甘蓝型油菜种皮色泽形成机理研究
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摘要
甘蓝型油菜(Brassica napus L,2n=38, AACC)属于十字花科(Cruclferae)芸薹属(Brassica)植物,是经过白菜(B. rapa)和甘蓝(B. oleracea)2个二倍体物种通过种间杂交形成的异源四倍体,是世界上重要油料作物之一,人类营养价值丰富的食用油来源之一,也是植物蛋白饲料重要的来源之一。甘蓝型油菜在植物分类学上和拟南芥同属十字花科植物,具有共同的祖先,亲缘关系比较近,并且基因组间存在广泛的共线性,基因序列和保守结构域也具有很高的一致性。已完成的白菜基因组测序工作和即将完成的甘蓝基因组测序工作,对甘蓝型油菜的比较基因组学研究、基因组遗传进化关系研究以及基因组结构特征的研究具有重要的理论指导意义,并为甘蓝型油菜的作物品质改良提供应用基础。
目前,大量的研究结果表明甘蓝型油菜黄籽品系与黑籽品系相比具有明显的优点,甘蓝型黄籽油菜不仅种皮薄、含油量高、蛋白含量高等优点,而且植酸、单宁、芥子碱和纤维素等有毒或抗营养物质含量较低,菜籽油的品质及饼粕的质量都有所提高。长期以来,国内外众多研究者对粒色相关性状进行了广泛研究,高产优质的黄籽油菜选育已成为当前油菜的重要目标之一。甘蓝型油菜在自然界中不存在天然的黄籽种质资源,甘蓝型油菜黄籽性状遗传复杂,且易受环境影响,目前获得的甘蓝型黄籽材料,其黄籽主效基因来自于白菜、甘蓝、芥菜型油菜、埃塞俄比亚芥诱变后代,但形成的机理仍然不清楚。
本研究通过分析黄黑籽甘蓝型油菜种皮发育过程中类黄酮及酚类化合物累积的动态变化规律以及不同发育时期种皮中相关结构基因和转录因子的差异表达,为揭示甘蓝型油菜种皮色素累积规律和相关基因的表达模式及调控机理提供了重要线索。另外,通过构建高密度遗传连锁图谱,用复合区间作图法在多个环境中重复检测到一个影响种皮色泽的主效QTL,通过将该区间与拟南芥和韩国提供的白菜第九染色体对应区间DNA序列进行了生物信息学分析,最终结合测定油菜籽粒色素合成高峰期的表达谱信息,找出稳定差异表达的基因并将此作为候选基因,构建各候选基因的正义表达和RNAi植物表达载体,采用根癌农杆菌介导法分别转化了甘蓝型黄籽油菜(正义表达)和甘蓝型黑籽油菜(RNAi)。主要研究结果如下：
1原花色素类多酚类物质的积累在黄黑籽甘蓝型油菜种皮中具有显著差异
通过用甲苯胺蓝染液(TBO)对不同发育时期的甘蓝型油菜黑籽中油821(ZY821)和黄籽GH06种子石蜡切片染色的结果表明：甘蓝型油菜种皮中色素的主要累积部位是种皮栅栏层和色素层,且原花色素类多酚物质初期最先在种脐部产生,随着种子的发育,在种皮栅栏层和色素层组织中色素积累逐渐增多,且黄黑籽间存在明显差异,在黑籽ZY821中原花色素类多酚类物质的含量明显高于黄籽GH06,说明原花色素类多酚类物质的积累是油菜粒色的重要原因之一。
2黄籽甘蓝型油菜中与表儿茶素及其衍生物合成相关的酶和基因功能衰减比黑籽强烈
通过LC-ESI-MS分析方法对甘蓝型油菜黑籽ZY821和黄籽GH06不同发育时期种皮中黄酮类衍生物含量进行的分析结果表明：35种黄酮类化合物的衍生物在黄黑籽甘蓝型油菜种皮中被检测到,大致可分为表儿茶素、槲皮素、异鼠李素和山萘酚等四类黄酮类化合物的衍生物,各种衍生物在黑籽ZY821中的含量明显高于黄籽GH06：但表儿茶素及其衍生物在黄籽GH06的整个发育时期中均未被检测到,且在整个发育过程中与表儿茶素合成相关的基因(BnUGT2和BnTT12)的表达水平明显低于黑籽,说明与表儿茶素及其衍生物合成相关的酶和基因功能衰减可能是引起色泽差异的关键因子。
3甘蓝型油菜种皮中不溶性原花色素的积累可能导致了黄黑籽在种皮颜色上的差异
甘蓝型油菜与拟南芥类似,在发育早期并不存在不溶性的原花色素的积累,而在种皮发育的中后期开始迅速积累,但黑籽油菜中不溶性原花色素的积累与黄籽不存在显著性差异,随着种子的成熟,种皮中大量的不溶性原花色素的聚合物在细胞壁上交联,促进了种皮颜色上的差异。
4甘蓝型油菜种皮色泽与相关性状的统计分析
不同环境中甘蓝型油菜种皮色泽与种子皮壳率、种皮中花色素、类黄酮、总酚和黑色素之间存在极显著的负相关,而皮壳率与四种种皮色素之间存在极显著的正相关,说明在甘蓝型油菜品种选育过程中通过黄籽性状选育可以同时多个相关性状进行遗传改良,同时这些性状之间可能存在共同或部分相同的代谢途径,或者是在同一遗传系统中受相同的上游调控因子调控。
5甘蓝型油菜种皮中类黄酮途径相关基因的表达差异可能是影响种皮色泽形成的根本原因
本研究通过qRT-PCR的方法研究了参与苯丙烷和类黄酮代谢途径相关的基因在黑籽甘蓝型油菜中油821和黄籽GH06七个不同发育时期种皮中的表达差异。结果表明：有9个结构基因(BnPAL、BnC4H、BnTT4、BnTT6、BnTT3、BnTT18、BnTT12、BnTT10和BnUGT2)和3个转录因子(BnTTG1、BnTTG2和BnTT8)在黄黑籽种皮中存在极显著表达差异,编码两种催化多种酚类及类黄酮终产物提供前体物质的形成关键酶的BnPAL和BnC4H两个基因和促进原花色素起始单体形成的重要基因BnTT12是引起黄黑籽种皮差异的关键基因。另外,BnTT4在授粉后28天达到最高峰值,而下游基因(BnTT5、BnTT6和BnTT7)晚于该基因一个时期达到最高峰值,BnTT3和BnTT18的最高峰值也晚于其上游基因一个时期达到最高峰值,说明BnTT4是甘蓝型油菜类黄酮代谢途径中影响下游基因的表达的一个重要基因,在类黄酮途径中存在上游基因对下游基因的调控。甘蓝型油菜种皮中的转录因子在黄黑籽之间表达模式与拟南芥类似,BnTTG1的表达受到BnTTG2的调控,而BnTT2、 BnTT8和BnTTG1协同作用又同时调控了甘蓝型油菜的类黄酮代谢过程。
6甘蓝型油菜遗传连锁图谱的构建
本研究利用重组自交系为作图群体,构建了一张包含1089个标记位点,其中包括451个SRAP标记,456个SSR标记,97个RAPD标记和75个IBP标记,覆盖19个连锁群,总长度约为2775cM,标记间的平均距离为2.54cM的甘蓝型油菜遗传图谱。每个连锁群上的标记数为6-184个,连锁群长度为47.22-243.46cM,连锁群密度为0.83cM/标记(A09)-7.87cM/标记(C02)。
