【作者】 孟庆长；

【导师】 喻德跃；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2006， 博士

【摘要】 种子发育研究是植物生物学和育种研究工作中的一个重要组成部分。大豆种子发育是一个非常复杂的、由多基因协调控制的过程。在这一过程中，转录水平上的调控起着重要的作用。NAC和LFY同源蛋白是其中两类非常重要的植物特异转录因子。NAC蛋白编码基因参与包括植物生长发育、胁迫应答等在内的多种植物生理生化过程。而编码LFY同源转录因子的基因主要参与植物开花时间的调控、花发育等重要的生殖发育过程。本研究的主要内容是参与大豆种子发育过程中编码NAC和LFY同源蛋白基因的克隆和鉴定以及大豆主要种子性状的QTL分析，以期为大豆的种子发育机理研究和遗传改良打下基础。首先，从大豆中克隆了6个编码NAC同源蛋白的基因序列并描述其分子特征。序列分析表明6个GmNAC基因编码的蛋白序列均包括一个位于N-末端的同源性较高且十分保守的NAC结构域和一个位于C-末端的高度可变区域。其中NAC结构域由A-E5个亚结构域组成。GmNAC基因结构十分相似，均由3个外显子和2个内含子序列组成，前两个外显子编码保守的NAC结构域，两个内含子在基因中的存在位置具有明显的保守性。RT-PCR分析表明GmNAC4和GmNAC6为组织组成性表达，而其它4个基因的表达都具有特异的表达模式。在4轮花器官中GmNAC3，GmNAC4和GmNAC6呈组成性表达，其它3个基因的表达模式略有差异。GmNAC基因在不同发育时期大豆种子中的表达具有协同性：种子发育前期基因表达量较少，在开花后30-35天种子中的表达量均达到最大值，随后又有所降低，呈“钟形”表达模式。SA，NaCl和机械伤害处理后，GmNAC基因在叶片中表达模式不尽相同。这表明，GmNAC基因参与非生物胁迫应答过程，但是所起的作用可能有所不同。亚细胞定位研究表明GmNAC5蛋白可以定位到细胞核中。系统发育分析表明GmNACl-GmNAC6可以分为5个不同的亚组，GmNAC3和GmNAC4属于AtNAC3亚组，GmNAC1，GmNAC2，GmNAC5，GmNAC6分别属于NAP，ATAF，NAM和TERN亚组。GmNAC基因的克隆和鉴定为进一步阐明和探讨大豆NAC转录因子的功能奠定了基础。采用RACE的方法从大豆花芽cDNA中克隆了大豆LFY同源cDNA序列（GmLFY）。成功开发出GmLFY基因特异的CAPS标记，利用遗传作图群体NJ（SP）BN将此标记定位到后文构建的大豆分子连锁图谱的C2连锁群上，位于2个SSR标记Sat₂13和Satt322之间，距离分别8.0和14.1cM。序列分析表明，GmLFY与拟南芥和豌豆等作物中LFY同源基因的同源性较高。GmLFY基因组序列和cDNA序列比较分析发现GmLFY基因由三个外显子和两个内含子组成，两个内含子在基因中存在的位置也具有明显的保守性。RT-PCR分析表明GmLFY基因主要表达于生殖器官，如花序，荚皮和正在发育的种子等。GmLFY主要在4轮花器官的第1轮和第4轮，即萼片和心皮中表达。GmLFY在不同发育时期大豆种子中的表达分析表明GmLFY在开花后15天表达水平较高，随后略有降低，后来又迅速升高，到开花后35-40天达最大值，随后又有所降低。原核表达结果表明GmLFY可以在大肠杆菌中正确表达并翻译。亚细胞定位研究表明在洋葱表皮细胞中GmLFY可以正确定位到细胞核中。系统发育分析表明GmLFY与豆科作物的LFY同源序列同源性较高，而且LFY同源蛋白的系统发育关系同物种的进化关系十分相似。利用遗传作图群体NJ（SP）BN，构建了一张基于SSR的大豆分子遗传图谱，图谱覆盖大豆基因组长度为2，854.9cM，包括24个连锁群，共有268个标记位点，标记间平均距离为10.65cM，连锁群上标记平均个数为11.17，平均每个连锁群长119.0cM。这张遗传图谱的构建为控制大豆重要农艺性状的基因定位打下基础。利用这张遗传图谱，结合2年的表型数据，用复合区间作图法（CIM）定位到10个大豆种子相关性状（蛋白质含量，油分含量和种子大小）的QTL，其中共有8个QTL的表型方差解释率在10％以上。在两年试验中重复检测到1个控制蛋白质含量和1个控制油分含量的QTL。大豆种子性状QTL定位对于大豆种子性状的遗传改良具有重要的理论和应用价值。更多还原

