【作者】 武晶；

【导师】 任正隆； 贾继增；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 小麦Rht-B1与Rht-D1基因以及水稻sd1(OSGA20ox2)基因的开发与利用对世界小麦与水稻的产量产生了巨大的推动作用,引发了一场举世闻名的“绿色革命”。然而人们对于小麦Rht-B1与Rht-D1基因也仅限于基因本身的研究,所以对这一重要基因位点的同源区段的序列组成结构进行分析研究,以及该基因在小麦多倍化进程中的遗传特点与进化规律的研究必将有助于我们更好的认识这一重要基因。此外人们对于小麦中对应的水稻“绿色革命”基因的组成与功能还不够了解,因此,分离小麦中的水稻“绿色革命基因”的同源基因GA20ox2,深入了解该基因在小麦生长发育中的作用机理,对于小麦的改良可能具有重要的现实意义。一方面本研究利用二倍体、四倍体、六倍体BAC文库资源对小麦Rht-D1(Rht-B1)基因位点进行分子进化研究,以及小麦A、B、D基因组之间的共线性研究,并与水稻、短柄草Rht-D1(Rht-B1)基因区段序列进行比较研究。另一方面本研究以水稻sd1(OSGA20ox2)基因为基础,在小麦中成功克隆TaGA20ox2并对其进行基因结构分析与遗传定位:通过这两方面的研究,本论文获得如下主要结果:1.首次对小麦“绿色革命基因”Rht-B1、Rht-D1在小麦A、B、D基因组中的同源区段序列组成结构进行了分析,发现该基因所在同源区段的基因密度为1个基因/33-50Kb,高于小麦基因组中基因的平均密度(1个基因/80-100Kb);该区段重复序列占序列总长度的57-85%,其中以LTR亚类的反转录转座子为主,DNA转座子以CACTA与MITE序列为主;序列分析发现了一些新的重复序列元件,包括一个新的LTR反转录转座子,4个新的MITE序列。2.Rht-A1、Rht-B1、Rht-D1所在A、B、D三个直向同源基因组区段序列比较发现:(1)基因的共线性远高于重复序列与其它基因间序列的共线性;(2)发现了一个共线性很好的WIS亚类的反转录转座子及两个3Kb的未知序列,这些序列可能为小麦属特有基因组序列;(3)重复序列及基因间序列大部分为基因组特异序列;(4)分别发现了1个B基因组、2个D基因组特异的候选基因或非共线性候选基因。分析认为这些共线性与非共线性的基因、重复序列与其它类型的基因间序列是造成小麦三个基因组的“部分同源”关系的基础。3.不同基因组之间的同源区段序列比较分析揭示了同一物种在进化过程中不同的基因组进化速度并不一致,该区段B基因组要先于A、D基因组分化;在A、D基因组的序列上发现一个共有反转录转座子Carmilla-1,但是8在B基因组该共线性区段没有发现这个转座子:比较分析Rht-A1、Rht-B1、Rht-D1基因序列在二倍体、四倍体、六倍体中的差异时发现B基因组的序列差异要明显大于A、D基因组;A、D基因组在同源区段的序列相似性要明显高于A、B以及B、D基因组的相似性。4.采用同源克隆结合BAC文库筛选的方法,依据水稻“绿色革命”基因GA20ox2基因序列获得了小麦中GA20ox2基因:TaGA20ox2-A1、TaGA20ox2-B1、TaGA20ox2-D1的基因组序列和cDNA序列;利用中国春缺体-四体材料将小麦中的TaGA20ox2基因分别定位于3A、3B、3D染色体上;根据TaGA20ox2-D1基因序列在国际作图亲本W7984与Opata85之间的差异设计InDel标记GA20ox2-3i,将TaGA20ox2-D1基因定位于xfba330与xgwm664标记之间,遗传距离分别为4.1cM和5.3cM。分子标记结果结合QTL定位信息可以初步推断TaGA20ox2-D1在小麦中的功能与水稻相似,都是控制株高的重要基因。更多还原

【Abstract】 The wheat Rht-B1,Rht-D1 and rice semidwarf-1(sd1) genes are well known as the "green revolution genes",and have contributed to the significant increase in wheat and rice production seen in the 1960s and 1970s.So investigation on the sequence structure and molecular evolution in the Rht-D1 locus region is very important to study on wheat Rht-B1 and Rht-D1 genes.On the other hand,we have a little knowledge about the composition and function of homologous sd1 gene in wheat,therefore, Cloning and identifying inheritance mode of this gene is significant to wheat genetic improvement.In this study,we investigated the molecular evolution of wheat Rht-D1 locus region by using sequenced Rht-D1 locus in six wheat genome from diploid,tetraploid and hexaploid species and compared the orthologous regions with Brachypodium and rice.Meanwhile,we obtained full length GA20ox genes from hexaploid wheat(Triticum aestivum) and located GA20ox genes on wheat genomes to give a better understanding of the character and evolutionary relationships of GA20ox genes in wheat.The results obtained are as follows:1.It is the first analyzing sequence structure in the homologus regions of Rht-D1 gene.The results