7甘蓝型油菜遗传图谱与公开发表的遗传图谱的比较分析
通过比较作图,与已公开发表的甘蓝型油菜的遗传连锁图谱进行了比较分析,结果表明该遗传图谱的连锁群与已发表的甘蓝型油菜的十九条染色体基本吻合,并且存在237个共有的标记位点,同时整合了根据白菜和甘蓝基因组信息设计的特异性引物196对,说明图谱具有了一定的通用性,但仍未达到饱和。
8甘蓝型油菜遗传图谱与白菜和拟南芥基因组的共线性分析
本研究将遗传连锁图谱上的SSR和IBP标记逐一与白菜基因组的Scaffold序列进行了比较,最终将甘蓝型油菜遗传连锁图谱上的436对引物标记锚定到白菜基因组的126个Scaffold上。甘蓝型油菜An与白菜Ar染色体组存在很好的共线性,但有些标记顺序存在重排现象,甘蓝型油菜A基因组来源于白菜。将锚定到白菜基因组Scaffold上的436对引物标记序列与拟南芥的全基因组进行了比对,仅有一对标记没有找到同源性,某些标记序列与拟南芥序列基本一致,在一定程度上说明了甘蓝型油菜与白菜和模式植物拟南芥具有共同的原始祖先。
9甘蓝型油菜种皮色泽的QTL分析
应用复合区间作图分析方法,对重组自交系SWU-2群体在6个环境中的种皮色泽进行了QTL初级定位和效应检测,共检测到18个种皮色泽相关的QTL,单个QTL解释性状表现的变幅在5.69%-64.17%之间。这些QTL分别位于第A01,A04,A07,A09,C03,C05和C08七个不同的连锁群上；控制种皮色泽的基因表现为主效+微效多基因控制的数量性状的特点,并受到环境条件影响。
10甘蓝型油菜在色素合成高峰期相关基因的差异表达谱数据分析
本研究以作图群体SWU-2的两个亲本(黑籽中油821和黄籽GH06)与一对黄黑籽近等基因系为材料,在种皮色素合成高峰期(授粉后42天)作基因表达谱分析,以黄籽为对照,共发现在种皮色泽主效QTL区间具有稳定上调差异表达的基因27个,这些基因大部分为一些与色素合成相关的调控因子和转运蛋白基因。
11甘蓝型油菜种皮色泽主效QTL区间的候选基因的筛选
将种皮色泽主效QTL区间与韩国提供的第九染色体对应的DNA序列进行生物信息学分析,确定该区段总长约为440kb左右,通过与我国测序的白菜、甘蓝基因组序列在NCBI (http://www.ncbi.nlm.nih.gov/)和白菜基因组数据库(http://www.brassicadb.org/brad/))中进行序列比对,该区间序列与白菜和甘蓝具有很高的同源性。提取该区间序列以拟南芥基因组数据集为模型进行基因预测,并在NCBI上进行BLASTP比对,进行基因注释,发现该区段内共存在约105个基因,结合甘蓝型油菜黄黑籽在籽粒色素合成高峰期差异表达谱以及种皮中类黄酮途径相关基因的差异表达的结果,最终在主效QTL区间筛选出了40个基因作为候选基因,这些基因大部分为一些与色素合成相关的调控因子和编码转运蛋白的基因。
12候选基因的克隆和功能研究
从上述候选基因中选择克隆了11个可能与甘蓝型油菜粒色相关的候选基因,构建了相应的正义表达和RNAi表达载体,将各载体采用根癌农杆菌法分别转化了根癌农杆菌LBA4404,形成了工程菌株。通过花序浸染法将表达载体的工程菌株分别转化甘蓝型黄籽油菜(正义表达)和甘蓝型黑籽油菜(RNAi)。目前已经获得了抗Basta的RNAi的再生植株,经过多重PCR检测结果证实获得了部分阳性植株,这些将为深入研究甘蓝型油菜粒色基因提供基础,为获得转基因甘蓝型油菜黄籽材料创造条件。
Brassica napus L.(2n=38, AACC) is Brassica of Cruclferae. B. napus is an amphidiploid species (AC genome, n=19) derived from interspecific of two diploid species, B. rapa (A genome, n=10) and B. oleracea (C genome, n=9). In the world, B. napus is not only one of the most important of oil crops, but is one of the most important oil crops worldwide. It is also an important source to edible oil and the vegetable protein feed. Besides, Brassica and the model plant Arabidopsis thaliana are belong to the same family, and have some similarities level at the genome sequence. The genes have high degree of sequence identity and remarkably conserved genome structure. Moreover, with the whole genome being sequenced of B. rapa and being sequencing of B. oleracea, we could analyze the genome synteny among them and get the differences and similarities in the structures of Brasica genome and genome structure. It also would provide a theoretical basis for the analysis of genetic evolution and practical implications for improvement of Brassica crops.
