【作者】 张兰；

【导师】 毛龙；

【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】 小麦是世界上重要的粮食作物之一，为人类的生存和发展提供了重要的能量来源和物质基础。本文研究了小麦重要农艺性状相关的两个基因TaqSH1和TaDEP1。其中TaqSH1影响小麦的落粒性，而TaDEP1则与小麦产量的重要组成因素千粒重相关。籽粒脱落是作物驯化过程中的一个重要事件。在小麦中，已知Q基因突变导致易落粒的特性改变为脆轴，关于小麦落粒的其它分子机制尚不清楚。水稻qSH1基因是影响水稻谷粒脱落的一个主要QTL。我们利用同源克隆的方法获得了水稻qSH1在六倍体小麦中的直系同源基因TaqSH1的B基因组全长。TaqSH1编码一个BEL1类蛋白，BEL1-like同源异型框区域在单双子叶植物中都很保守。我们利用缺体四体将TaqSH1定位在3B染色体上，这是控制小麦籽粒脱落的一个新的遗传位点。TaqSH1基因编码区在易落粒小麦近缘种与不易落粒小麦及其近缘种间有8个SNP的差异，其中3个SNP使氨基酸发生了改变。在拟南芥中过表达TaqSH1使植株表现出明显比野生型植株矮化，角果间距缩短变紧密，角果角度平直，角果上假隔膜变窄，角果成熟后花仍然不脱落的特点。扫描电镜观察显示，转基因植株花瓣离区细胞发育与野生型相比出现延迟，从而导致了花瓣脱落时间延后。表达检测表明，转基因植株中IDA、HAESA、KNAT1、KNAT6、SHP1、SHP2等在拟南芥离区形成、花脱落等过程中发挥着重要作用的基因的表达量明显下降，而TaqSH1在拟南芥中的正源基因RPL的表达量没有变化。酵母双杂交结果表明，TaqSH1基因分别与KNAT1、KNAT6互作，这有可能是BEL1与KNOX结构域互作的结果。我们根据以上结果推测TaqSH1可能在小麦谷粒离区的发育中发挥作用，其工作原理在单双子叶间具有一定的保守性。本文研究的第二个基因与小麦的产量相关。水稻DEP1(DENSE AND ERECT PANICLE1)位点是控制产量性状的一个主效QTL。DEP1最后一个外显子的一个碱基发生突变，形成提前终止，被命名为dep1。dep1基因能够控制穗型、增强植株分生组织活力，缩短花序节间距离，增加穗粒数，进而提高水稻产量。我们在小麦中克隆到了TaDEP1基因并将其定位在第五同源群。我们在染色体5B上TaDEP1拷贝的第一个内含子上开发了一个InDel标记。这个标记(我们将其命名为dep1-5b)精细定位在5B染色体长臂GS34和S67两个标记间约10cM的区域内。对标记dep1-5b在262份小麦微核心种质中的检测结果进行关联分析发现dep1-5b与两年的小麦微核心种质中的千粒重相关。当把小麦微核心种质分成地方品种和栽培品种时，dep1-5b只和地方品种中的千粒重相关联，与栽培品种中的千粒重性状相关性不显著。水稻中具有长、短（提前终止）两个DEP1(dep1)等位基因，短的等位基因dep1表达量高时水稻产量增加。我们对其它草类的DEP1基因结构进行观察，发现除玉米外几乎所有的草类包括普通小麦的祖先(乌拉尔图、粗山羊草、拟斯卑尔脱山羊草)、短柄草、高粱都只有长度缩短的等位基因。玉米第2、第7染色体上分别有长、短两个DEP1(dep1)基因。此观察结果与关联分析结果一致，即经过驯化后能使产量增加的短的等位基因dep1被保留下来，长的DEP1逐渐丢失；同时TaDEP1基因在小麦全部微核心种质中与千粒重显著相关，而在经人工选择后的栽培品种中与千粒重相关性不显著，说明这个短的等位基因dep1是驯化的结果。更多还原

【Abstract】 Wheat (Triticum aestivum L.) is one of the most important crops in the world and provides mostcalories consumed by human beings. We studied two important agronomic trait genes in wheat:TaqSH1and TaDEP1which are associated with seed shattering and thousand grain weight.Seed shattering is one of the major domestication traits of crops. In wheat, except for the Q genewhose mutation renders free threshing and changing of rachis fragility, not much is known about themolecular mechanism for this process. We report here the cloning and characterization of TaqSH1, theortholog of the rice seed shattering gene qSH1. TaqSH1encodes a BEL1-like protein that is conservedamong monocots and eudicots. TaqSH1is located on the homoeologous group3, a potential new geneticlocus for seed threshability in wheat. Over expression of TaqSH1in Arabidopsis resulted in dwarfedplants. The inflorescences of transgenic plants were more compact with larger pedicel angles. ScanningElectron Microscopy showed that the transgenic siliques had narrower replums with altered dehiscencezones. In addition, petal abscission was significantly delayed due to the slow down of abscission zonecell development. Real-time PCR assays showed that over expression of TaqSH1down regulatedseveral known Arabidopsis abscission related genes, such as IDA, HAESA, KNAT1and KNAT6in thetransgenic plants. Taken together, our data suggest that TaqSH1may represent another example ofconserved mechanisms across monocots and eudicots for fruit/grain abscission and should havepotential application in genetic manipulation of wheat seed shattering.The second gene was related to rice DEP1(DENSE AND ERECT PANICLE1) gene. DEP1was amajor quantitative trait loci (QTLs) that controlled grain yield. There are truncated deletions in theC-terminal of the mutant DEP1allele (dep1). Dep1is an important regulator that can enhance meristemactivity, reduce inter-node distance, increase per spike grain number, and eventually increase the riceyield. We report here further characterization of the DEP1gene (TaDEP1) in wheat. Chromosomalmapping showed that TaDEP1was located on chromosomes of homoeologous group5. An InDelmarker was developed from its first intron and was mapped to chromosome5B. This marker (tentativelynamed dep1-5b) was used to fine map TaDEP1into a~10cM region between genetic markers GS34and S67on the5B long arm. We then genotyped262wheat accessions from the wheat core germplasmcollection (CWGC) using dep1-5b. Association analysis revealed that dep1-5b was significantlyassociated with higher one-thousand grain weight (TGW) in the two year dataset among CWGC. Whenwe divided the CWGC accessions into subpopulations of landraces and cultivars, we found that dep1-5bwas significantly associated with grain yield only in the landraces but not in the cultivars. Two kinds ofalleles are found in rice with the dep1alleles contributing to grain yield increase. We observed the genestructures in other grasses, and found that shorter/truncated alleles were present in nearly all grassspecies surveyed, including the progenitors of bread wheat (T. uratu, Ae. tauschii, and T. turgidum),Brachypodium and sorghum contrasting to the existence of both long and short alleles in rice.Interestingly, we found two alleles (long and short) respectively located in chromosome2and7ofmaize. The observation was consistent with association analysis result. Shorter allele in most of the grass species was reserved by domestication and longer allele lost; TaDEP1was significantly associatedwith TGW in the CWGC but not in the cultivars of artificial selection. It indicated that theshorter/truncated allele was possibly a domesticated gene.更多还原