大麦耐镉及镉积累相关QTL定位与候选基因筛选及调控研究
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摘要
镉是一种有毒重金属,其在环境中的迁移性强,极易被植物吸收积累,并通过食物链危害人体健康。明确大麦耐镉与镉积累机理是培育耐镉与低镉积累新品种和制订耐镉与低镉积累调控措施的基础。本研究以本实验室前期筛选获得的镉耐性与镉积累不同的大麦基因型为材料,探讨镉吸收和分布的规律及基因型差异的生理机理；分析不同籽粒镉积累大麦基因型籽粒蛋白质表达谱差异,鉴定大麦籽粒低镉积累相关的目标蛋白；克隆籽粒镉积累相关基因,并进行大麦遗传转化；构建大麦悬浮细胞系在细胞水平上分析其耐镉性差异及镉积累目标基因的表达情况；以耐镉和镉敏感基因型杂交产生F1构建的DH群体为材料,构建遗传图谱,进行耐镉相关性状QTL定位及环境互作研究。同时,探讨了外源乙酰半胱氨酸(N-acetylcysteine, NAC)对大麦镉吸收与耐镉性影响的基因型差异及生理机制,为低镉积累育种与生产提供理论依据和技术指导。主要研究结果如下：
1.镉胁迫对大麦幼苗光合特性及镉吸收与分布的影响及基因型差异
以镉耐性与积累不同的大麦基因型(浙农8号和W6nk2)为材料,水培试验,对照,5、50和500μM不同浓度镉胁迫试验,研究了镉胁迫对大麦幼苗生长、光合特性及镉吸收与分布的影响及基因型差异。结果表明,镉胁迫显著降低麦苗株高、根长、叶绿素含量、地上部和根系干重；基因型之间差异显著,耐镉基因型浙农8号受害较轻,镉敏感基因型W6nk2抑制严重。根尖镉荧光定位分析结果显示,镉主要滞留在根系的质外体,尤其是细胞壁中,其中耐性基因型更为明显；同一植株内由根尖到茎部的镉含量呈逐渐下降趋势,且处理时间越长,镉积累量越高,并有从根尖向茎部移动的趋势。镉胁迫显著影响大麦的光合作用及叶绿素荧光,且不同基因型之间差异显著,镉敏感基因型W6nk2受抑制严重,耐镉基因型浙农8号表现出相对较强的抗性。
2.不同籽粒镉积累大麦基因型籽粒蛋白表达谱的比较研究
利用双向电泳和MALDI-TOF质谱技术,分析比较了籽粒镉积累不同的大麦基因型(浙农8号和VV6nk2)籽粒蛋白表达差异,检测到29个差异表达蛋白质点,浙农8号与W6nk2相比,籽粒蛋白质高表达蛋白点17个,表达受抑制蛋白点12个。差异表达蛋白以蛋白酶抑制剂类相关蛋白为主,包括高表达蛋白α淀粉酶/胰岛素抑制剂CM,Z-型丝氨酸蛋白酶抑制剂和丝氨酸蛋白酶抑制剂Z7；表达受抑制蛋白包括胰蛋白酶抑制剂蛋白BTI-CMe2.1蛋白、胰蛋白酶抑制剂蛋白BTI-CMe2.2蛋白和胰蛋白酶抑制剂。其次是热休克蛋白HSP70和Ras致癌基因家族成员Rab4蛋白等胁迫相关蛋白在浙农8号中高表达；而在W6nk2中有脱氢抗坏血酸还原酶等胁迫相关蛋白高表达。另外还有贮藏类蛋白,比如球蛋白,推定的燕麦蛋白前体在浙农8号中高表达。这些差异表达蛋白可能与大麦籽粒镉积累与耐性密切相关,本研究鉴定得到的蛋白可为深入阐明大麦镉积累机理提供新的契机。
3.大麦遗传图谱的构建及苗期耐镉相关性状的QTL定位
以萎缩不知(耐镉)和苏引麦2号(镉敏感)杂交F1构建的含有108个株系的大麦DH群体为材料,利用SSR和SNP标记,构建遗传图谱,该图谱包含41个SSR标记和15个SNP标记,图谱总长396.9cM,标记间平均距离8.1cM。水培镉胁迫试验,以不加镉为对照,分析苗期耐镉相关农艺与生理性状的耐镉指数CTI,检测到农艺性状相关加性QTLs9个,表型贡献率介于3.30-9.49%之间；检测到酶活和MDA含量相关QTL22个,分布在大麦7条染色体上,其加性效应都达到了显著水平。其中地上部和根系各有11个QTLs,平均表型变异为5.6%。另外,检测到一对控制根系GPX活性的上位性QTL。在第2染色体上标记区间(?)BC2-Chitinase检测到了4个QTLs,其中单株叶片数、根系CAT活性及根系干重都位于0.0cM处,推测为同一QTL,说明控制这些性状的基因很有可能与ABC转运蛋白相关。基因与环境互作条件下,检测到苗期镉含量相关QTLs6个；其中,地上部检测到了2个,分别位于2H和7H上,且这2个QTLs存在极显著地加性×加性上位性互作效应,加性效应值为-23.68,环境镉效应引起的加性效应值为-21.21,可解释表型变异0.57%。根系检测到4个,分别位于1H、5H、6H和7H上。地上部和根系分别检测到7个和11个微量元素含量相关QTLs,总遗传贡献率分别为16.37%和24.95%,由于环境互作效应引起的总遗传变异分别为12.41%和14.6%。
4.大麦不同生育期耐镉及镉积累相关性状QTL定位
盆栽试验,研究了镉胁迫对不同生育期大麦生长及生理性状的影响及株系间差异,检测大麦不同生育期耐镉与镉积累相关QTLs。检测到不同生育期镉含量相关QTLs11个,检测到2对上位性QTLs,分别控制籽粒镉积累及根系Zn含量。另外,检测到地上部镉累积量相关的具有显著加性与环境互作效应的QTL1个。检测到63个与微量元素含量相关QTLs,其中苗期、分蘖期、拔节期和灌浆期4个生育期叶片微量元素含量相关QTLs25个,收获期不同器官微量元素相关QTLs38个；其中17个具有显著地加性与环境互作效应。定位到了8个与农艺和产量CTI相关的QTLs,表型变异在1.54-10.79%之间。而且,遗传标记区间EBmac0615-EBmatc0054, EBmac0557-EBmac0615和SNP标记ABC转运蛋白检测到了镉及数个微量元素含量相关QTLs。
5.大麦籽粒镉积累相关基因表达分析、克隆及大麦转化
利用基因芯片技术,分析了籽粒镉积累差异显著的大麦基因型(高积累：浙农8号；低积累：W6nk2)幼苗在镉胁迫下的基因表达谱,结果显示,镉胁迫显著影响大麦转录水平,基因型间存在显著差异；对2基因型镉胁迫下差异表达基因比对发现,5Cd处理诱导低镉积累基因型W6nk2的运输载体相关基因上调表达,如ZIP、ABC转运蛋白、ATPase、以及Zn、Fe离子运输载体等基因上调表达,说明W6nk2籽粒镉低积累特性可能与这些转运载体基因的上调表达相关；荧光定量PCR验证这些候选基因,得到了一致的表达。进一步利用Gateway克隆技术构建了ZIP基因家族在大麦里发现的4个成员ZIP3、ZIP5、ZIP7和ZIP8的基因沉默表达载体,阳性克隆载体经PCR验证、酶切和测序鉴定；为了进一步研究ZIP家族不同成员之间及在不同大麦基因型间的表达差异和功能,用农杆菌介导法转化此基因到品质较好的模式转化大麦Golden promise,以期进一步研究ZIP基因对大麦镉转运与积累的调控与影响。
