【作者】 张吉宇；

【导师】 王彦荣； German Spangenberg；

【作者基本信息】 兰州大学 ， 草业科学， 2008， 博士

【摘要】 野生植物携带有潜在的特异基因和变异基因,通过功能基因组的研究和基因挖掘,可揭示此类植物适应环境胁迫的特异机制,为农作物和草类植物的基因改良奠定基础。无芒隐子草（Cleistogenes songorica）为旱生性多年生草类,其极端抗旱性及作为沙漠草原恢复重建种的潜在价值已逐渐得到认可。本研究从收集我国极抗旱野生无芒隐子草种质资源、分析种质抗旱特性基础上,构建其干旱胁迫诱导cDNA文库;通过高通量测序技术平台获得大量cDNA序列;通过高性能生物信息学平台挖掘无芒隐子草抗旱功能基因组信息,进行抗旱相关基因的表达产物分析、转基因模式植物（拟南芥）的功能验证,确定目标基因的功能,为这些基因在草类作物遗传改良中的利用奠定基础。主要结论如下。收集到我国16份野生无芒隐子草种质资源,采用RAPD标记分析了无芒隐子草种群遗传多样性。用筛选得到的7个随机引物,共扩增出52条DNA片段,其中23条为多态性片段。分子变异分析（AMOVA）结果显示,种群间的变异占总变异的53.19%,种群内不同个体间的变异占总变异的46.81%,种群间的变异存在着显著性差异（P＜0.001）。聚类分析将无芒隐子草16个种群大致划分为3个聚类组:第一个聚类组包括10个种群,即:pop1～pop5、pop7、pop13～pop16;第2个聚类组包括pop6、pop8、pop11和pop12;第3个聚类组包括pop9和pop10。以无芒隐子草遗传差异较大的三个种群CS01,CS09和CS15为材料,通过观察其幼苗体内水分、光合作用参数、叶绿素荧光参数和光合色素等特征,研究其在持续干旱及复水期间忍耐极端干旱的保护机制。证实这三个种群响应干旱胁迫能力存在差异。干旱胁迫过程中,无芒隐子草仍保持相对较高的叶片相对含水量（75%）和叶水势（-3.2 MPa）,属于高水势耐旱型植物;光合器官和光合色素含量在干旱胁迫下受到严重影响;而光化学器官的功能影响不大,除PSⅡ最大光化学效率Fv/Fm略有下降外,其它光化学参数都变化不大。无芒隐子草在持续缺水的环境下,光合速率持续下降,而胞间CO₂浓度变化却不明显,两者变化方向不一致,故初步判断无芒隐子草净光合速率下降归因于叶肉细胞同化能力的降低。采用SMART技术,构建了无芒隐子草的轻度干旱胁迫根和叶、极度干旱胁迫根和叶共4个cDNA文库,文库Cs01L、Cs02R、Cs03L和Cs04R的重组率分别为78.6%、100%、78.6%和71.4%,插入片段大小在0.2～2.8kb之间。采用5’端随机测序的策略对无芒隐子草不同材料来源的全长cDNA文库进行了测序,共获得5664条序列。去除载体和污染序列,并将50bp以下序列剔除后共计获得3579条高质量序列,总序列长度达到2,191,166bp,平均每个EST长度为613bp。把获得的高质量EST使用phrap进行组装和去冗余后获得1499个重叠群,包括805个独立非冗余序列和694个多成员非冗余序列。基因功能注释（GO）采用与UniProt蛋白质数据库的匹配,60%（899条）无芒隐子草序列得到GO功能注释术语。这些GO术语中包含分子功能、生物过程、细胞组分三个层次,分别占22%、46%和32%。功能注释显示胁迫响应基因63个,其中与抗旱密切相关的基因25个。使用国际上通用的SSR引物设计软件,对1499个无芒隐子草EST（长度共计1.03Mb）进行了SSR分子标记的鉴定,共找到161个SSR特异分子标记。采用RACE法等相关技术进一步克隆无芒隐子草22个抗旱相关基因全长序列,已经获得全长序列10个。geNorm程序获得无芒隐子草7个内参基因的表达稳定度由高到低依次为:CsEIF5₂＞CsEIF5₁＞CsGAPDH＞CsActin-11＞CsUBQ＞CsTUA2＞CsHistone,以表达相对最稳定的3个内参基因GAPDH、CsEIF5₁、CsEIF5₂获得定量PCR实验中相对定量或绝对定量数据的所有标准化因子,以标准化因子对全部数据进行标准化。通过RT—PCR、qPCR相对定量来鉴定无隐子芒草22个抗旱相关基因的表达,发现有8个基因的表达是由干旱胁迫诱导的,并且受干旱胁迫的差异调控较显著,在胁迫过的根和叶中的表达都较对照增加3倍以上;qPCR绝对定量进一步研究7个基因在不同组织、不同胁迫处理（干旱胁迫0天、4天、6天、8天、10天、以及恢复浇水后1天和4天共7个关键时间点）的表达情况,揭示了这些基因在适应干旱环境过程中的表达调控机制。CsALDH基因在干旱胁迫下根系中相对表达量是对照的17倍。相对定量和绝对定量结果显示CsLEA2基因只在胁迫诱导下才表达,此基因在胁迫4天后的根中低水平表达,胁迫6天后根和叶中的表达量逐渐增大,干旱胁迫最大的第8天和第10天表达量达到了最大值;无芒隐子草恢复浇水后,基因表达量显著降低,直至浇水后第4天植物恢复正常生长时此基因表达消失。应用Gateway克隆技术,将CsALDH和CsLEA2基因分别与强组成型启动子CaMV 35S和胁迫诱导型启动子rd29A融合,构建了这两个基因的两种植物表达载体,并获得了拟南芥转基因植株。更多还原

