【作者】 高文瑞；

【导师】 麻浩；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 随着全球性的气候异常和生态平衡的破坏,干旱、沙化、盐碱土地面积急剧增加,水资源短缺已成为全人类面临的一个严重生态问题。干旱也已成为一种世界性的重大农业灾害,其对农作物造成的损失在所有的非生物胁迫中居首位,仅次于生物胁迫病虫害造成的损失。鹰嘴豆是最早被人类驯化利用的豆科植物之一。在豆科类作物中居第3位,是世界上栽培面积较大的食用豆类作物之一。鹰嘴豆具有耐旱、抗逆、水分利用效率高等特点,是进行作物耐旱性改良的重要基因资源。本研究首先从我国新疆的鹰嘴豆品种中筛选鉴定耐旱品种,然后在构建鹰嘴豆幼苗水分胁迫下两个平行cDNA文库及其EST序列分析的基础上,克隆与水分胁迫响应的重要基因,并研究其表达模式,结果如下：1、鹰嘴豆干旱胁迫条件下生理生化的研究与耐旱性筛选鉴定通过室内试验,以耐旱性强弱不同的12个鹰嘴豆品种为材料,对干旱胁迫下鹰嘴豆叶片的相对含水量、丙二醛含量、脯氨酸含量等生理生化指标进行研究,探讨了这些生理生化指标与耐旱性的关系,同时通过系统聚类的方法对12个鹰嘴豆品种进行了耐旱性综合评价。结果表明,耐旱性较强的品种叶片保水能力强,水分亏缺小,脯氨酸增加倍数大,能保持较高的细胞膜相对完整性,细胞膜脂过氧化作用较小,净光合速率下降幅度较小,蒸腾速率和气孔导度下降幅度较大,水分利用效率较高。12个品种可划分为高耐旱性、中耐旱性和低耐旱性3个类群。对耐旱性强的209和耐旱性弱的88-1进行干旱胁迫下叶片和根中内源激素变化的研究表明,60 mM PEG 4000胁迫24 h后两个品种的ABA与IAA含量均显著增加,但209的增幅大于88-1；GA和ZR含量均显著降低,且209的降幅大于88-1。2、鹰嘴豆叶片cDNA文库的构建在前期鉴定鹰嘴豆耐旱性基础上,选取耐旱性较好的品种209用于构建PEG 4000模拟的干旱胁迫（MH1）和正常生长（MH2）的两个平行的cDNA文库。通过梯度稀释与细菌平板记数法计算出MH1库容量为4.9×105,重组率为92%；MH2库容量7.5×105,重组率为90%。经过菌落PCR鉴定,两个文库中外源基因的插入片段基本上均达到1 Kb以上。因此构建的cDNA文库均符合文库标准,可以进行下一步大规模的EST测序。这为筛选抗旱相关的重要基因提供了有效的平台,为基因克隆等奠定了良好的基础。3、两个鹰嘴豆叶片cDNA文库大规模测序及EST序列分析对前期构建的两个非均一化的cDNA文库（一个是PEG 4000胁迫处理文库,另一个是对照文库）进行了EST测序。每个文库随机挑选2500个左右的克隆进行测序,并对测序结果进行生物信息学分析。经过IDEG6在线生物软件分析,结果表明有92个基因的表达差异显著,且这些基因参与了不同的生物学过程。许多上调表达的基因都与干旱耐性相关,而下调表达的基因大多数与光合作用相关。为验证文库分析的结果,挑选了5个差异表达的基因进行实时荧光定量PCR（qPCR）分析,结果表明,qPCR结论与EST分析的结果一致。本结果将为鹰嘴豆耐旱分子基础的研究做出重要的贡献。4、鹰嘴豆S—腺苷甲硫氨酸（SAM）代谢途径中几个重要基因的克隆及其表达研究在对前期构建的两个鹰嘴豆叶片cDNA文库5’随机测序和序列拼接注释的基础上,克隆了鹰嘴豆SAM代谢途径中的CpSAMs、CpMS及CpSAMDC3个重要基因,并对这三个基因进行了相关的生物信息学分析。结果表明这3个基因都包含了完整的开放阅读框,其中CpSAMs基因编码的蛋白包含了两个腺苷甲硫氨酸信号结构域,CpMS基因编码的蛋白含有Meth_synt₁和Meth_synt₂两个结构域,CpSAMDC基因编码的蛋白包含了酶原剪切位点和PEST两个结构域。同时为了研究SAM代谢途径与鹰嘴豆耐旱的相关性,对该途径中CpSAMs、CpMS、CpSAHH和CpSAMDC等4个基因进行了半定量RT-PCR分析。结果表明这4个基因于干旱胁迫的初始阶段在根和叶中的表达量都上升,在根、茎、叶中的表达存在组织特异性。因此,SAM代谢途径可能参与鹰嘴豆干旱胁迫响应。更多还原

【Abstract】 With deterioration of global climate and destruction of the balance of biogeocenose, the area of aridity, desertification and salinization rapidly increased. The shortage of water resources has been a serious ecological problem which faced by human being. Drought has also been a global agricultural calamity and is first abiotic stress factor led to reduction of yield.The cultivated chickpea, Cicer arietinum L., was one of the first grain legumes to be domesticated in the ancientry. Today, chickpea is the third most important pulse crop in the world. Due to displaying considerable drought tolerance, adverse resistance and high water used efficiency, chickpea is being used as a important gene resource for improving drought tolerance of crop. In this study, strong drought tolerant varieties were screened and identified from 12 chickpea varieties from Xinjiang Autonomous Region. On the basis of construction of two cDNA libraries of chickpea leaves under water stress and analysis of expressed sequence tags （ESTs）, cloning and expression pattern of some important water-stress responded genes involved in SAM pathway were studied. The main conclusions are as follows:1. Effects of drought stress on physiological-biochemical indexes and identification of drought tolerance of chickpea cultivarsThrough laboratory experiment study, relative water content, malondialdehyde content, proline content and other physiological-biochemical indexes of leaves of 12 chickpea cultivars under drought stress were investigated. The relationship between these physiological-biochemical indexes and drought tolerance were discussed. By system cluster analysis, the 12 chickpea varieties could be classified into 3 drought tolerant ranks:the strong drought tolerant, the medium drought tolerant, and the weak drought tolerant. The results showed that the strong drought tolerant varieties