Studies have demonstrated that in the same genetic background, yellow rapeseed seed has both thinner seed coat and higher seed oil and protein, and lower meal lignin content, cellulose and polyphenol than black seed varieties. In addition, other toxic materials or antinutritives, such as glucosinolates, phytic acid, tannin and sinapine, is lower than the black seed and easy to be digested by the animals and hindering its usage as feed. Therefore, development of yellow seeded lines and cultivars is one of the major breeding objectives in rapeseed, and the inheritance of seed coat colour has been investigated for many years in B. napus by several researchers around the world. However, there are no stable lines of yellow-seeded oilseed germplasm, and the major genes for the yellow-seeded were come from the B. rapa, B. oleracea, B. carinata, B. juncea and their mutation progeny in B. napus. Besides, the inheritance of seed coat color in B. napus is quite complicated, and affected by environmental factors greatly. Moreover, the molecular mechanism of yellow seed trait formation of Brassica is remaining poorly understood.
In the process of seed coat development, we analyzed laws of the variations in accumulation of flavonol content and phenolic compounds, and identified differential expression of the transcription factors and main structural genes. It is useful to explain the laws of pigments accumulation and expression patterns or regulation mechanism of gene in B. napus. In addition, based on the constructed linkage groups, a stable major QTL was detected by composite interval mapping using Windows QTL Cartographer2.5, and affecting the seed coat colour of B. napus in different different environments. Moreover, according to the results of comparative genomic and bioinformatics analysis of the major QTL region with Arabidopsis chromosome sequences and related DNA sequences in chromosome A09of B. rapa, and analysis of gene expression response in the peak period of seed coat pigments synthesis in B. napus, some stable differential expression of genes were found and acted as the candidate gene. Then we constructed the overexpression vector and RNAi expression vector of engineering strains and transform by the transformation protocol technique. Some conclusions are as follows:
1Accumulation of Proantnocyanins and polyphenols showing the significant difference in seed coat of the yellow-and black-seeded B. napus
The paraffin sections of the different development stages of seed in yellow-(GH06) and black-seeded (ZY821) of B. napus were stained with toluidine blue O. The results showed that the pigments mainly accumulated in the layer of palisade and pigment of seed coat. These compounds began to accumulate in the hilum of seed during the early development stages. The pigments will gradually increase and show the significant difference in the layer of palisade and pigment of seed coat during the seed mature. Moreover, proantnocyanins and polyphenols content is higher in the black-seeded than in the yellow-seeded of B. napus.
2The function of enzymes and gene for the synthesis of epicatechin and their derivates may be strongly attenuated in B. napus
In this research, thirty-five phenolic compounds were detected in the flavonoid extracts from ZY821and GH06seed coat according to the retention time analysis of LC-UV-MS, including that13and one compounds could be detected only in ZY821and GH06, respectively. Four types of flavonols derivates were identified, including epicatechin, quercetin, isorhametin, and kaemferol derivates. But in the whole development stages, epicatechin derivates cannot be detected in seed coat of yellow seed coat parent, and the expression level of related genes (BnUGT2and BnTT12) for epicatechin synthesis was also significantly lower in yellow-seeded than in black-seeded. The result showed that the function of enzymes and gene for the synthesis of epicatechin and their derivates may be more strongly attenuated in yellow-seeded than in black-seeded in B. napus.
3In-PAs accumulation may be involved in the difference in seed coat colour of B. napus
Similar to Arabidopsis, in-PAs cannot accumulate in the B. napus in early developmental stages of seed coat. Whereas, in-PA began to speedily accumulate and not showed the significant difference between the yellow-and black-seeded in B. napus during the middle-to-late stages. With the accumulation of in-PAs in cell walls, the seed coat colour gradually forming differences in B. napus.
4Correlation analysis among some traits of seed coat
Correlation analysis were carried out among the seed coat colour, seed hull content and seed coat pigments of RILs populations in B. napus L. The correlation studies showed seed coat colour had significant negative correlation with seed hull content and seed coat pigments in the different environments, including the anthocyanidin content, flavonoid content, total phenol content and melanin content. The results showed that it is possible that we can improve some quality traits according to select the yellow-seed traits. Moreover, the syntheses of seed coat colour and seed coat pigments are probably controlled by the same genes or have the same pathway partially.
