Plant Cell,2013, 25(5):1541-1554
60. Cheung F, Trick M, Drou N, Lim Y R, Park J Y, Kwon S J, Kim J A, Scott R, Pires JC, Paterson AH, Town C, Bancroft I. Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence. Plant Cell,2009,21(7):1912-1928
61. Cheung WY, Friesen L, Rakow GFW, Seguin-Swartz G, Landry BS. A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]. Theor Appl Genet, 1997,94(6):841-851
62. Clough SJ, Bent AE. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal,1998,16(6):735-743
63. Costaglioli P, Joubes J, Garcia C, Stef M, Arveiler B, Lessire R, Garbay B. Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray. analysis. Biochim Biophys Acta,2005,1734(3):247-258
64. DeBono A, Yeats TH, Rose J, Bird D, Jetter R, Kunst L, Samuelsa L. Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface. Plant Cell,2009,21(4):1230-1238
65. Decottignies A, Gotteau A. Complete inventory of the yeast ABC proteins. Nature Genet,1997,15(2):137-45
66. Dietrich CR, Perera MADN, Yandeau-Nelson MD, Meeley RB, Nikolau BJ, Schnable PS. Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development. Plant Journa,2005,1.42:844-61
67. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus,1990,12:13-15
68. Dun XL, Zhou ZF, Xia SQ, Wen J, Yi B, Shen JX, Ma CZ, Tu JX, Fu TD. BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus. Plant J,2011,68(3):532-545
69. Farnham MW. Glossy and nonglossy near-isogenic lines USVL115-GL, USVL115-NG, USVL188-GL, and USVL188-NG of broccoli. Hortscience,2010,45(4):660-662
70. Gable K, Garton S, Napier J A, Dunn TM. Functional characterization of the Arabidopsis thaliana orthologue of Tsc13p, the enoyl reductase of the yeast microsomal fatty acid elongating system. JExp Bot,2004,55(396):543-545
71. Gao M, Li G, Quiros CF. Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana. Theor Appl Genet,2005,111(5):949-955
72. Gao M, Li G, Potter D, McCombie WR, Quiros CF. Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana. Plant cell reports,2006,25(6):592-598
73. Gonzalez A, Ayerbe L. Effect of terminal water stress on leaf epicuticular wax load, residual transpiration and grain yield in barley. Euphytica,2010,172 (3):341-349
74. Greer S, Wen M, Bird D, Wu X, Samuels L, Kunst L, Jetter R. The cytochrome P450 enzyme CYP96A15 is the mid-chain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis thaliana. Plant Physiol,2007,145(3):653-67
75. Haslam TM, Manas-Fernandez A, Zhao LF, Kunst L. Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths. Plant Physiol 2012,160(3):1164-1174
76. Haslam TM, Kunst L. Extending the story of very-long-chain fatty acid elongation. Plant Science 2013,210:93-107
77. Heyn FW. Marker genes in Brassica napus. Eucarpia Cruciferae Newsletter,1977,2: 9
78. Hong CP, Kwon SJ, Kim JS, Yang TJ, Park BS, Lim YP. Progress in Understanding and Sequencing the Genome of Brassica rapa. Int J Plant Genomics,2008,58:28-37
79. Hooker TS, Lam P, Zheng H, Kunst L. A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis. Plant Cell,2007,19(3):904-913
80. Hu TT, Pattyn P, Bakker EG, Cao J, Cheng JF, Clark RM, Fahlgren N, Fawcett JA, Grimwood J, Gundlach H, Haberer G, Hollister JD, Ossowski S, Ottilar RP, Salamov AA, Schneeberger K, Spannagl M, Wang X, Yang L, Nasrallah ME, et al. The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nature Genetics,2011,43(5):476-81
81. Hwang KT, Weller CL, Cuppett SL, Hanna MA. Changes in composition and thermal transition temperatures of grain sorghum wax during storage. Industrial Crops Products,2004,19(2):125-132
82. Hatakeyama K, Suwabe K, Tomita RN, Kato T, Nunome T, Fukuoka H, Matsumoto S. Identification and Characterization of Crrla, a Gene for Resistance to Clubroot Disease(Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS ONE,2013, 8(1):e54745.