6.不同大麦基因型悬浮细胞系的建立及耐镉性差异分析
以籽粒镉积累不同的大麦基因型(高积累：浙农8号；低积累：W6nk2),和耐镉性不同的大麦基因型(耐镉：萎缩不知；镉敏感：东17)为材料,在成功建立分散均匀、稳定的胚性悬浮细胞系的基础上,分析镉胁迫对大麦悬浮细胞生长影响及其基因型差异,及镉胁迫对不同基因型大麦悬浮细胞系生长的影响；并进一步利用大麦不同基因型单细胞悬浮系来验证本实验前期结果所得基因ZIP蛋白的表达情况。结果表明,以直径约2mm的幼胚为外植体,可以成功建立上述4基因型大麦悬浮细胞系；50μM Cd处理,4基因型大麦悬浮细胞活力都显著下降,随着Cd处理时间延长,大麦细胞活力逐步下降；且敏感基因型下降更为明显。SDS-PAGE电泳和Western杂交结果表明,ZIP7蛋白在籽粒低镉积累基因型W6nk2中的表达要强于浙农8号,这一结果与前面实验结果相符。
7.外源NAC对大麦镉毒害的缓解效应及基因型差异
以耐镉基因型萎缩不知和镉敏感基因型东17为材料,水培试验,研究外源NAC对镉所致大麦损伤的缓解作用。结果表明,50μM Cd处理下,添加200μM NAC (Cd+NAC)显著减少大麦幼苗对镉的吸收和积累；缓解大麦幼苗镉毒害症状,2个基因型株高、根长及生物量都比Cd处理显著提高。外源NAC显著影响细胞超微结构及活性氧代谢,NAC显著缓解镉胁迫引起的叶绿体和根系细胞结构的损伤,基本恢复叶绿体片层结构的损伤,嗜锇粒数量显著减少,有效提高了根系分生组织细胞核膜的稳定性和完整性。外源NAC对镉胁迫引起的大麦氧化损伤有明显的缓解效应,显著提高抗氧化酶活性,减少叶片O2·-和·OH、MDA累积,缓解镉对大麦根尖细胞活力的损伤。
Cadmium (Cd), a non-essential trace element, is one of the most phytotoxic heavy metals and widespread pollutant in the environment. Cd is easily absorbed and accumulated in plants and of toxicity at low concentrations, thus may induce potential human health risk via food chain. It is crucial important to understand the underlying mechanism associated with Cd tolerance and accumulation in plants, for developing Cd-resistant/tolerant and low accumulated cultivars or using agronomic practice to inhibit the involvement of Cd in food chains. The purpose of the present study is to elucidate physiological mechanism of genotypic difference in Cd uptake and distribution, using barley genotypes differing in Cd tolerance and accumulation, to identify specific proteins and relevant genes for Cd-tolerance/low accumulation. Meanwhile, genotypic difference were systemically investigated by employing the newly constructed suspension cell lines. With the aid of analyzing available SSR and SNP markers and the establishment/construction of genetic linkage map, Cd tolerance/accumulation relevant gene(s) were tagged/localized, and the genetic relationship was investigated between Cd accumulation and some morphological and physiological indexes, using DH population derived by F1hybrids of genotypes differing in Cd accumulation and tolerance. In addition, we investigated the possibility to reduce Cd uptake and accumulation in barley plants by application of exogenous N-acetyl cysteine (NAC) and its protective effect against Cd-induced growth inhibition, oxidative stress, and damage in ultrastructure and cell viability of roots tips in the two different genotypes differing in Cd tolerance. The main results were summarized follows:
1. Genotypic differences in photosynthesis performance and Cd uptake and distribution in response to Cd toxicity in barley seedlings