5Differential genes expression of flavonoid biosynthesis pathway may be the key factor for formation seed coat colour
Using the method of qRT-PCR, we analyzed and identified differential expression of the main structural genes and transcription factors of biosynthesis pathway of flavonoid. The results indicated that the expression of nine structural genes(BnPAL, BnC4H, BnTT4, BnTT6, BnTT3, BnTT18, BnTT12, BnTT10and BnUGT2) and three transcription factors (BnTTG1、BnTTG2and BnTT8) showed significant differences between the yellow-and black-seeded. BnPAL, BnC4H and BnTT12encode enzymes specifically involved in the steps of the pathway leading to the biosythesis of precursor of polyphenols and flavonoids compounds and proantnocyanins, and may be the key genes for formation seed coat colour. The BnTT4expression levels reached on the tops at the28DAP, and involved in the expression of downstream genes (BnTT5, BnTT6and BnTT7). Likewise, downstream genes of BnTT3and BnTT18were also regulated by upstream gene. This result indicated that the BnTT4is the first key gene in the flavonoid pathway in B. napus. Moreover, downstream genes were regulated by the upstream genes in the flavonoid biosynthetic pathway. In addition, BnTTGl can also be directly regulated by BnTTG2, and the co-expression of BnTT2, BnTT8and BnTTG1regulates flavonoid biosynthesis pathway in B. napus. Therefore, the function of and the expression patterns of transcription factors in B. napus were also similar with Arabidopsis thaliana.
6Linkage map construction
The recombinant inbred lines (RILs) population was used for the mapping population and derived from a cross between black-seeded male parent cultivar ZY821and yellow-seeded female parent line GH06was established by selfing for seven successive generations with single seed propagating from F2. A high-density genetic linkage map was constructed using JoinMap4.0.1089loci (456for SSR,97for RAPD,451for SRAP and75for IBP) were mapped on19linkage groups, covering2775cM of B. napus genome and the average distance between two adjacent markers was2.54cM. The numbers of markers were varied from6to184on each linkage group. The length of linkage group varied from47.22cM to243.46cM and the average genetic distance was between0.83cM/marker (A09)-7.87cM/marker (C02).
7Comparative analysis of genetic mapping with published genetic mapping of B. napus
In this research, comparative mapping and analysis of linkage groups between the RILs and been published in B. napus, and the results showed that the nineteen chromosomes of linkage map showed the consistence of others.237markers of them were found and indicated that the linkage mapping could be universally used in the researches of B. napus. Meanwhile,196markers were integrated to the linkage maps and designed according to the genome sequences of B. rapa and B. oleracea.
8Comparative the linkage map of B. napus with the genome of B. rapa and Arabidopsis thaliana
The sequences of all SSRs and IBP makers located on the linkage map were compared with the genome of B. rapa and Arabidopsis thaliana, respectively.237sequences had homologous with the B. rapa and located on the126Scaffold of genome. Extensive macrocolinearity and rearrangement have been found between the A subgenomes of B. napus (An) and B. rapa (Ar). The syntenic between An and Ar subgenomes showed that there were highly homologous in B. napus and B. rapa. Whereas, one marker of436sequences were not found the homologous in Arabidopsis thaliana genome, and some sequences of them were consistent with gene sequences of Arabidopsis thaliana. The result provided strong evidence to support the hypothesis that B.napus, B. rapa and the model plant Arabidopsis originated from the common ancestor.
9QTL analysis seed coat colour
This study was to identify QTL which controlled seed coat colour components in RILs (SWU-2) using the composite interval mapping (CIM) method.18of QTLs were detected on7different linkage groups. One major QTLs explaining41.38%-64.17%of phenotypic variation were found and located in the linkage groups A09in different environments. In addition, the minor QTL were also detected and located in the groups A07, C03and C08in different environments, accounting for5.69%-11.04%of phenotypic variation, respectively. Three QTLs with relative smaller effects were also found in the linkage groups A01, A04and C05, respectively. These results have indicated that the seed coat colour was controlled by major and many minor-effect genes, with a pattern of quantitative trait inheritance, and the expression of the QTL was affected by environmental factors.
10Analysis of gene expression in the peak period of seed coat pigments synthesis in B. napus
In this study, gene expression profiles were analyzed in the peak period of seed coat pigments synthesis (42DAP) using to two parents (ZY821and GH06) and two near-isogenic lines of black-and yellow-seeded in B. napus. There are27genes expressed much higher in black-seeded than in yellow-seeded. These genes are the regulatory factors and transporter protein of gene in the biosynthesis of seed coat pigments, and might be candidates for the seed coat colour gene.
11Selection of candidate gene for seed coat colour in the major QTL region
Comparative genomic and bioinformatics analysis of the major QTL region with related DNA sequences in chromosome A09of B. rapa supplied by Korea, and covering about440kb interval in chromosome A09of B. rapa. The sequence was submitted to the NCBI website (http://www.ncbi.nlm.nih.gov/) and BRAD database (http://www.brassicadb.org/brad/) for BLAST search. The sequences of major QTL region were found high homologues in the B. rapa and B. oleracea. We extracted the interval sequences and made gene forecast according to the Arabidopsis thaliana genome. The sequence was also submitted to the NCBI website for BLASTP and completed the gene annotation.105genes were found in this region and selected40genes acting as the candidate gene for seed coat colour, according to the results of gene expression in the peak period of seed coat pigments synthesis and differential genes expression of flavonoid biosynthesis pathway. Most of these genes are the regulation factors for pigments biosynthesis and structure gene for coding transporters.