83. Islam M A, Du H, Ning J, Ye H, Xiong. Characterization of Glossyl homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Molecular Biology,2009,70(4):443-456
84. Jambhulkar SJ, Raut RN. Inheritance of flower color and leaf waxiness in Brassica carinata A. Braun. Cruciferae Newsletter,1995,17:66-67
85. Jeffee CE. The fine structure of the plant cuticle. In:Riederer M, Muller C eds., Biology of the Plant Cuticle, Annual Plant Review. Blackwell Publishers,2006.11-110
86. Jenks MA, Tuttle H A, Eigenbrode SD, Feldmann KA. Leaf Epicuticular Waxes of the Eceriferum Mutants in Arabidopsis. Plant Physiol,1995,108(1):369-377
87. Karaba A. Improvement of water use efficiency in rice and tomato using Arabidopsis wax biosynthetic genes and transcription factors. [Ph. D. Dissertation]. The Netherlands:Wageningen University,2007
88. Kim H, Lee SB, Kim HJ, Min MK, Hwang I, Suh MC. Characterization of glycosylphosphatidylinositol-anchored lipid transfer protein 2 (LTPG2) and overlapping function between LTPG/LTPG1 and LTPG2 in cuticular wax export or accumulation in Arabidopsis thaliana. Plant Cell Physiol,2012,53(8):1391-1403
89. Kinnunen H, Huttunen S, Laakso K. UV-absorbing compounds and waxes of scots pine needles during a third growing season of supplemental UV-B. Environ Pollution, 2001,112 (2):215-220
90. Klein I, Salkadi B, Varadi A. An inventory of the human ABC proteins. Biochem Biophys Acta,1999,1461:237-262
91. Klypina N, Hanson SF. Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs. Plant Sci,2008,175(3):312-20
92. Koch k, Ensikat H J. The hydrophobic coatings of plant surfaces:Epicuticular wax crystals and their morphologies, crystallinity and molecular self-assembly. Micron, 2008,39:759-772.
93. Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol,2001,21(1):109-125
94. Kolattukudy P E. Enzymatic synthesis of fatty alcohols in Brassica oleracea. Arch.Biochem. Biophys,1971,142(2):701-709
95. Kosambi DD. The estimation of map distances from recombination values. Ann Eugen,1944,12(1):172-175
96. Kuhlmann F, Mueller C. UV-B impact on aphid performance mediated by plant quality and plant changes induced by aphids. Plant Biol,2010,12 (4):676-684
97. Kunst L, Samuels AL. Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res,2003,42(1):51-80
98. Kunst L, Samuels L. Plant cuticles shine:advances in wax biosynthesis and export. Curr Opin Plant Biol,2009,12(6):721-727
99. Kunst L, Taylor DC, Underhill E W. Fatty acid elongation in developing seed of Arabidopsis thaliana. Plant Physiol Biochem,1992,30(4):425-434
100. Ladaniya MS, Shyam S, Mahalle B. Suboptimum low temperature storage of 'Nagpur'mandarin as influenced by wax coating and intermittent warming. Indian J Horti,2005,62(1):1-7
101. Lagercrantz U, Lydiate D. Comparative genome analysis in Brassica. Genetics, 1996,144(4):1903-1909
102. Lam P, Zhao LF, McFarlane HE, Aiga M, Lam V, Hooker TS, Kunst L. RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis. Plant Physiol,2012,159(4):1385-1395
103. Lander E, Green P, Abrahamson J, Barlow A, Daley M, Lincoln S, Newburg L. MAPMAKER:an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics,1987,1(2):174-181
104.Landry BS, Hubert N, Etoh T, Harada JJ, Lincoln SE. A genetic map for Brassica napus based on restriction fragment length polymorphisms detected with expressed DNA sequences. Genome,1991,34(4):543-552.
