【作者】 刘文婷；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 种质资源的扩增、改良与创新是现代育种体系的首要环节，是公益性、战略性的长期研究课题。Mu因子是目前为止玉米中发现的转座活性最高、诱变能力最强的转座因子，虽然对其转座的调控机理还知之不多，但利用Mu因子的插入诱变，构建大型突变体库已成为创造玉米种质、获得突变基因，分离、克隆玉米重要功能基因进而开展功能基因组学研究的重要方法之一，同时Mu因子也将会成为其他异源物种基因诱变和基因分离的工具。 本研究以Q105、WW51、115F、V26-2、919J为Mutator转座子供体材料，与本地优良玉米自交系受体材料Hz85、W328杂交构建我国第一个基于Mu转座子的的大型插入突变体库，并初步评价其遗传基础，为我国玉米功能基因组研究搭建材料和技术平台，获得的主要结果如下： （1） 获得了4218个单株的插入诱变F₁群体（M1）。田间鉴定M1单株的生物学特征及农艺学性状的表型突变，室内考察突变单株自交果穗M2籽粒性状的突变类型，并以斑点籽粒出现的频率评价Mu因子的转座频率。结果表明：在所构建的Mu介导的玉米插入突变体库中，以Hz85（黄色籽粒、普通玉米）为母本的M1群体的平均田间表型突变频率为0.154，以W328（BzBz糊粉层紫红色、糯玉米）为母本的M1群体的平均田间表型突变频率为0.071；以W328×Mu的M2果穗斑点籽粒出现的频率评价Mu转座频率为0.122。 （2） 利用优化的MuTAIL-PCR技术扩增、分析了99条转座子插入位点的侧翼序列，归并整理后获得59条（每条约400核菅酸）非重复靶位点序列。 （3） 对59条序列非重复的Mu因子插入产生的9-bp靶位点正向重复序列进行同源性比较分析，表明Mu的插入有序列热点。经生物信息学分析后注释了27条与玉米、水稻等植物核苷酸数据库中匹配值较高的功能序列。利用比较基因组学方法将36条单一基因序列定位在玉米遗传图谱上，有多条Mu插入靶位点序列定位在单个标记位点上。分析发现8条Mu插入靶位点序列的预测功能与其相应的定位标记的功能具有一致性。 Mu插入突变体库的构建与遗传评价为进一步利用Mu转座子发掘玉米基因，深入开展玉米功能基因组学研究搭建重要的技术与材料平台。更多还原

【Abstract】 The innovation, improvement and expansion of maize germplasm are a chief step in modern breeding systems and are a long-term stratagem for the commonweal. Mu elements are the transposons with the highest transposition activity and mutation capability discovered so far. Although the mechanism of mutator transposon was not known very clearly, it is useful in constructing large mutant population of maize for creating maize germplasm, obtaining mutant genes, isolating and cloning important maize functional genes. Mu element will be used to mutate and isolate genes in other heterogenous species.In this research, A total of 4218 Ml plants were generated from seeds of crosses between the active mutator transposon donor parents (Q105, WW51, 115F, V26-2 and 919J) and the recipient parents(Hz85 and W328 with B(?) gene). The phenotypes of M1 plants were observed in the field and self-pollinated to establish the mutator insertion-mutagenized M2 seeds pool. The reversion frequency of mutator in the genome was calculated based on the spotted aleurone phenotype of the M2 seeds. The results showed that:(1) The mutation frequency of Ml phenotypes in the filed was 0.154 in the population of Hz85×Mu; the mutation frequency of Ml phenotypes in the filed was 0.071 in the population of W328×Mu; the reversion frequency of the Mu elements was 0.122 in the M2 population of W328×Mu.(2) 99 flanking sequences of mutator transpostion were amplified by modified MuTAIL-PCR, among which 59 sequences about 400 base pair were non-redundant and bioinformatically analyzed.(3) Results showed that 27 sequences could be annotated comparing with the non-redundant nucleotide database of Maize, Rice and Arabidopsis, and that 36 sequences could be located on the genetic map of maize. Hotspot sequences of mutator transposition were found after comparing the homologies among the 9-bp target site duplication of the mutator insertion. Several flanking sequences of mutator insertion were mapped at a single marker locus on the genetic map of maize. The putative functions of 8 flanking sequences of mutator transposition were found to be consistent with the functions of their corresponding marker locus.The mutator insertional mutants pool in maize constructed in the current research will provide a valuable platform for gene discovery and functional genomics study in maize.更多还原