12Molecular cloning and functional identification of candidate gene
In this research, we cloned11candidate genes fragments of B. napus. Through transformation of Agrobacterium tumefaciens LBA4404strain, we have received the overexpression vector and RNAi expression vector of engineering strains, respectively. With the transformation protocol technique, we transform the yellow-seed and black-seeded lines with the overexpression vector and RNAi expression vector of candidate genes, respectively. Nowly, we have got some Basta-resistantce plants and transgenic positive plants by multiple PCR identification. The results will provide the foundation for further research of genetic mechanism of yellow-seeded gene, and will be helpful to create the germplasm resources of yellow-seeded in B. napus.
目前,大量的研究结果表明甘蓝型油菜黄籽品系与黑籽品系相比具有明显的优点,甘蓝型黄籽油菜不仅种皮薄、含油量高、蛋白含量高等优点,而且植酸、单宁、芥子碱和纤维素等有毒或抗营养物质含量较低,菜籽油的品质及饼粕的质量都有所提高。长期以来,国内外众多研究者对粒色相关性状进行了广泛研究,高产优质的黄籽油菜选育已成为当前油菜的重要目标之一。甘蓝型油菜在自然界中不存在天然的黄籽种质资源,甘蓝型油菜黄籽性状遗传复杂,且易受环境影响,目前获得的甘蓝型黄籽材料,其黄籽主效基因来自于白菜、甘蓝、芥菜型油菜、埃塞俄比亚芥诱变后代,但形成的机理仍然不清楚。
本研究通过分析黄黑籽甘蓝型油菜种皮发育过程中类黄酮及酚类化合物累积的动态变化规律以及不同发育时期种皮中相关结构基因和转录因子的差异表达,为揭示甘蓝型油菜种皮色素累积规律和相关基因的表达模式及调控机理提供了重要线索。另外,通过构建高密度遗传连锁图谱,用复合区间作图法在多个环境中重复检测到一个影响种皮色泽的主效QTL,通过将该区间与拟南芥和韩国提供的白菜第九染色体对应区间DNA序列进行了生物信息学分析,最终结合测定油菜籽粒色素合成高峰期的表达谱信息,找出稳定差异表达的基因并将此作为候选基因,构建各候选基因的正义表达和RNAi植物表达载体,采用根癌农杆菌介导法分别转化了甘蓝型黄籽油菜(正义表达)和甘蓝型黑籽油菜(RNAi)。主要研究结果如下：
1原花色素类多酚类物质的积累在黄黑籽甘蓝型油菜种皮中具有显著差异
通过用甲苯胺蓝染液(TBO)对不同发育时期的甘蓝型油菜黑籽中油821(ZY821)和黄籽GH06种子石蜡切片染色的结果表明：甘蓝型油菜种皮中色素的主要累积部位是种皮栅栏层和色素层,且原花色素类多酚物质初期最先在种脐部产生,随着种子的发育,在种皮栅栏层和色素层组织中色素积累逐渐增多,且黄黑籽间存在明显差异,在黑籽ZY821中原花色素类多酚类物质的含量明显高于黄籽GH06,说明原花色素类多酚类物质的积累是油菜粒色的重要原因之一。
2黄籽甘蓝型油菜中与表儿茶素及其衍生物合成相关的酶和基因功能衰减比黑籽强烈
通过LC-ESI-MS分析方法对甘蓝型油菜黑籽ZY821和黄籽GH06不同发育时期种皮中黄酮类衍生物含量进行的分析结果表明：35种黄酮类化合物的衍生物在黄黑籽甘蓝型油菜种皮中被检测到,大致可分为表儿茶素、槲皮素、异鼠李素和山萘酚等四类黄酮类化合物的衍生物,各种衍生物在黑籽ZY821中的含量明显高于黄籽GH06：但表儿茶素及其衍生物在黄籽GH06的整个发育时期中均未被检测到,且在整个发育过程中与表儿茶素合成相关的基因(BnUGT2和BnTT12)的表达水平明显低于黑籽,说明与表儿茶素及其衍生物合成相关的酶和基因功能衰减可能是引起色泽差异的关键因子。
3甘蓝型油菜种皮中不溶性原花色素的积累可能导致了黄黑籽在种皮颜色上的差异
甘蓝型油菜与拟南芥类似,在发育早期并不存在不溶性的原花色素的积累,而在种皮发育的中后期开始迅速积累,但黑籽油菜中不溶性原花色素的积累与黄籽不存在显著性差异,随着种子的成熟,种皮中大量的不溶性原花色素的聚合物在细胞壁上交联,促进了种皮颜色上的差异。
4甘蓝型油菜种皮色泽与相关性状的统计分析
不同环境中甘蓝型油菜种皮色泽与种子皮壳率、种皮中花色素、类黄酮、总酚和黑色素之间存在极显著的负相关,而皮壳率与四种种皮色素之间存在极显著的正相关,说明在甘蓝型油菜品种选育过程中通过黄籽性状选育可以同时多个相关性状进行遗传改良,同时这些性状之间可能存在共同或部分相同的代谢途径,或者是在同一遗传系统中受相同的上游调控因子调控。
5甘蓝型油菜种皮中类黄酮途径相关基因的表达差异可能是影响种皮色泽形成的根本原因
本研究通过qRT-PCR的方法研究了参与苯丙烷和类黄酮代谢途径相关的基因在黑籽甘蓝型油菜中油821和黄籽GH06七个不同发育时期种皮中的表达差异。结果表明：有9个结构基因(BnPAL、BnC4H、BnTT4、BnTT6、BnTT3、BnTT18、BnTT12、BnTT10和BnUGT2)和3个转录因子(BnTTG1、BnTTG2和BnTT8)在黄黑籽种皮中存在极显著表达差异,编码两种催化多种酚类及类黄酮终产物提供前体物质的形成关键酶的BnPAL和BnC4H两个基因和促进原花色素起始单体形成的重要基因BnTT12是引起黄黑籽种皮差异的关键基因。另外,BnTT4在授粉后28天达到最高峰值,而下游基因(BnTT5、BnTT6和BnTT7)晚于该基因一个时期达到最高峰值,BnTT3和BnTT18的最高峰值也晚于其上游基因一个时期达到最高峰值,说明BnTT4是甘蓝型油菜类黄酮代谢途径中影响下游基因的表达的一个重要基因,在类黄酮途径中存在上游基因对下游基因的调控。甘蓝型油菜种皮中的转录因子在黄黑籽之间表达模式与拟南芥类似,BnTTG1的表达受到BnTTG2的调控,而BnTT2、 BnTT8和BnTTG1协同作用又同时调控了甘蓝型油菜的类黄酮代谢过程。