【Abstract】 Seed development is one of the vital parts of plant biology and breeding project. Forsoybean it’s an especially complexed process regulated and controlled by many diverse genes.During this process the regulation by transcription factors at the transcriptional level playsimportant roles. NAC-like and LFY-like proteins both are identified as two types of plant-specific transcription factors. Genes encoding NAC TFs are involved in many physiologicaland biochemical processes including plant growth and development, responsiveness to stress;while those encoding LFY-like TFs are implicated in the regulation of reproductivedevelopment, such as flowering time control and floral development et al.In this study, we focus on the cloning and identification of genes encoding NAC andLFY-like TFs involved in seed development, and mapping QTLs controlling seed characters insoybean.Firstly, six NAC-like genes from soybean, designated as GmNAC1 to GmNAC6, werecloned and characterized. Sequence analysis showed that six GmNAC proteins uniformlycontained a conserved NAC domain at N-terminus with high homology and a diverged C-terminus. Also, NAC domains in GmNAC protein were found to contain five differentsubdomains A-E. They shared conservative structures of genomic organization: each containedtwo introns and three exons and the first two exons encoded NAC domain. Moreover, insertionsites of introns were much conserved among six proteins. RT-PCR analysis indicated that eachGmNAC gene exhibited a specific expression pattern in tissues examined, revealing thedifferent roles for GmNAC genes in soybean development. GmNAC2, 3, 4, and 6 were detectedto express in most tested tissues while GmNAC1 and GmNAC5 were limited to few tissues. Infloral organs, except for GmNAC3, GmNAC4, and GmNAC6, which were expressedconstitutively, other three GmNAC genes showed unique expression patterns. Furthermore,expression patterns of GmNAC genes were characterized during seed filling and coordinatedexpression was observed between GmNAC genes. Most of them were expressed at a low levelat the early stages of seed development and reached a maximum level between 30-35DAF thendecreased afterward. After treatment of SA, NaCI and wounding, GmNAC genes were regulated differently. Subcellular localization of GmNAC5 in onion epidermic cells suggested GmNAC5was targeted to the nucleus. Based on phylogenetic analysis, six GmNAC proteins were classedinto five subgroups. GmNAC1, GmNAC2, GmNAC5, and GmNAC6 belonged to the NAP,ATAF, NAM, and TERN subgroup, respectively; while GmNAC3 and GmNAC4 wereclassified into the AtNAC3 subgroup.These results provide potent basis for futureinvestigations of NAC-like genes’ roles in seed development and other physiological processesin soybean.Secondly, a LEAFY homologue in soybean, GmLFY, was cloned from cDNA preparedfrom young inflorescences by rapid amplification of cDNA ends （RACE） method. A GmLFY-specific CAPS marker was developed and then was mapped on molecular linkage group C2 in amapping population NJ（SP）BN. The marker was located between two SSR markers Sat₂13and Satt322 with the distance of 8.0 and 14.1cM, respectively. Comparative analysis of GmLFYgenomic and cDNA sequences showed that GmLFY gene had three exons and two introns withthe similar spliced sites as other LFY-like genes. RT-PCR analysis indicated that GmLFY waspredominantly expressed in reproductive organs such as inflorescences, pods without seeds, anddeveloping seeds. Moreover, GmLFY was mainly expressed in whorl 1 and whorl 4, the sepalsand pistils, of the floral organs. Expression pattern of GmLFY in developing seeds was alsoinvestigated. GmLFY was expressed at a middle level in seeds at 15 days after flowering （DAF）and decreased in 20 DAF seeds. Thereafter, it increased, reaching its maximum expression levelbetween 35 and 40 DAF and decreased gradually after that time. Result of expression in E.colishowed that GmLFY could be expressd and translated in prokaryote; Subcellular localization ofGmLFY in onion epidermic cells suggested GmLFY was targeted to the nucleus. Phylogeneticanalysis showed that GmLFY belonged to the family of legume together with LjLFY of Lotusjaponicus, MtUNI of Medicago truncatula and UNI of Pisum sativum.In soybean seed traits such as protein content, oil content, and seed size are the majortargets in breeding programs. Using a mapping population NJ（SP）BN derived from Bogao xNG94-156, an SSR-based molecular genetic map with 268 loci was developed. This linkagemap contained 24 molecular linkage groups and spaned 2,854.9cM of the soybean genome withan average interval distance of 10.65cM, the average markers per group of 11.17cM and theaverage length of the LGs of 119.0cM. Using CIM method, QTLs for each seed trait wereanalyzed. A total of 10 QTLs controlling protein content, oil content, and seed size in soybeanwere identified in the years of 2004 and 2005. Analysis and comparison of detected QTLsshowed that one QTL for protein content （qpcD1a₁） and one for oil content （qocC2₁） were stable across two years. Our results will be contributing for genetic improvement for seed traitswith marker-assisted selection in soybean.更多还原