【作者】 王莉；

【导师】 杨守萍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2012， 硕士

【摘要】 基因芯片技术是研究不同品种及处理间基因差异表达的有效手段,它既能直观反映出基因组中各基因间的相互关系,也能表现出不同条件下基因的转录调控水平,从而通过基因组转录效率来获得共同表达的基因信息,有助于筛选目的基因。本研究运用基因芯片技术对大豆质核互作雄性不育系NJCMS2A与其保持系NJCMS2B间差异表达基因进行研究,寻找大豆雄性不育相关基因,为利用基因工程方法创造大豆雄性不育新材料提供基础。获得主要结果如下：1.通过基因芯片数据比较大豆质核互作雄性不育系NJCMS2A及其保持系NJCMS2B的基因表达情况,筛选出NJCMS2A与NJCMS2B之间差异2倍以上的基因2176条,其中上调表达基因1631条、下调表达基因545条。利用GO(Gene Ontology)分类,对P<0.01、差异2.5倍以上的174条差异表达基因进行进一步的功能分类,结果表明这些基因主要涉及催化活性、物质运输、代谢过程、生物调节、细胞繁殖、细胞器组成等。根据基因的ID,确定差异表达基因的EST序列,再进行大量的比对分析、查找资料,从这174个基因中发现一些与大豆雄性不育可能有重要关系的基因包括ATP酶相关基因、多聚半乳糖醛酸酶相关基因等。2.分别从NJCMS2A和NJCMS2B中克隆了Gmatp9基因,序列比较分析表明在NJCMS2A与NJCMS2B中Gmatp9基因序列存在2个碱基的差异,荧光定量PCR分析结果显示Gmatp9基因在NJCMS2A中的表达量显著高于NJCMS2B,与基因芯片的分析结果一致,推测Gmatp9基因可能与NJCMS2A雄性不育有关。3.开展了Gmatp9基因的拟南芥遗传转化研究,通过分子鉴定获得T3代转基因拟南芥植株,花粉活力检测结果显示,转Gmatp9基因的拟南芥植株中花粉活力有所下降,至于Gmatp9基因与大豆雄性不育的关系如何,还有待于进一步研究。更多还原

【Abstract】 Gene chip technology is an effective approach to study the differential gene expression of different varieties and treatments, which not only can reflect the interaction of each gene, but also can reflect the transcription regulation level of genes under different conditions. Thus, genetic information of the common expressed genes obtained through genome transcription efficiency can facilitate the screening of target genes. In this study, the differential expression genes between soybean cytoplasmic-nuclear male-sterile line NJCMS2A and its maintainer NJCMS2B were investigated using gene chip technology to find out the soybean male-sterile genes and provide basis for creating new soybean male-sterile materials by using genetic engineering. The main results obtained are as follows:1. cDNA microarray was carried out on soybean cytoplasmic-nuclear male-sterile line NJCMS1A and its maintainer line NJCMS1B to obtain their gene expression situation.2176genes with more than2-fold differential expression between NJCMS2A and NJCMS2B, of which1631genes were up-regulated differential expression genes and545genes were down-regulated differential expression genes. Function analysis was carried out using GO (Gene Ontology) classification technique on the174genes with more than2.5-fold differential expression (P<0.01). It was found that these differential expression genes were primarily related to catalytic activity, material transport, metabolism, biological regulation, cell proliferation, and cell construction. EST sequences of the differential expression genes were identified according to the gene ID, which were analyzed comparatively. Some of these174genes were considered to be related to soybean male sterility, including genes related to ATP, polygalacturonase, and so on.2. The genes Gmatp9were cloned from the NJCMS2A and NJCMS2B, respectively. Sequence comparison analysis showed that there were2different bases between the sequences of genes Gmatp9from NJCMS2A and NJCMS2B. The fluorescence quantitative PCR analysis showed that the gene expression of Gmatp9amount in NJCMS2A was significantly higher than that of NJCMS2B, which was consistent with the results of gene chip analysis. Thus, it was speculated that gene Gmatp9may be associated with the male sterility of NJCMS2A.3. The study on Arabidopsis thaliana genetic transformation of gene Gmatp9was carried out. Transgenic Arabidopsis plants of T3generation were obtained through molecular identification. Pollen viability test results showed that pollen viability of Arabidopsis plants with the transgenic Gmatp9decreased. Further research was needed to found out the relationship of gene Gmatp9and male sterility of soybean.更多还原