6甘蓝型油菜遗传连锁图谱的构建
本研究利用重组自交系为作图群体,构建了一张包含1089个标记位点,其中包括451个SRAP标记,456个SSR标记,97个RAPD标记和75个IBP标记,覆盖19个连锁群,总长度约为2775cM,标记间的平均距离为2.54cM的甘蓝型油菜遗传图谱。每个连锁群上的标记数为6-184个,连锁群长度为47.22-243.46cM,连锁群密度为0.83cM/标记(A09)-7.87cM/标记(C02)。
7甘蓝型油菜遗传图谱与公开发表的遗传图谱的比较分析
通过比较作图,与已公开发表的甘蓝型油菜的遗传连锁图谱进行了比较分析,结果表明该遗传图谱的连锁群与已发表的甘蓝型油菜的十九条染色体基本吻合,并且存在237个共有的标记位点,同时整合了根据白菜和甘蓝基因组信息设计的特异性引物196对,说明图谱具有了一定的通用性,但仍未达到饱和。
8甘蓝型油菜遗传图谱与白菜和拟南芥基因组的共线性分析
本研究将遗传连锁图谱上的SSR和IBP标记逐一与白菜基因组的Scaffold序列进行了比较,最终将甘蓝型油菜遗传连锁图谱上的436对引物标记锚定到白菜基因组的126个Scaffold上。甘蓝型油菜An与白菜Ar染色体组存在很好的共线性,但有些标记顺序存在重排现象,甘蓝型油菜A基因组来源于白菜。将锚定到白菜基因组Scaffold上的436对引物标记序列与拟南芥的全基因组进行了比对,仅有一对标记没有找到同源性,某些标记序列与拟南芥序列基本一致,在一定程度上说明了甘蓝型油菜与白菜和模式植物拟南芥具有共同的原始祖先。
9甘蓝型油菜种皮色泽的QTL分析
应用复合区间作图分析方法,对重组自交系SWU-2群体在6个环境中的种皮色泽进行了QTL初级定位和效应检测,共检测到18个种皮色泽相关的QTL,单个QTL解释性状表现的变幅在5.69%-64.17%之间。这些QTL分别位于第A01,A04,A07,A09,C03,C05和C08七个不同的连锁群上；控制种皮色泽的基因表现为主效+微效多基因控制的数量性状的特点,并受到环境条件影响。
10甘蓝型油菜在色素合成高峰期相关基因的差异表达谱数据分析
本研究以作图群体SWU-2的两个亲本(黑籽中油821和黄籽GH06)与一对黄黑籽近等基因系为材料,在种皮色素合成高峰期(授粉后42天)作基因表达谱分析,以黄籽为对照,共发现在种皮色泽主效QTL区间具有稳定上调差异表达的基因27个,这些基因大部分为一些与色素合成相关的调控因子和转运蛋白基因。
11甘蓝型油菜种皮色泽主效QTL区间的候选基因的筛选
将种皮色泽主效QTL区间与韩国提供的第九染色体对应的DNA序列进行生物信息学分析,确定该区段总长约为440kb左右,通过与我国测序的白菜、甘蓝基因组序列在NCBI (http://www.ncbi.nlm.nih.gov/)和白菜基因组数据库(http://www.brassicadb.org/brad/))中进行序列比对,该区间序列与白菜和甘蓝具有很高的同源性。提取该区间序列以拟南芥基因组数据集为模型进行基因预测,并在NCBI上进行BLASTP比对,进行基因注释,发现该区段内共存在约105个基因,结合甘蓝型油菜黄黑籽在籽粒色素合成高峰期差异表达谱以及种皮中类黄酮途径相关基因的差异表达的结果,最终在主效QTL区间筛选出了40个基因作为候选基因,这些基因大部分为一些与色素合成相关的调控因子和编码转运蛋白的基因。
12候选基因的克隆和功能研究
从上述候选基因中选择克隆了11个可能与甘蓝型油菜粒色相关的候选基因,构建了相应的正义表达和RNAi表达载体,将各载体采用根癌农杆菌法分别转化了根癌农杆菌LBA4404,形成了工程菌株。通过花序浸染法将表达载体的工程菌株分别转化甘蓝型黄籽油菜(正义表达)和甘蓝型黑籽油菜(RNAi)。目前已经获得了抗Basta的RNAi的再生植株,经过多重PCR检测结果证实获得了部分阳性植株,这些将为深入研究甘蓝型油菜粒色基因提供基础,为获得转基因甘蓝型油菜黄籽材料创造条件。
Brassica napus L.(2n=38, AACC) is Brassica of Cruclferae. B. napus is an amphidiploid species (AC genome, n=19) derived from interspecific of two diploid species, B. rapa (A genome, n=10) and B. oleracea (C genome, n=9). In the world, B. napus is not only one of the most important of oil crops, but is one of the most important oil crops worldwide. It is also an important source to edible oil and the vegetable protein feed. Besides, Brassica and the model plant Arabidopsis thaliana are belong to the same family, and have some similarities level at the genome sequence. The genes have high degree of sequence identity and remarkably conserved genome structure. Moreover, with the whole genome being sequenced of B. rapa and being sequencing of B. oleracea, we could analyze the genome synteny among them and get the differences and similarities in the structures of Brasica genome and genome structure. It also would provide a theoretical basis for the analysis of genetic evolution and practical implications for improvement of Brassica crops.