A hydroponic experiment was carried out to examine genotypic differences in photosynthesis performance and Cd uptake and distribution in response to different Cd levels (0,5,50,500μM Cd), using two barley genotypes differing in Cd-tolerance and accumulation (c.f. W6nk2, Cd-sensitive and low-grain-accumulation; Zhenong8, Cd-tolerant and high accumulator). The results showed that plant height, root length, and biomass accumulation of shoot/root were significantly reduced in plants grown in5,50,500μM Cd compared with control. There was highly significant difference in the reduction of these growth parameters between the two genotypes, Zhenong8showed much less reduction, suggesting its high tolerance to Cd toxicity, while W6nk2with the greater reduction. Significant genotypic difference in Cd concentration was also found. Zhenong8contained higher Cd concentration in plant tissue than W6nk2. The results of Cd-specific fluorescence localization in root tip showed that Cd was mainly in apoplast, particularly within the cell wall, this phenomenon was more obvious in tolerant genotype Zhenong8. Within the same plant Cd concentration from the root tip to the shoot was gradually decreased, with the treatment time extended the Cd accumulation was higher, and there was a upward moving trend from the root tip to shoot. Speculating that the roots have the binding role to Cd spread, and therefore the roots were poisoned heavyly. Cd stress significantly affected photosynthesis and chlorophyll fluorescence of barley, and there were significant differences between different genotypes, Cd-sensitive genotype W6nk2severely inhibited, while Cd-tolerant genotype Zhenong8showed relatively strong resistance.
2. Comparison study of grain protein expression between two different grain Cd accumulate genotype
We compared grain protein expression in two barley genotypes differing in Cd tolerance and accumulation using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spcetrometry analysis. A total of29protein were identified differentially expression, of these alerted proteins,17were significantly higher expressed and12were significantly suppressed in Zhenong8compared with W6nk2grains. Overall, these differential expression protein were mainly on proteinase inhibitor, involving in higher expressed protein α-amylase/trypsin inhibitor CM, protein z-type serpin, serpin-Z7(HorvuZ7), and significantly suppressed protein BTI-CMe2.1protein, BTI-CMe2.2protein. Followed by stress-related protein, such as up-regultaed protein HSP70and RAB, member of RAS oncogene family-like4, suppressed protein dehydroascorbate reductase. There were also storage protein, embryo globulin and putative avenin-like a precursor were higher expressed in Zhenong8. It was indicated that these proteins might play an important role in Cd accumulation and tolerance or detoxification, and these protein identified may provide new opportunities for elucidating the barley Cd accumulation mechanism.