105. Lee SB, Jung SJ, Go YS, Kim HU, Kim JK, Cho HJ, Park OK, Suh MC. Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress. Plant J,2009,60(3):462-475
106. Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L. Identification of the wax ester synthase/acyl-coenzyme A:Diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol,2008,148(1): 97-107
107. Li X, Chen L, Hong M, Zhang Y, Zu F, Wen J, Yi B, Ma CZ, Shen JX, Tu JX, Fu TD. A Large Insertion in bHLH Transcription Factor BrTT8 Resulting in Yellow Seed Coat in Brassica rapa. Plos One,2012,7(9):e44145
108. Li-Beisson Y., et al. Acyl-lipid metabolism. In:The Arabidopsis Book. American Society of Plant Biologists,2010.8:e0133
109. Lu SY, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA. Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J,2009, 59(4):553-564
110. Lukens L, Zou F, Lydiate D, Parkin I, Osbom T. Comparison of Brassica oleracea genetic map with the genome of Arabidopsis thaliana. Genetics,2003,164(1):359-372
111. Lundqvist U, von Wettstein-Knowles P. Dominant mutations at Cer-yy change barley spike wax into leaf blade wax. Carlsberg Res Communication,1982, 47(1):29-43
112.Mackova J. The role of plant cuticle in water loss protection. [Ph.D. Thesis]. University of South Bohemia,2010
113. Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L. CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell,1999,11:825-38
114. Millar AA, Kunst L. Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J,1997,12(1):121-131
115. Mo JG, Li WQ, Wang JS. Inheritance and Agronomic Performance of the Wax-less. Character in Brassica napus L. Plant Breeding,1992,108(3):256-259
116. Mo JG, Li WQ, Yu Q, Bodnaryk R P. Inheritance of the wax-less character of Brassica napus Nilla glossy. Canadian Journal of Plant Science,1995,75:893-894
117. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell,1990,2(4):279-289
118. North C, Priestley WG. A lossy-leaved mutant of Brussels sprout. Horticultural Research,1962,1:95-99
119.Oshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M. MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri. Plant Cell,2013, 25(5):1609-24
120.Panjabi P, Jagannath A, Bisht NC, Padmaja KL, Sharma S, Gupta V, Pradhan AK, Pental D. Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers:homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes. BMC Genomics,2008,9:113
121. Park JJ, Jin P, Yoon J, Yang JI, Jeong HJ, Ranathunge K, Schreiber L, Franke R, Lee IJ, An G. Mutation in Wilted Dwarf and Lethal 1 (WDL1) causes abnormal cuticle formation and rapid water loss in rice. Plant Mol Biol,2010,74(1-2):91-103
122. Parkin IAP, Gulden SM, Sharpe AG, Lukens L, Trick M, Osbom TC, Lydiate D J. Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana. Genetics,2005,171:765-781
123. Parkin IAP, Sharpe AG, Lydiate DJ. Patterns of genome duplication within the Brassica napus genome. Genome,2003,46:291-303
124. Pascal S, Bernard A, Sorel M, Pervent M, Vile D, Haslam RP, Napier JA, Lessire R, Domergue F, Joubes J. The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process. Plant J, 2013,73(5):733-746
125. Pighin JA, Zheng HQ, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL. Plant cuticular lipid export requires an ABC transporter. Science,2004, 306(5696):702-704
126. Porra RJ, Thompson WA, Kriedemann PE. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents:verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta,1989,975(3): 384-394
127. Post-Beittenmiller D. Biochemistry and molecular biology of wax production in plants. Annu Rev Plant Physiol Plant Mol Biol,1996,47:405-430.
