【作者】 马振；

【导师】 吴昌银；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 利用突变体来克隆基因是功能基因组研究的重要策略,目前很多研究机构都在用各种方法创建突变体库,并且从突变体中分离到了很多重要的基因。本研究利用水稻中的一个反转录转座子Tos17创建水稻突变体库。Tos17是一个“复制-粘贴”型的转座子,在组培的情况下被激活产生新的拷贝插入到基因组中,而分化成植株后Tos17的转座活性消失,新的拷贝能稳定遗传。为了证明利用Tos17创建突变体来分离克隆基因的方法的有效性,本研究对T1代家系进行了正向遗传学的筛选,目的是寻找与Tos17插入位点共分离的家系,为基因分离和克隆奠定基础。此外通过接头PCR的方法分离Tos17侧翼序列,不仅能更加深入的了解Tos17在水稻基因组中的分布,而且根据侧翼序列提供的基因信息,可以广泛的开展反向遗传学的研究。主要研究结果如下：1.共计产生独立的Tos17突变体15,543株。通过对Tos17拷贝数的Southern检测,发现中花11中Tos17原始拷贝数为4,经过三个月的组培时间,平均每株含有新Tos17插入拷贝1.4个,整个突变体库中约含有插入位点21760个。2.利用接头PCR的方法和TAIL-PCR的方法分离到能精确定位到水稻染色体上的Tos17侧翼序列601条,其中非重复的侧翼序列为524条。3.从侧翼序列的分析的结果来看,Tos17偏向于插入较大的染色体,插入数目和染色体的长度存在显著的正相关(r=0.717,P<0.01)。而在基因组区间内,我们发现Tos17显著偏爱于插入基因区(SR=10.85),而显著不偏爱于插入基因间隔区(-8.88)和转座因子相关区(-3.68)。在基因区域内,Tos17偏爱插入基因区(SR=4.55)而不偏向插入ATG上游1kb区(SR=-4.62)和翻译终止密码子下游500bp(SR=-3.25)这两个区。在基因编码区内,Tos17显著偏爱插入基因的外显子区域(SR=2.48)。4.2008年夏季对Tos17插入突变体T1代1,881个家系进行了筛选,结果显示整个田间有突变表型的频率为5.69%,主要突变表型有矮化、不育、白化、黄化、晚抽穗等。对10个家系中的12个插入位点进行了PCR验证,发现有10个Tos17插入位点是可以确认是正确的。更多还原

【Abstract】 Mutant for cloning novel gene is an important strategy for functional gene research. A lot of notable genes had been isolated by mutants created by various methods. In this study, we generated a Tos17 insertional mutant library using Zhonghua 11 (japonica cv.) as a recipint. Tosl 7 is a’copy-paste’mode retrotransposon which is activated by tissue culture, becoming inactive in regenerated plants. T1 Tos17 mutants had been screened by forward genetic strategy to identify the mutant which phenotype co-segregate with Tos17 insertion sites and provide a base for gene isolation and cloning. The flanking sequences of Tos17 were isolated by Adapter-PCR and TAIL-PCR. Through the analysis the flanking sequence of Tos17, we can characterize the Tos17 distribution pattern in rice genome and carry out reverse genetic study of the corresponding genes.The main results are described as follows:1. A total of 15,543 independent Tos17 mutants had been created in Zhonghua 11. Our Southern blot result showes that there are four native copies in Zhonghua 11. After 3 months cultivation, each To line had 1.4 new copy numbers in the generation plants on average. The total insertion site was evaluated about 21,760 in the whole genome.2. Employing Adapter-PCR and TAIL-PCR methods,601 Tos17 flanging sequences had been isolated which can be well mapped onto the rice genome. And 524 Tos17 flanking sequences are unique.3. The analysis of 524 independent flanking sequences shows that Tos17 insertions were biased towards large chromosome. The insertion sites are highly correlated with the chromosome size (r=0.717, P<0.01). Within chromosomes, Tos17 strongly favored the genic sequences (SR=10.85) and strongly disfavored the intergenic (SR=-8.88) and TE-related (SR=-3.68) sequences. In rice genes, strong bias towards coding sequences (SR=4.55), but not towards the 5’upstream (SR=-4.62) and 3’downstream regions(SR=-3.25). In the coding region, Tosl7 strongly favored the exon region (SR=2.48).4.1,881 T1 Tos17 insertional lines had been screened morphologically under normal growth condition in 2008, the result showed that the mutant frequency was 5.69%. The main mutant phenotypes were:dwarf, sterility, albino leaf, yellow leaf, delayed heading, et al.12 insertion sites from 10 mutant lines had been test by PCR and 10 insertion sites were correct.更多还原