3. Genetic linkage map construction of barley DH population and Cd tolerant related QTL mapping at seedling stage
A mapping population of108lines (DH), derived from F1coming from a cross between Cd tolerant genotype Weisuobuzhi and Cd sensitive genotype Suyinmai2, were used in this investigation. The linkage map consisted of41SSR and15SNP markers-spanning396.9centimorgans (cM), with an average linkage distance between markers of8.1cM, and clustered into7molecular linkage groups (LGs). Hydroponic and pots experiments were carried out to study the barley growth differences and Cd tolerant related traits among different lines under Cd treatment during different develop stages, and mapped Cd related QTLs. Nine QTLs were detected, associated with Cd tolerance index (CTI) of SPAD value and growth traits on chromosomes1,2,5and7. The individual contribution of each QTL to the phenotypic variation was ranging3.30-9.49%. Twenty two QTLs controlling antioxidant enzymes and MDA content located on all seven chromosomes. Half of them were related in the shoots and half detected in the roots, with the average phenotypic variance5.6%. One pairs epistatic QTLs were detected, eg. Cd tolerance index (CTI) of root glutathione peroxidase (GPX) activity. In addition, four QTLs, which were the leaf numbers per plant, root CAT activity and root dry weight, dry weight at harvest were detected in the interval ABC2-Chitinase on2H, the position was0.0cM, suggesting that these QTLs maybe the same QTL and these traits were associated with ABC transporter. Under genetype and environment interaction conditions,6QTLs for Cd concentration/accumulation were mapped on chromosomes1,2,5,6,7. Two QTLs, qRCd1-1and qACd1-1, overlapped by the same genetic region of ABC1-GMS021, and2additive QTLs of qShCd2-3and qShCd7-2exist epistatic effect. There were7and11QTLs for microelement concentrations in shoots and roots, respectively, with the total contribution of16.37%and24.95%. The total phenotypic variation caused by environment interaction effect was12.41%and14.6%respectively.
4. Analysis of QTLs associated with Cd accumulation and tolerance at different growth stages
In pot experiment, eleven QTLs for Cd concentration with significantly additive (A) effect at different stages and in different organs were detected, some of them represented a single locus affecting different traits and/or the same trait under both control and Cd treatments. Two pairs epistatic QTLs were detected, i.e. one was kernel Cd accumulation with positive epistatic effect, being significant in additive×additive interactions by Cd environment interaction effects; the other was root Zn concentration related epistatic QTLs. In addition, one shoot Cd accumulation related QTL with significantly additive (A) effect and/or additive environment interaction (AE) effect was identified. For63microelements related QTLs,25and38were of leaves and different organs, respectively, and17of them with significant AE effect. Eight QTLs associated with CTI of agronomic and yield traits were detected with phenotypic variation1.54-10.79%. Furthermore, genetic regions EBmac0615-EBmatc0054, EBmac0557-EBmac0615and the novel SNP marker ABC transporters assigned to Cd and several microelement concentrations.
5. Identification of relevant genes for Cd-tolerance and low accumulation, the candidate gene clone and transformation of high and low grain Cd accumulating barley
Using cDNA microarray technology to analyze gene expression under5μM Cd stress and select Cd tolerant and low Cd accumulate related genes in two barley genotypes differing in Cd tolerance and accumulation. The result showed that there was significant difference on expression response of genes related to Cd stress between two genotypes, the up-regulated transcript levels of genes relating to metal transporters in plants were observed, such as ZIP, ABC transporter, P-type ATPase and iron-phytosiderophore transporter in leaves of W6nk2after Cd exposure, which might contribute to its low Cd accumulation characterization. Real time PCR result also confirmed these gene's expression. Thus, we islolated some of them (e.g. ZIP family) from both barley genotypes and transformed them into Golden Promise, our hypothesis was that using RNAi technology may prove the low Cd accumulating gene by suppression of this gene expression in transgenic plants with elevated Cd concentration.
6. Establishment of suspension cells in different barley genotypes and analysis of its Cd tolerance
Here the well-diffused and stable suspension cells were successfully constructed for the four barley genotypes differing in cadmium-tolerance and accumulation (Weisuobuzhi and Dong17were Cd tolerant and sensitive, Zhenong8and W6nk2were high and low Cd accumulate genotypes, respectively). The biological effects and the expression of protein ZIP7under Cd stress on four barley cells and the genotype difference were systemically investigated by the newly constructed suspension cell lines. The result showed that stable single suspension cells of four barley genotypes were successfully constructed using immature embryo with diameter2mm as explant. After50μM Cd treatment, the ratio of live suspension cells of four barley genotypes were decreased significantly compared with control. With increase in treating time, the survival of cells decreased, especially the Cd sensitive genotype Dong17and W6nk2. SDS-PAGE gel combined with Western blotting results indicated that the expression of protein ZIP7was upregulated after Cd treatment in W6nk2compared with Zhenong8, this result testified previous result by microarrays and RT-PCR.