128. Priestley WG, Wills AB. Glossy-leaf in collard (Brassica oleracea L. var. acephala D. C.):An incomplete dominant. Euphytaca,1966,15:389-394
129. Priestly WG, Complementary'glossy' genes in Brussels sprout and their application to the production of F1 cultivars. Horticulture Research,1967,7:74-75
130. Pulsifer IP, Kluge S, Rowland O. Arabidopsis long-chain acyl-CoA synthetase 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiol Biochem,2012,51:31-39
131. Qaderi MM, Yeung EC, Reid DM. Growth and physiological responses of an invasive alien species, silene noctiflora, during two developmental stages to four levels of ultraviolet-B radiation. Ecoscience,2008,15 (2):150-159
132. Rana D, van den Boogaart T, O'Neill CM, Hynes L, Bent E, Macpherson L, Park J P, Kim YP, Bancroft I. Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives. Plant J,2004,40:725-733
133. Riederer M, Miiller C. Biology of the Plant Cuticle.2006,145-175
134.Rossak M, Smith M, Kunst L. Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana. Plant Mol Biol,2001,46(6): 717-725
135. Rowland O, Lee R, Franke R, Schreiber L, Kunst L. The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1. FEBS Lett,2007, 581:3538-44
136. Rowland O, Zheng HQ, Hepworth SR, Lam P, Jetter R, Kunst L. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol,2006,142(3):866-877
137.Roudier F, Gissot L, Beaudoin F, Haslam R, Michaelson L, Marion J, Molino D, Lima A, Bach L, Morin H, Tellier F, Palauqui JC, Bellec Y, Renne C, Miquel M, Dacosta M, Vignard J, Rochat C, Markham JE, Moreau P, Napier J, Faure JD. Very-long-chain fatty acids are involved in polar auxin transport and developmental patterning in Arabidopsis. Plant Cell,2010,22:364-375
138. Russin JS, Guo BZ, Tubajika KM, Brown RL, Cleveland TE, Wid strom NW. Comparison of Kernel Wax from Corn Genotypes Resistant or Susceptible to Aspergillus flavus. Biochem Cell Biol,1997,87 (5):529-533
139. Ryder CD, Smith LB, Teakle GR, King GJ. Contrasting genome organisation:two regions of the Brassica oleracea genome compared with collinear regions of the Arabidopsis thaliana genome. Genome,2001,44:808-817
140. Sakuradani E, Zhao L F, Haslam T M, Kunst L. The CER22 gene required for the synthesis of cuticular wax alkanes in Arabidopsis thalianais allelic to CER1. Planta, 2013,237(3):731-738
141. Samdur MY, Manivel P, Jain VB, Chikani BM, Gor HK, Desai S, Misra JB. Genotypic differences and water-deficit induced enhancement in epicuticular wax load in peanut. Crop Sci,2003,43(4):1294-1299
142. Samuels AL, Kunst L, Jetter R. Sealing plant surfaces:cuticular wax formation by epidermal cells. Annu Rev Plant Biol,2008,59:683-707
143. Sangtarash MH, Qaderi MM, Chinnappa CC, Reid DM. Differential sensitivity of canola(Brassica napus) seedlings to ultraviolet-B radiation, water stress and abscisic acid. Environ Exp Bot,2009,66 (2):212-219
144. Schnurr J, Shockey J, Browse J. The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell,2004,16:629-642
145. Schranz ME, Lysak MA, Mitchell-Olds T. The ABC's of comparative genomics in the Brassicaceae:building blocks of crucifer genomes. Trends Plant Sci,2006,11: 535-542
146. Shockey JM, Fulda MS, Browse JA. Arabidopsis contains nine long-chain acyl-Coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant Physiol,2002,129:1710-1722
