【作者】 常建忠；

【导师】 景蕊莲；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 干旱、高盐、低温等非生物逆境胁迫严重威胁着世界粮食的产量。小麦(Triticum aestivum L.)是世界上最主要粮食作物之一，干旱是限制小麦产量最主要的非生物胁迫因素。发掘小麦抗旱基因资源是改良小麦抗旱性、培育抗旱新品种的根本途径。在小麦种质资源中蕴藏着丰富的等位变异，分离鉴定出与抗旱相关的功能等位变异不仅有助于深入了解小麦抗旱分子机制，而且还可以依据鉴定出的优异等位变异开发功能标记，为小麦抗旱分子标记辅助选择育种奠定基础。关联分析分辨率高、研究周期短，可直接鉴定与表型紧密关联的功能性位点，是等位基因发掘的有效手段。本研究以小麦逆境应答基因TaSAP1为候选基因，进行了两方面的研究。一是通过与小麦D基因组供体种粗山羊草(Aegilops tauschii)基因组序列比对，在普通小麦中获得TaSAP1的侧翼序列，然后在30份多态性较高的小麦材料中研究TaSAP1-A1多态性，并根据多态性位点开发分子标记，进行单倍型分析，在自然群体中与表型数据进行基于标记和单倍型的关联分析；二是通过瞬时表达和稳定表达的方式对TaSAP1-A2启动子功能进行初步研究。主要研究结果如下：1.通过与小麦D基因组供体种粗山羊草基因组序列比对，在普通小麦中获得TaSAP1-A1和TaSAP1-A2两种序列，序列长度分别为5.1kb和4.1kb。TaSAP1-A1和TaSAP1-A2编码区长度均为510bp，启动子长度差异较大，分别为3.4kb和2.5kb。与cDNA序列比对发现TaSAP1编码区没有内含子，而在5′UTR存在两个内含子，长度分别为153bp和758bp，位于-967bp~-814bp和-771bp~-13bp。2.以30份小麦为材料，分析TaSAP1-A1序列多态性，在约5.1kb的序列中检测到46个多态性位点，包括43个单核苷酸多态性(SNP)位点和3个插入/缺失突变(InDel)位点，其中在启动子区有39个多态性位点，intron-2中有2个SNP位点，3′端非编码区具有5个多态性位点，TaSAP1-A1编码区未发现多态性位点。滑动窗口分析表明启动子上游-3,499bp到-1,498bp区域的序列多态性最高，π值为0.00631；然后是3′端非编码区和intron-2，π值分别为0.00133和0.00100。3.基于TaSAP1-A1启动子区3个多态性位点InDel5-1810、SNP-2606和InDel39-1637开发了3个分子标记，分别命名为T7AM5、T7AM2606和T7AM39。其中T7AM5和T7AM2606是CAPS标记，T7AM39是等位基因特异PCR标记。利用T7AM39将TaSAP1-A1定位于小麦7A染色体标记Xwmc530和Xbarc174之间，与两个标记距离分别为2.1cM和13.9cM。利用3个分子标记可以将自然群体300材料划分为6种单倍型，分别为HapI~HapVI，其中HapI和HapII在群体中所占比例最大，分别占45.6%和31.5%，HapVI所占比例最小为2.9%。4.关联分析表明3个分子标记T7AM5、T7AM2606和T7AM393与5个农艺性状显著关联，包括穗长、穗下节长、每穗总小穗数、穗粒数和千粒重。T7AM5在4种环境下与穗长显著关联，其中3种环境为旱地，在6种环境下与穗下节长显著关联，其中4种为旱地；T7AM39和T7AM2606与千粒重显著关联。不同单倍型与千粒重、穗长和每穗小穗数显著相关，其中HapIII对千粒重和穗长具有正向贡献，千粒重在所有环境下都呈现HapIII>HapII>HapI的规律。分析不同年代育成品种中HapIII的频率，结果表明HapIII在育种过程中经历了不断的正向选择。HapIII在不同小麦生态区的分布表明其在育种中的应用不受生态环境限制。5.序列分析表明TaSAP1-A2启动子区存在逆境相关顺式作用元件，如ABRE、MBS、ARE等。瞬时表达分析表明TaSAP1-A2启动子在小麦愈伤组织中属于诱导型启动子，且5′UTR内含子对启动子活性具有重要作用。5′缺失片段活性分析表明只有最小片段P6在拟南芥中具有转录激活活性，而TaSAP1-A2全启动子活性被片段P(5-6)抑制。P6具有组成型转录激活活性，其活性与CaMV35S相当。在拟南芥中启动子片段P-(5-6)的活性可以被干旱和低温诱导增强。研究结果表明P6和P-(5-6)在小麦等作物基因工程相关研究中具有潜在的应用价值。更多还原

【Abstract】 Drought, salinity and low temperature are the major abiotic stresses that dramatically threaten thefood supply in the world. Wheat is one of the most important staple food crops in the world, drought isconsidered to be the major limiting factor seriously impacting the wheat production. Exploringdrought-response gene resources in germplasm is a fundamental approach for improving droughttolerance in wheat. There are abundant allelic variations in wheat germplasm. Identifying thesefunctional alleles for drought tolerance will help to further understand the drought-resistancemechanisms. Functional markers can also be developed based on the identified functional variations,which provide the basis for marker-assisted selection for drought improvement. Association mappingpossesses the advantages of higher mapping resolution and shorter research time, therefore it isconsideredas a powerful approach for identifying functional alleles in germplasm resources.In this study, TaSAP1gene was chosen as the candidate gene to perform two aspects of research.(1)Genomic region flanking TaSAP1was obtained by a BLASTsearch against the Aegilops tauschii draftgenome sequence. Thirty wheat accessions were selectedfor polymorphism analysis.Based on thevariations in promoter region of TaSAP1-A1, molecular markers were developed for genotyping.Association analysis were performed to identifyfunctional alleles and haplotypes;(2) The function ofTaSAP1-A2was preliminarily studied by using transient expression and stable expression. The mainresults are summarized as follows:1.Two fragments, designated as TaSAP1-A1(5.1kb)and TaSAP1-A2(4.1kb), were