7. Effects of NAC application on Cd toxicity and its genotypic difference
A greenhouse hydroponic experiment was carried out, using two barley genotypes differing in Cd tolerance, to evaluate the alleviation effect of exogenous NAC against Cd-induced oxidative stress, barley growth inhibition, Cd and other nutrients uptake and ultrastructure damage. Addition of200μM NAC in50μM Cd culture medium significantly alleviated Cd-induced growth inhibition, Cd toxic symptoms was alleviated by NAC application, and the increases in plant height, root length and dry weight over its Cd treatment were much higher than that in Dong17. Meanwhile, exogenous NAC dramatically depressed ROS, MDA accumulation and antioxidant enzymes, compared with50μM Cd treatment, meliorated Cd-induced damages on leaf and root ultrastructure, the damage of the lamellar structure and shape of chloroplasts almost returned to the control level, a significant reduction in the number of osmiophilic plastolobuli, effectively increased antioxidant enzymes activity, improved the stability and integrity of the nuclear membrane of root meristem, and increased the cell viability in roots tips.
1.镉胁迫对大麦幼苗光合特性及镉吸收与分布的影响及基因型差异
以镉耐性与积累不同的大麦基因型(浙农8号和W6nk2)为材料,水培试验,对照,5、50和500μM不同浓度镉胁迫试验,研究了镉胁迫对大麦幼苗生长、光合特性及镉吸收与分布的影响及基因型差异。结果表明,镉胁迫显著降低麦苗株高、根长、叶绿素含量、地上部和根系干重；基因型之间差异显著,耐镉基因型浙农8号受害较轻,镉敏感基因型W6nk2抑制严重。根尖镉荧光定位分析结果显示,镉主要滞留在根系的质外体,尤其是细胞壁中,其中耐性基因型更为明显；同一植株内由根尖到茎部的镉含量呈逐渐下降趋势,且处理时间越长,镉积累量越高,并有从根尖向茎部移动的趋势。镉胁迫显著影响大麦的光合作用及叶绿素荧光,且不同基因型之间差异显著,镉敏感基因型W6nk2受抑制严重,耐镉基因型浙农8号表现出相对较强的抗性。
2.不同籽粒镉积累大麦基因型籽粒蛋白表达谱的比较研究
利用双向电泳和MALDI-TOF质谱技术,分析比较了籽粒镉积累不同的大麦基因型(浙农8号和VV6nk2)籽粒蛋白表达差异,检测到29个差异表达蛋白质点,浙农8号与W6nk2相比,籽粒蛋白质高表达蛋白点17个,表达受抑制蛋白点12个。差异表达蛋白以蛋白酶抑制剂类相关蛋白为主,包括高表达蛋白α淀粉酶/胰岛素抑制剂CM,Z-型丝氨酸蛋白酶抑制剂和丝氨酸蛋白酶抑制剂Z7；表达受抑制蛋白包括胰蛋白酶抑制剂蛋白BTI-CMe2.1蛋白、胰蛋白酶抑制剂蛋白BTI-CMe2.2蛋白和胰蛋白酶抑制剂。其次是热休克蛋白HSP70和Ras致癌基因家族成员Rab4蛋白等胁迫相关蛋白在浙农8号中高表达；而在W6nk2中有脱氢抗坏血酸还原酶等胁迫相关蛋白高表达。另外还有贮藏类蛋白,比如球蛋白,推定的燕麦蛋白前体在浙农8号中高表达。这些差异表达蛋白可能与大麦籽粒镉积累与耐性密切相关,本研究鉴定得到的蛋白可为深入阐明大麦镉积累机理提供新的契机。
3.大麦遗传图谱的构建及苗期耐镉相关性状的QTL定位
以萎缩不知(耐镉)和苏引麦2号(镉敏感)杂交F1构建的含有108个株系的大麦DH群体为材料,利用SSR和SNP标记,构建遗传图谱,该图谱包含41个SSR标记和15个SNP标记,图谱总长396.9cM,标记间平均距离8.1cM。水培镉胁迫试验,以不加镉为对照,分析苗期耐镉相关农艺与生理性状的耐镉指数CTI,检测到农艺性状相关加性QTLs9个,表型贡献率介于3.30-9.49%之间；检测到酶活和MDA含量相关QTL22个,分布在大麦7条染色体上,其加性效应都达到了显著水平。其中地上部和根系各有11个QTLs,平均表型变异为5.6%。另外,检测到一对控制根系GPX活性的上位性QTL。在第2染色体上标记区间(?)BC2-Chitinase检测到了4个QTLs,其中单株叶片数、根系CAT活性及根系干重都位于0.0cM处,推测为同一QTL,说明控制这些性状的基因很有可能与ABC转运蛋白相关。基因与环境互作条件下,检测到苗期镉含量相关QTLs6个；其中,地上部检测到了2个,分别位于2H和7H上,且这2个QTLs存在极显著地加性×加性上位性互作效应,加性效应值为-23.68,环境镉效应引起的加性效应值为-21.21,可解释表型变异0.57%。根系检测到4个,分别位于1H、5H、6H和7H上。地上部和根系分别检测到7个和11个微量元素含量相关QTLs,总遗传贡献率分别为16.37%和24.95%,由于环境互作效应引起的总遗传变异分别为12.41%和14.6%。
4.大麦不同生育期耐镉及镉积累相关性状QTL定位
盆栽试验,研究了镉胁迫对不同生育期大麦生长及生理性状的影响及株系间差异,检测大麦不同生育期耐镉与镉积累相关QTLs。检测到不同生育期镉含量相关QTLs11个,检测到2对上位性QTLs,分别控制籽粒镉积累及根系Zn含量。另外,检测到地上部镉累积量相关的具有显著加性与环境互作效应的QTL1个。检测到63个与微量元素含量相关QTLs,其中苗期、分蘖期、拔节期和灌浆期4个生育期叶片微量元素含量相关QTLs25个,收获期不同器官微量元素相关QTLs38个；其中17个具有显著地加性与环境互作效应。定位到了8个与农艺和产量CTI相关的QTLs,表型变异在1.54-10.79%之间。而且,遗传标记区间EBmac0615-EBmatc0054, EBmac0557-EBmac0615和SNP标记ABC转运蛋白检测到了镉及数个微量元素含量相关QTLs。
5.大麦籽粒镉积累相关基因表达分析、克隆及大麦转化
利用基因芯片技术,分析了籽粒镉积累差异显著的大麦基因型(高积累：浙农8号；低积累：W6nk2)幼苗在镉胁迫下的基因表达谱,结果显示,镉胁迫显著影响大麦转录水平,基因型间存在显著差异；对2基因型镉胁迫下差异表达基因比对发现,5Cd处理诱导低镉积累基因型W6nk2的运输载体相关基因上调表达,如ZIP、ABC转运蛋白、ATPase、以及Zn、Fe离子运输载体等基因上调表达,说明W6nk2籽粒镉低积累特性可能与这些转运载体基因的上调表达相关；荧光定量PCR验证这些候选基因,得到了一致的表达。进一步利用Gateway克隆技术构建了ZIP基因家族在大麦里发现的4个成员ZIP3、ZIP5、ZIP7和ZIP8的基因沉默表达载体,阳性克隆载体经PCR验证、酶切和测序鉴定；为了进一步研究ZIP家族不同成员之间及在不同大麦基因型间的表达差异和功能,用农杆菌介导法转化此基因到品质较好的模式转化大麦Golden promise,以期进一步研究ZIP基因对大麦镉转运与积累的调控与影响。
6.不同大麦基因型悬浮细胞系的建立及耐镉性差异分析
以籽粒镉积累不同的大麦基因型(高积累：浙农8号；低积累：W6nk2),和耐镉性不同的大麦基因型(耐镉：萎缩不知；镉敏感：东17)为材料,在成功建立分散均匀、稳定的胚性悬浮细胞系的基础上,分析镉胁迫对大麦悬浮细胞生长影响及其基因型差异,及镉胁迫对不同基因型大麦悬浮细胞系生长的影响；并进一步利用大麦不同基因型单细胞悬浮系来验证本实验前期结果所得基因ZIP蛋白的表达情况。结果表明,以直径约2mm的幼胚为外植体,可以成功建立上述4基因型大麦悬浮细胞系；50μM Cd处理,4基因型大麦悬浮细胞活力都显著下降,随着Cd处理时间延长,大麦细胞活力逐步下降；且敏感基因型下降更为明显。SDS-PAGE电泳和Western杂交结果表明,ZIP7蛋白在籽粒低镉积累基因型W6nk2中的表达要强于浙农8号,这一结果与前面实验结果相符。