147. Shen WY, Li JQ, Dauk M, Huang Y, Periappuram C, Wei YD, Zou JT. Metabolic and Transcriptional Responses of Glycerolipid Pathways to a Perturbation of Glycerol 3-Phosphate Metabolism in Arabidopsis.J BIOL CHEM,2010,285(30): 22957-22965
148. Sieber P, Schorderer M, Ryser U, Buchala A, Kolattukudy P, Metraux J P, Nawrath C. Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell,2000, 12:721-738
149. Singh D, Singh A B, and Singh S P. Note on inheritance of a new mutant with glaucous surface in Indian colza(Brassica campestris L. var. sarson Prain). Indian Journal of Agricultural Sciences,1969,39:1133-1134
150. Singh, D. Bloomless mutant in Brassica campestris var. sarson. Sci. Cult.1954,19: 454
151.Slocum MK, Figdore SS, Kennard WC, Suzuki JY, Osborn TC. Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea. Theor Appl Genet,1990,80(1):57-64
152. Snowdon RJ, Friedrich T, Friedt W, Kohler W. Identifying the chromosomes of theA-and C-genome diploid Brassica species B. rapa (syn. campestris) and oleracea in their amphidiploid B. napus. Theor Appl Genet,2002,104(4):533-538
153. Snowdon RJ, Kohler W, Friedt W, Kohler A. Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids. Theor Appl Genet,1997,95(8): 1320-1324
154. Song KM, Suzuki JY, Slocum MK, Williams PM, Osborn TC. A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci. Theor Appl Genet,1991,82(3):296-304
155. Stringam GR. Genetics of two bloomless mutants in Brassica campestris. Canadian Journal of Genetic and Cytology,1976,18(2):225-230
156. Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F. Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol,2005,139(4):1649-1665
157. Tanaka T, Tanaka H, Machida C, Watanabe M, Machida Y. A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis. The Plant Journal,2004,37(1):139-146
158. Tatlioglu T. Inheritance of waxlessness in kohlrabi(Brassica oleracea L. var. gongylodes) and its utilization in hybrid seed production. Plant Breeding,1989,102: 215-221
159. Thampson KF. Cytoplasmic male sterility in oil-seed rape. Heredity,1972,29:253-257
160. Todd J, Post-Beittenmiller D, Jaworski JG. KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase. Plant J.1999,17(2):119-30
161.Truco M, Quiros C. Structure and organization of the B genome based on a linkage map in Brassica nigra. Theor Appl Genet,1994,89(5):590-598
162. van der Krol AR, Mur LA, Beld M, Mol JN, Stuitje AR. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell,1990,2(4):291-2899
163. Vioque J, Kolattukudy PE. Resolution and purification of an aldehyde-generating and an alcohol-generating fatty acyl-CoA reductase from pea leaves (Pisum sativum L).Arch Biochem Biophys,1997,340(1):64-72
164. Von WKP. New alleles of Cer-yy and cer-b. Barley Genet Newsl.1991,20:66-68
165. Wang H, Hao J, Chen X, Hao Z, Wang X, Lou Y, Peng Y, Guo Z. Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol Biol,2007,65(6):799-815
166. Wang J, Long Y, Wu B, Liu J, Jiang C, Shi L, Zhao J, King GJ, Meng J. The evolution of Brassica napus FLOWERING LOCUS T paralogues in the context of inverted chromosomal duplication blocks. BMC evolutionary biology,2009,9:271
167. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F, Huang S, Li X, Hua W, Wang J, Wang X, Freeling M, Pires JC, Paterson AH, Chalhoub B, Wang B, et al. The genome of the mesopolyploid crop species Brassica rapa. Nature genetics,2011,43(10):1035-1039