Studies have demonstrated that in the same genetic background, yellow rapeseed seed has both thinner seed coat and higher seed oil and protein, and lower meal lignin content, cellulose and polyphenol than black seed varieties. In addition, other toxic materials or antinutritives, such as glucosinolates, phytic acid, tannin and sinapine, is lower than the black seed and easy to be digested by the animals and hindering its usage as feed. Therefore, development of yellow seeded lines and cultivars is one of the major breeding objectives in rapeseed, and the inheritance of seed coat colour has been investigated for many years in B. napus by several researchers around the world. However, there are no stable lines of yellow-seeded oilseed germplasm, and the major genes for the yellow-seeded were come from the B. rapa, B. oleracea, B. carinata, B. juncea and their mutation progeny in B. napus. Besides, the inheritance of seed coat color in B. napus is quite complicated, and affected by environmental factors greatly. Moreover, the molecular mechanism of yellow seed trait formation of Brassica is remaining poorly understood.
In the process of seed coat development, we analyzed laws of the variations in accumulation of flavonol content and phenolic compounds, and identified differential expression of the transcription factors and main structural genes. It is useful to explain the laws of pigments accumulation and expression patterns or regulation mechanism of gene in B. napus. In addition, based on the constructed linkage groups, a stable major QTL was detected by composite interval mapping using Windows QTL Cartographer2.5, and affecting the seed coat colour of B. napus in different different environments. Moreover, according to the results of comparative genomic and bioinformatics analysis of the major QTL region with Arabidopsis chromosome sequences and related DNA sequences in chromosome A09of B. rapa, and analysis of gene expression response in the peak period of seed coat pigments synthesis in B. napus, some stable differential expression of genes were found and acted as the candidate gene. Then we constructed the overexpression vector and RNAi expression vector of engineering strains and transform by the transformation protocol technique. Some conclusions are as follows:
1Accumulation of Proantnocyanins and polyphenols showing the significant difference in seed coat of the yellow-and black-seeded B. napus
The paraffin sections of the different development stages of seed in yellow-(GH06) and black-seeded (ZY821) of B. napus were stained with toluidine blue O. The results showed that the pigments mainly accumulated in the layer of palisade and pigment of seed coat. These compounds began to accumulate in the hilum of seed during the early development stages. The pigments will gradually increase and show the significant difference in the layer of palisade and pigment of seed coat during the seed mature. Moreover, proantnocyanins and polyphenols content is higher in the black-seeded than in the yellow-seeded of B. napus.
2The function of enzymes and gene for the synthesis of epicatechin and their derivates may be strongly attenuated in B. napus
In this research, thirty-five phenolic compounds were detected in the flavonoid extracts from ZY821and GH06seed coat according to the retention time analysis of LC-UV-MS, including that13and one compounds could be detected only in ZY821and GH06, respectively. Four types of flavonols derivates were identified, including epicatechin, quercetin, isorhametin, and kaemferol derivates. But in the whole development stages, epicatechin derivates cannot be detected in seed coat of yellow seed coat parent, and the expression level of related genes (BnUGT2and BnTT12) for epicatechin synthesis was also significantly lower in yellow-seeded than in black-seeded. The result showed that the function of enzymes and gene for the synthesis of epicatechin and their derivates may be more strongly attenuated in yellow-seeded than in black-seeded in B. napus.
3In-PAs accumulation may be involved in the difference in seed coat colour of B. napus
Similar to Arabidopsis, in-PAs cannot accumulate in the B. napus in early developmental stages of seed coat. Whereas, in-PA began to speedily accumulate and not showed the significant difference between the yellow-and black-seeded in B. napus during the middle-to-late stages. With the accumulation of in-PAs in cell walls, the seed coat colour gradually forming differences in B. napus.
4Correlation analysis among some traits of seed coat
Correlation analysis were carried out among the seed coat colour, seed hull content and seed coat pigments of RILs populations in B. napus L. The correlation studies showed seed coat colour had significant negative correlation with seed hull content and seed coat pigments in the different environments, including the anthocyanidin content, flavonoid content, total phenol content and melanin content. The results showed that it is possible that we can improve some quality traits according to select the yellow-seed traits. Moreover, the syntheses of seed coat colour and seed coat pigments are probably controlled by the same genes or have the same pathway partially.