【Abstract】 Native plants are a potentially rich resource for discovery of novel genes and gene variants.Functional genomics and genes discovery specifically targeting native plants can provide a view on the mechenism of abiotic stress tolerance and derive improved crops and forages with enhanced tolerance to abiotic.Cleistogenes songorica is a xerophytic perennial grass.Although it had very important value,the value of drought tolerance and as a potential plant for regeneration of desert-steppe areas have only recently been recognized.In this project,native C.songorica germplasm were collected for drought tolerance mechanism understanding and drought responsive genes discovery. Expressed sequence tags（ESTs）were generated from cDNA libraries prepared from water stressed leaf and root.We analyzed the expression level of drought reponsive genes in C.songorica and after transformed in arabidopsis,which will provide basic information of those genes for genetic improved forage grasses.The progress was as followings.In this study,16 native C.songorica germplasm were collected in China.Genetic diversity of the C.songorica populations were studied at RAPD levels.A total of 192 individuals of 16 populations were analyzed using 7 primers,producing 52 scorable bands,23 of which were polymorphic.An analysis of molecular variance（AMOVA） revealed a much larger genetic variation among populations（53.19%）than between them （46.81%）.A significant proportion was attributable to differences among populations（P＜0.001,tested using a 1000 permutations）,but not within populations.The UPGMA Cluster analysis showed two groups according to community type and altitude.The first group was including 10 populations of pop1～pop5,pop7 and pop13～pop16,while 6 populations of pop6、pop8～pop12 were included in second group.Three highly divergent populations of CS01,CS09 and CS15 were imposed drought stress by withholding irrigation and then rewatering.Leaf water content, photosynthesis,chlorophyll florescence and pigment content were studied for our understanding of the mechanisms involved in plant response and tolerance to water stress. During the withholding irrigation period,leaf relative water content is 75%,and leaf water potential is -3.2 Mpa,so C.songorica can be seen as a drought tolerant plant with high level of leaf water potential.Drought induced a decline of photosynthesis and pigment content rather than a direct damage to PSⅡ.Only Fv/Fm was observed to decline during the drought stress experiment,other PSⅡphotochemistry parameters were not affected.Different changes existed between photosynthesis and intercellular CO₂ concentration,which tell us that the decline of the photosynthesis in C.songorica attribute to the decline of assimilation capacity of mesophyll cells.Using SMART method,4 cDNA libraries were constructed from leaf and root of C. songorica with light and severe drought stress.The recombination rate of Cs01L,Cs02R, Cs03L and Cs04 library was 78.6%,100%,78.6%and 71.4%respectively,with the insert fragment size of 0.2～2.8kb.Randomly selected cloned were used to establish a genomic resouce of expressed sequence tags（ESTs）.A total of 5664 sequence were generated with 3579 of them being of suffient quality for further analysis.This produced a database of 2,191,166 bases of C.songorica sequence.The 3579 ESTs were assembled into 1499 contigs with 805 singleton unigenes and 694 multi-memeber unigenes using the Phrap program.Gene Ontology（GO）functional annotation was assigned to the unigenes by sequence similarity to entries within Uniprot using BLAST,with 60%（899） of the unigenes annoted with GO term.Categories were assigned on the basis of molecular function,biological process and cellular component with 22%,46%and 32% respectively.63 unigenes were annoted as stress responsive genes,including 25 drought responsive unigenes.Using SSRPrimer program,we searched for potential microsatellite （SSR）sequences in the 1499 candidate gene set.In total,161 SSR were identified from C. songorica sequence databases.Using RACE method,10 full length of drought responsive genes were cloned. GeNorm software was used to analyze gene expression stability.The expression stability of 7 house keeping genes were CsEIF5₂＞CsEIF5₁＞CsGAPDH＞CsActin-11＞CsUBQ＞CsTUA2＞CsHistone.GeNorm analysis indicated that GAPDH,CsEIF5₁ and CsEIF5₂ are the most stable genes,Hence,after calculation of a nomalization factor from 3 most stable genes,the normalized expression levels can be calculated by dividing the raw quantities for each sample by the the appropriate normalization factor.By screening the expression level of 22 drought response genes using RT—PCR and relative quantification of qPCR,8 genes were observed to induce by drought stress and differentially expressed under different stress treatment.To understand the mechanism of expression and regulation of 7 genes,absolute quantification was employed to analyze its expression under diverse tissues and stress treatment（0d,4d,6d,8d,10d after withholding watering and 1d,4d after rewatering）.CsALDH gene was expressed about 17 folds in drought stressed root tissue than in the controls.CsLEA2 gene was only observed to express in drought stressed plants.It began to express slightly in root tissue under 4 days of withholding watering,then increased after 6 days and got the maximum expression after 8 days and 10 days of withholding watering.The expression level was decreased significantly after 1 day of rewatering and no expression was detected after 4 days ofrewatering.Using Gateway cloning technology,CsALDH and CsLEA2 genes were constructed into plant expression vectors contaning constitutive expression promotor CaMV 35S or stress induced promotor rd29A,which were thansferred to Arabidopsis plant.更多还原