7.外源NAC对大麦镉毒害的缓解效应及基因型差异
以耐镉基因型萎缩不知和镉敏感基因型东17为材料,水培试验,研究外源NAC对镉所致大麦损伤的缓解作用。结果表明,50μM Cd处理下,添加200μM NAC (Cd+NAC)显著减少大麦幼苗对镉的吸收和积累；缓解大麦幼苗镉毒害症状,2个基因型株高、根长及生物量都比Cd处理显著提高。外源NAC显著影响细胞超微结构及活性氧代谢,NAC显著缓解镉胁迫引起的叶绿体和根系细胞结构的损伤,基本恢复叶绿体片层结构的损伤,嗜锇粒数量显著减少,有效提高了根系分生组织细胞核膜的稳定性和完整性。外源NAC对镉胁迫引起的大麦氧化损伤有明显的缓解效应,显著提高抗氧化酶活性,减少叶片O2·-和·OH、MDA累积,缓解镉对大麦根尖细胞活力的损伤。
Cadmium (Cd), a non-essential trace element, is one of the most phytotoxic heavy metals and widespread pollutant in the environment. Cd is easily absorbed and accumulated in plants and of toxicity at low concentrations, thus may induce potential human health risk via food chain. It is crucial important to understand the underlying mechanism associated with Cd tolerance and accumulation in plants, for developing Cd-resistant/tolerant and low accumulated cultivars or using agronomic practice to inhibit the involvement of Cd in food chains. The purpose of the present study is to elucidate physiological mechanism of genotypic difference in Cd uptake and distribution, using barley genotypes differing in Cd tolerance and accumulation, to identify specific proteins and relevant genes for Cd-tolerance/low accumulation. Meanwhile, genotypic difference were systemically investigated by employing the newly constructed suspension cell lines. With the aid of analyzing available SSR and SNP markers and the establishment/construction of genetic linkage map, Cd tolerance/accumulation relevant gene(s) were tagged/localized, and the genetic relationship was investigated between Cd accumulation and some morphological and physiological indexes, using DH population derived by F1hybrids of genotypes differing in Cd accumulation and tolerance. In addition, we investigated the possibility to reduce Cd uptake and accumulation in barley plants by application of exogenous N-acetyl cysteine (NAC) and its protective effect against Cd-induced growth inhibition, oxidative stress, and damage in ultrastructure and cell viability of roots tips in the two different genotypes differing in Cd tolerance. The main results were summarized follows:
1. Genotypic differences in photosynthesis performance and Cd uptake and distribution in response to Cd toxicity in barley seedlings
A hydroponic experiment was carried out to examine genotypic differences in photosynthesis performance and Cd uptake and distribution in response to different Cd levels (0,5,50,500μM Cd), using two barley genotypes differing in Cd-tolerance and accumulation (c.f. W6nk2, Cd-sensitive and low-grain-accumulation; Zhenong8, Cd-tolerant and high accumulator). The results showed that plant height, root length, and biomass accumulation of shoot/root were significantly reduced in plants grown in5,50,500μM Cd compared with control. There was highly significant difference in the reduction of these growth parameters between the two genotypes, Zhenong8showed much less reduction, suggesting its high tolerance to Cd toxicity, while W6nk2with the greater reduction. Significant genotypic difference in Cd concentration was also found. Zhenong8contained higher Cd concentration in plant tissue than W6nk2. The results of Cd-specific fluorescence localization in root tip showed that Cd was mainly in apoplast, particularly within the cell wall, this phenomenon was more obvious in tolerant genotype Zhenong8. Within the same plant Cd concentration from the root tip to the shoot was gradually decreased, with the treatment time extended the Cd accumulation was higher, and there was a upward moving trend from the root tip to shoot. Speculating that the roots have the binding role to Cd spread, and therefore the roots were poisoned heavyly. Cd stress significantly affected photosynthesis and chlorophyll fluorescence of barley, and there were significant differences between different genotypes, Cd-sensitive genotype W6nk2severely inhibited, while Cd-tolerant genotype Zhenong8showed relatively strong resistance.