5Differential genes expression of flavonoid biosynthesis pathway may be the key factor for formation seed coat colour
Using the method of qRT-PCR, we analyzed and identified differential expression of the main structural genes and transcription factors of biosynthesis pathway of flavonoid. The results indicated that the expression of nine structural genes(BnPAL, BnC4H, BnTT4, BnTT6, BnTT3, BnTT18, BnTT12, BnTT10and BnUGT2) and three transcription factors (BnTTG1、BnTTG2and BnTT8) showed significant differences between the yellow-and black-seeded. BnPAL, BnC4H and BnTT12encode enzymes specifically involved in the steps of the pathway leading to the biosythesis of precursor of polyphenols and flavonoids compounds and proantnocyanins, and may be the key genes for formation seed coat colour. The BnTT4expression levels reached on the tops at the28DAP, and involved in the expression of downstream genes (BnTT5, BnTT6and BnTT7). Likewise, downstream genes of BnTT3and BnTT18were also regulated by upstream gene. This result indicated that the BnTT4is the first key gene in the flavonoid pathway in B. napus. Moreover, downstream genes were regulated by the upstream genes in the flavonoid biosynthetic pathway. In addition, BnTTGl can also be directly regulated by BnTTG2, and the co-expression of BnTT2, BnTT8and BnTTG1regulates flavonoid biosynthesis pathway in B. napus. Therefore, the function of and the expression patterns of transcription factors in B. napus were also similar with Arabidopsis thaliana.
6Linkage map construction
The recombinant inbred lines (RILs) population was used for the mapping population and derived from a cross between black-seeded male parent cultivar ZY821and yellow-seeded female parent line GH06was established by selfing for seven successive generations with single seed propagating from F2. A high-density genetic linkage map was constructed using JoinMap4.0.1089loci (456for SSR,97for RAPD,451for SRAP and75for IBP) were mapped on19linkage groups, covering2775cM of B. napus genome and the average distance between two adjacent markers was2.54cM. The numbers of markers were varied from6to184on each linkage group. The length of linkage group varied from47.22cM to243.46cM and the average genetic distance was between0.83cM/marker (A09)-7.87cM/marker (C02).
7Comparative analysis of genetic mapping with published genetic mapping of B. napus
In this research, comparative mapping and analysis of linkage groups between the RILs and been published in B. napus, and the results showed that the nineteen chromosomes of linkage map showed the consistence of others.237markers of them were found and indicated that the linkage mapping could be universally used in the researches of B. napus. Meanwhile,196markers were integrated to the linkage maps and designed according to the genome sequences of B. rapa and B. oleracea.
8Comparative the linkage map of B. napus with the genome of B. rapa and Arabidopsis thaliana
The sequences of all SSRs and IBP makers located on the linkage map were compared with the genome of B. rapa and Arabidopsis thaliana, respectively.237sequences had homologous with the B. rapa and located on the126Scaffold of genome. Extensive macrocolinearity and rearrangement have been found between the A subgenomes of B. napus (An) and B. rapa (Ar). The syntenic between An and Ar subgenomes showed that there were highly homologous in B. napus and B. rapa. Whereas, one marker of436sequences were not found the homologous in Arabidopsis thaliana genome, and some sequences of them were consistent with gene sequences of Arabidopsis thaliana. The result provided strong evidence to support the hypothesis that B.napus, B. rapa and the model plant Arabidopsis originated from the common ancestor.
9QTL analysis seed coat colour
This study was to identify QTL which controlled seed coat colour components in RILs (SWU-2) using the composite interval mapping (CIM) method.18of QTLs were detected on7different linkage groups. One major QTLs explaining41.38%-64.17%of phenotypic variation were found and located in the linkage groups A09in different environments. In addition, the minor QTL were also detected and located in the groups A07, C03and C08in different environments, accounting for5.69%-11.04%of phenotypic variation, respectively. Three QTLs with relative smaller effects were also found in the linkage groups A01, A04and C05, respectively. These results have indicated that the seed coat colour was controlled by major and many minor-effect genes, with a pattern of quantitative trait inheritance, and the expression of the QTL was affected by environmental factors.
10Analysis of gene expression in the peak period of seed coat pigments synthesis in B. napus
In this study, gene expression profiles were analyzed in the peak period of seed coat pigments synthesis (42DAP) using to two parents (ZY821and GH06) and two near-isogenic lines of black-and yellow-seeded in B. napus. There are27genes expressed much higher in black-seeded than in yellow-seeded. These genes are the regulatory factors and transporter protein of gene in the biosynthesis of seed coat pigments, and might be candidates for the seed coat colour gene.
11Selection of candidate gene for seed coat colour in the major QTL region
Comparative genomic and bioinformatics analysis of the major QTL region with related DNA sequences in chromosome A09of B. rapa supplied by Korea, and covering about440kb interval in chromosome A09of B. rapa. The sequence was submitted to the NCBI website (http://www.ncbi.nlm.nih.gov/) and BRAD database (http://www.brassicadb.org/brad/) for BLAST search. The sequences of major QTL region were found high homologues in the B. rapa and B. oleracea. We extracted the interval sequences and made gene forecast according to the Arabidopsis thaliana genome. The sequence was also submitted to the NCBI website for BLASTP and completed the gene annotation.105genes were found in this region and selected40genes acting as the candidate gene for seed coat colour, according to the results of gene expression in the peak period of seed coat pigments synthesis and differential genes expression of flavonoid biosynthesis pathway. Most of these genes are the regulation factors for pigments biosynthesis and structure gene for coding transporters.
12Molecular cloning and functional identification of candidate gene
In this research, we cloned11candidate genes fragments of B. napus. Through transformation of Agrobacterium tumefaciens LBA4404strain, we have received the overexpression vector and RNAi expression vector of engineering strains, respectively. With the transformation protocol technique, we transform the yellow-seed and black-seeded lines with the overexpression vector and RNAi expression vector of candidate genes, respectively. Nowly, we have got some Basta-resistantce plants and transgenic positive plants by multiple PCR identification. The results will provide the foundation for further research of genetic mechanism of yellow-seeded gene, and will be helpful to create the germplasm resources of yellow-seeded in B. napus.
引文
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