have stronger water retaining capacity, smaller water deficit, larger increase multiple of proline, higher relative integrity of the membrane, smaller membrane lipid peroxidation, smaller decline rate of net photo synthetic, larger decline rate of stomata conductance and transpiration, higher water use efficiency.The IAA, GA3, ZR and ABA contents in chickpea under drought conditions were determined by the method of ELISA and the relationship between these endogenous hormones and drought tolerance were discussed. The results showed, after 60 mM PEG 4000-treated 24 h, contents of ABA and IAA increased significantly and contents of GA3 and ZR decreased significantly in 209 （strong drought tolerant） and 88-1 （weak drought tolerant）. The stronger the drought tolerance of the variety is, the more the decrease of GA3 and ZR contents, the less the increase of ABA and IAA contents.2. Construction and identification of two cDNA libraries of chickpea leavesOn the basis of identification of drought tolerance, two non-normalized cDNA libraries were constructed from the seedling leaves of a strong drought-tolerant chickpea cultivar 209 under PEG 4000-treated （MH1） and-nontreated （MH2） conditions. Through serial dilution and bacteria plate count method, the content of cDNA library and the recombination were calculated. Results showed the content of the MH1 was 4.9×105 and the recombination was 92%, and the content of the MH2 was 7.5×105 and the recombination was 90%. The results of identification of colony PCR showed that the inserts of the two cDNA libraries were basically more than 1 Kb. The two libraries were constructed successfully and could be used for large-scale sequencing. It will provide an effective platform of screening for drought tolerant-related genes and gene clone.3. Large-scale sequencing and EST sequence analysis of two chickpea leaf cDNA librariesAbout 2500 clones from each library were selected randomly for sequencing analysis. Based on IDEG6 online software analysis,92 genes were differentially expressed, and these genes were involved in diverse biological progresses, such as metabolism, transcription, signal transduction, protein synthesis and others. Most of the up-regulated genes were related to drought tolerance, and the down-regulated genes were mainly involved in photosynthesis. The differential expression patterns of 5 functional unigenes were confirmed by quantitative real-time PCR （qPCR）. The results will be helpful in understanding the molecular basis of drought tolerance in chickpea. 4. Gene cloning and expression pattern study of some important genes in S-adenosylmethionine （SAM） metabolic pathwayUnder the basis of 5’EST sequencing, assembling and annotation of the two chickpea leaf libraries, CpSAMs、CpMS and CpSAMDC, which involved in SAM metabolic pathway, were cloned and analyzed by bioinformatics. In order to study the relationship between SAM metabolic pathway and responses of chickpea to drought, the expression patterns of CpSAMs、CpMS、CpSAHH and CpSAMDC were confirmed by semi-quantitative PCR（qPCR）. The results showed the three cloned genes have complete open reading frames. CpSAMs contains two conserved SAMs motifs, CpMS contains Meth_synt₁ and Meth_synt₂ motifs and CpSAMDC contains PEST regions and proenzyme cleavage site motif. The gene expression of CpSAMs, CpMS, CpSAHH and CpSAMDC were up-regulated during the initial stages of drought stress and the three genes also have differentially expression patterns in chickpea roots, stems and leaves. Taken together, it can be speculated that SAM metabolic pathway may be involve in chickpea response to drought stress.更多还原