obtained by aBLAST search against the Aegilops tauschii draft genome sequence. Both of them had a coding regionof510bp. Sequence analysis indicated that the difference between TaSAP1-A1and TaSAP1-A2wasmainly located in the promoter region.Thesequences were3.4kb and2.5kbrespectively. Alignment ofthe cDNA andgenomic DNA sequence revealed two introns in the5′UTR of TaSAP1. Intron-1was153bp in length and located between-967bp and-814bp from ATG, and intron-2was758bp locatedbetween-771bp and-13bp.2.Forty-six variants in the entire region of TaSAP1-A1, comprising of43SNPs and3indels, wereidentified among the30accessions. These include39polymorphisms in thepromoter region, two SNPsin intron-2in the5′UTR, and five variants, including four SNPs and one indel, in the3′flanking region.No variation was observed in the coding region. Sliding-window analysis indicated the highest variationoccurred in the upstream promoter region(-3,499bp to-1,498bp)where the nucleotide diversitywas0.00631, followed by the3′flanking region(π=0.00133) and intron-2(π=0.00100).3.Based on three polymorphic sites in the promoter region, including InDel5-1810, SNP-2606andInDel39-1637, three marker T7AM5, T7AM2606and T7AM39were developed. T7AM5andT7AM2606were CAPS markers, and T7AM39was an allele-specific PCR marker. TaSAP1-A1wasmapped to a region flanked by Xwmc530(2.1cM) and Xbarc174(13.9cM) on chromosome7A usingmarker T7AM39. A total of six haplotypes (HapI-HapVI) were detected among the300wheataccessions based on the three markers. Among them, HapI and HapII were the main haplotypes, accounting for45.3%and31.5%of accessionsrespectively. The proportion of HapVI was the lowest(2.9%).4.Marker-trait association indicated T7AM5, T7AM2606and T7AM39were significantlyassociated withfive traits including spike length, peduncle length, number of spikelets per spike, numberof grains per spike and1000-grain weight. T7AM5was significantly associated with spike length in fourenvironments among which three were drought-stressed. It was again significantly associated withpeduncle length in six environments, four of which were drought-stressed. T7AM39and T7AM2606were mainly associated with1000-grain weight. Moreover, haplotype significantly affected someagronomic traits, such as1000-grain weight, spike lengthand number of spikelets per spike. HapIIImade a positive contribution to TGW and SL, and the average TGW of HapIII was the highest in allenvironments with the pattern of HapIII>HapII>HapI.The frequency of HapIII was an increasingtrend during the breeding process,showed it was positivly selected. The geographical distribution ofHapIII in China indicated the use of HapIII was not limited byecological environments, such as lightand temperature.5.Sequence analysis revealed several putative stress-response cis-acting regulatory elements inTaSAP1-A2promoter, such as ABRE, MBS and ARE. Transient expression in wheat calli proved thatthepromoter of TaSAP1-A2was the stress-inducible promoter. Introns in5′UTR of TaSAP1-A2wereinvolved in stress-induction. Five5′deletions were evalued by stable expression in Arabidopsis. Nopromoter activity was detected in Arabidopsis for all the fragments, except for P6. Further studyindicated a fragment named P(5-6) inhibited the promoter activity of TaSAP1-A2. P6possessesconstitutive transcriptional activation, and its activity was comparable to that of CaMV35S. The activityof P-(5-6) could be enhanced by drought and low temperature. The results showed that P6and P-(5-6)promoters have potential value in genetic engineering of crop plants.更多还原