showed gene density is one gene/33-50Kb in this region,which is high to average gene density in the wheat genome(one gene/80-100Kb).Repetitive sequences accounted for about 57-85%of the homologus region sequences.LTR retrotransposons are major types of the repetitive sequences, whereas CACTA and MITE are major of the DNA transposons.Additionally,a new LTR retrotransposons and four MITE sequences were reported in this paper.2.The sequences of a physical contig covering the Rht-D1 locus from the A genome of durum wheat were compared with the orthologous regions from the B and D genome of hexaploid wheat.The results suggested:(1) the colinearity of gene were much better than repetitive or intergenic other sequences.(2) it was found that a WIS element and two three-Kb unknown sequences were retained good colinearity, these sequences maybe unique for wheat genome.(3) most of the repetitive or intergenic other sequences are genome-specific sequence.(4) a gene of B genome specific and two genes of D genome specific were to be found.From this results,it is deduced that collinearity or no collinearity genes, repetitive sequences and intergenic other sequences lead to homeologous between wheat subgenomes. 3.By comparison of homology sequences among different genome,it was found that wheat subgenomes have the different evolution speed.The fact that Carmilla-1 element shared by the A and D genomes,but not by the B genome,indicated that the A and D genomes share a common ancestor that had previously diverged from the B genome ancestor.Several other lines of evidence discovered in this study,including the difference of Rht-B1 sequence between tetraploid and hexaploid more higher than Rht-A1 sequence between tetraploid and hexaploid,and higher sequence homology in the Rht-1 regions between the A and D genomes than the B genome,also support this conclusion.4.A novel gene encoding GA20-oxidase was cloned from hexaploid wheat(Triticum aestivum) using the homologous cloning method.Sequences analysis showed that wheat GA20-oxidase have three sequences:TaGA20ox2-A1、TaGA20ox2-B1 and TaGA20ox2-D1.Sequence alignment proved wheat GA20-oxidase genes have the highest homology with rice GA20-oxidase(80%cDNA sequence identity).A series of ’Chinese Spring’ nulli-terasomic stocks were employed to ascertain the chromosomal location of wheat GA20-oxidase gene.The results showed that TaGA20ox2-Al、TaGA20ox2-B1 and TaGA20ox2-D1 were on wheat chromosome 3A、3B and 3D.Using a population of recombinant inbred lines from the cross W7984×Opata85,TaGA20ox2-D1 was mapped on chromosome 3D,and flanked by SSR markers Xfba330 and Xgwm664 at 4.1 and 5.3 cM,respectively.In this region a plant height loci was reported.Based on these results,we think that TaGA20ox2-D1 gene maybe have an impact on wheat height development.更多还原