168. Wang YH, Wan LY, Zhang LX, Zhang ZJ, Zhang HW, Quan RD, Zhou SR, Huang RF. An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice. Plant Mol Biol,2012,78(3):275-288
169. Weng H, Molina I, Shockey J, Browse J. Organ fusion and defective cuticle function in alacsl lacs2 double mutant of Arabidopsis. Planta,2010,231:1089-1100
170. Wu R, Li S, He S, Wassmann F, Yu C, Qin G, Schreiber L, Qu L J, Gu H. CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis. Plant Cell,2011,23(9):3392-3411
171. Xia SQ, Cheng L, Zu F, Dun XL, Zhou ZF, Yi B, Wen J, Ma CZ, Shen JX, Tu JX, Fu TD. Mapping of BnMs4 and BnRf to a common microsyntenic region of Arabidopsis thaliana chromosome 3 using intron polymorphism markers. TheorAppl Genet,2012,124(7):1193-1200
172. Yadav AK, Singh H, Yadava TP. Inheritance of non-waxy trait in Indian mustard and its reaction to the aphids. Journal of Oilseeds Research,1985,2:120-123
173. Yan J, Yang X, Shah T, Sanchez-VH, Li JS, Warburton M, Zhou Y, Crouch J H, Xu Y B. High-throughput SNP genotyping with the Golden Gate assay in maize. Molecular Breeding,2009,25(3):441-451
174. Yang MG, Yang QY, Fu TD, Zhou YM. Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance. Plant Cell Rep,2011,30(3):373-388
175. Yang TJ, Kim JS, Lim KB, Kwon SJ, Kim JA, Jin M, Park JY, Lim MH, Kim HI, Kim SH, Lim YP, Park BS. The Korea Brassica Genome Project:A glimpse of the Brassica genome based on comparative genome analysis with Arabidopsis. Comparative and Functional Genomics,2005,6(3):138-146
176. Yeats TH, Rose JK. The formation and function of plant cuticles. Plant Physiol, 2013,163(1):5-20
177. Yi B, Zeng FQ, Lei SL, Chen YN, Yao XQ, Zhu Y, Wen J, Shen JX, Ma CZ, Tu JX, Fu TD. Two duplicate CYP704B1 homologous genes BnMsl and BnMs2 are required for pollen exine formation and tapetal development in Brassica napus. The Plant Journal,2010,63(6):925-938
178. Yu H, Xie W, Li J, Zhou F, Zhang Q. A whole-genome SNP array (RICE6K) for genomic breeding in rice. Plant Biotechnol J,2013, doi:10.1111/pbi.12113
179. Yu J, Zhao M, Wang X, Tong C, Huang S, Tehrim S, Liu Y, Hua W, Liu S. Bolbase: a comprehensive genomics database for Brassica oleracea. BMC Genomics,2013, 14(1):664
180. Zhang JY, Blancaflor EB, Sumner L W,Wang ZY. Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance. Plant Mol Biol,2007,64(3):265-278
181. Zhang JY, Broeckling CD, Blancaflor EB, Sledge M, Sumner LW, Wang ZY. Overexpression of WXP1, a putative Medicago truncatulaAP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa(Medicago sativa). Plant J,2005,42(5):689-707
182. Zhang X, Liu ZY, Wang P, Wang QS, Yang S, Feng H. Fine mapping of BrWaxl, a gene controlling cuticular wax biosynthesis in Chinese cabbage(Brassica rapa L. ssp. pekinensis). Mol Breeding,2013, DOI 10.1007/s11032-013-9914-0
183. Zhang J, Lu Y, Yuan Y, Zhang X, Geng J, Chen Y, Cloutier S, McVetty PB, Li G. Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa. Plant Mol Biol.2009,69(5):553-63
184. Zhao L, Katavic V, Li F, Haughn GW, Kunst L. Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis. Plant J,2010,64(6): 1048-1058
185. Zheng H, Rowland O, Kunst L. Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell,2005,17(5):1467-1481
186. Zinsou V, Wydra K, Ahohuendo B, Schreiber L. Leaf waxes of cassava(Manihot esculenta Crantz) in relation to ecozong and resistance to Xanthomonas blight. Euphytica,2006,149(1):189-198