2. Comparison study of grain protein expression between two different grain Cd accumulate genotype
We compared grain protein expression in two barley genotypes differing in Cd tolerance and accumulation using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spcetrometry analysis. A total of29protein were identified differentially expression, of these alerted proteins,17were significantly higher expressed and12were significantly suppressed in Zhenong8compared with W6nk2grains. Overall, these differential expression protein were mainly on proteinase inhibitor, involving in higher expressed protein α-amylase/trypsin inhibitor CM, protein z-type serpin, serpin-Z7(HorvuZ7), and significantly suppressed protein BTI-CMe2.1protein, BTI-CMe2.2protein. Followed by stress-related protein, such as up-regultaed protein HSP70and RAB, member of RAS oncogene family-like4, suppressed protein dehydroascorbate reductase. There were also storage protein, embryo globulin and putative avenin-like a precursor were higher expressed in Zhenong8. It was indicated that these proteins might play an important role in Cd accumulation and tolerance or detoxification, and these protein identified may provide new opportunities for elucidating the barley Cd accumulation mechanism.
3. Genetic linkage map construction of barley DH population and Cd tolerant related QTL mapping at seedling stage
A mapping population of108lines (DH), derived from F1coming from a cross between Cd tolerant genotype Weisuobuzhi and Cd sensitive genotype Suyinmai2, were used in this investigation. The linkage map consisted of41SSR and15SNP markers-spanning396.9centimorgans (cM), with an average linkage distance between markers of8.1cM, and clustered into7molecular linkage groups (LGs). Hydroponic and pots experiments were carried out to study the barley growth differences and Cd tolerant related traits among different lines under Cd treatment during different develop stages, and mapped Cd related QTLs. Nine QTLs were detected, associated with Cd tolerance index (CTI) of SPAD value and growth traits on chromosomes1,2,5and7. The individual contribution of each QTL to the phenotypic variation was ranging3.30-9.49%. Twenty two QTLs controlling antioxidant enzymes and MDA content located on all seven chromosomes. Half of them were related in the shoots and half detected in the roots, with the average phenotypic variance5.6%. One pairs epistatic QTLs were detected, eg. Cd tolerance index (CTI) of root glutathione peroxidase (GPX) activity. In addition, four QTLs, which were the leaf numbers per plant, root CAT activity and root dry weight, dry weight at harvest were detected in the interval ABC2-Chitinase on2H, the position was0.0cM, suggesting that these QTLs maybe the same QTL and these traits were associated with ABC transporter. Under genetype and environment interaction conditions,6QTLs for Cd concentration/accumulation were mapped on chromosomes1,2,5,6,7. Two QTLs, qRCd1-1and qACd1-1, overlapped by the same genetic region of ABC1-GMS021, and2additive QTLs of qShCd2-3and qShCd7-2exist epistatic effect. There were7and11QTLs for microelement concentrations in shoots and roots, respectively, with the total contribution of16.37%and24.95%. The total phenotypic variation caused by environment interaction effect was12.41%and14.6%respectively.
4. Analysis of QTLs associated with Cd accumulation and tolerance at different growth stages
In pot experiment, eleven QTLs for Cd concentration with significantly additive (A) effect at different stages and in different organs were detected, some of them represented a single locus affecting different traits and/or the same trait under both control and Cd treatments. Two pairs epistatic QTLs were detected, i.e. one was kernel Cd accumulation with positive epistatic effect, being significant in additive×additive interactions by Cd environment interaction effects; the other was root Zn concentration related epistatic QTLs. In addition, one shoot Cd accumulation related QTL with significantly additive (A) effect and/or additive environment interaction (AE) effect was identified. For63microelements related QTLs,25and38were of leaves and different organs, respectively, and17of them with significant AE effect. Eight QTLs associated with CTI of agronomic and yield traits were detected with phenotypic variation1.54-10.79%. Furthermore, genetic regions EBmac0615-EBmatc0054, EBmac0557-EBmac0615and the novel SNP marker ABC transporters assigned to Cd and several microelement concentrations.
5. Identification of relevant genes for Cd-tolerance and low accumulation, the candidate gene clone and transformation of high and low grain Cd accumulating barley
Using cDNA microarray technology to analyze gene expression under5μM Cd stress and select Cd tolerant and low Cd accumulate related genes in two barley genotypes differing in Cd tolerance and accumulation. The result showed that there was significant difference on expression response of genes related to Cd stress between two genotypes, the up-regulated transcript levels of genes relating to metal transporters in plants were observed, such as ZIP, ABC transporter, P-type ATPase and iron-phytosiderophore transporter in leaves of W6nk2after Cd exposure, which might contribute to its low Cd accumulation characterization. Real time PCR result also confirmed these gene's expression. Thus, we islolated some of them (e.g. ZIP family) from both barley genotypes and transformed them into Golden Promise, our hypothesis was that using RNAi technology may prove the low Cd accumulating gene by suppression of this gene expression in transgenic plants with elevated Cd concentration.
6. Establishment of suspension cells in different barley genotypes and analysis of its Cd tolerance
Here the well-diffused and stable suspension cells were successfully constructed for the four barley genotypes differing in cadmium-tolerance and accumulation (Weisuobuzhi and Dong17were Cd tolerant and sensitive, Zhenong8and W6nk2were high and low Cd accumulate genotypes, respectively). The biological effects and the expression of protein ZIP7under Cd stress on four barley cells and the genotype difference were systemically investigated by the newly constructed suspension cell lines. The result showed that stable single suspension cells of four barley genotypes were successfully constructed using immature embryo with diameter2mm as explant. After50μM Cd treatment, the ratio of live suspension cells of four barley genotypes were decreased significantly compared with control. With increase in treating time, the survival of cells decreased, especially the Cd sensitive genotype Dong17and W6nk2. SDS-PAGE gel combined with Western blotting results indicated that the expression of protein ZIP7was upregulated after Cd treatment in W6nk2compared with Zhenong8, this result testified previous result by microarrays and RT-PCR.
7. Effects of NAC application on Cd toxicity and its genotypic difference
A greenhouse hydroponic experiment was carried out, using two barley genotypes differing in Cd tolerance, to evaluate the alleviation effect of exogenous NAC against Cd-induced oxidative stress, barley growth inhibition, Cd and other nutrients uptake and ultrastructure damage. Addition of200μM NAC in50μM Cd culture medium significantly alleviated Cd-induced growth inhibition, Cd toxic symptoms was alleviated by NAC application, and the increases in plant height, root length and dry weight over its Cd treatment were much higher than that in Dong17. Meanwhile, exogenous NAC dramatically depressed ROS, MDA accumulation and antioxidant enzymes, compared with50μM Cd treatment, meliorated Cd-induced damages on leaf and root ultrastructure, the damage of the lamellar structure and shape of chloroplasts almost returned to the control level, a significant reduction in the number of osmiophilic plastolobuli, effectively increased antioxidant enzymes activity, improved the stability and integrity of the nuclear membrane of root meristem, and increased the cell viability in roots tips.
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