【作者】 王晖；

【导师】 王玉平；

【作者基本信息】 四川农业大学 ， 遗传学， 2012， 硕士

【摘要】 雄性不育是一种植物中普遍存在的现象,水稻雄性不育因杂交水稻的成功应用而备受关注。育种家们通过各种方法获得不育材料,研究其不育机制,欲从中得到控制不育性状的基因,通过对不育基因的克隆,改造和利用以获得高配合力的不育系,应用于杂交稻育种中,具有重要的价值。水稻雄性不育的遗传机制复杂,涉及花药发育,物质代谢合成等方面。本研究所用到的四川隐性核不育材料H2S,之前通过图位克隆的方法将其控制育性的基因定位在第6染色体上标记Inde140520和RM20366之间一段45kb的区间内。分析该区间内的7个候选基因,将其初步锁定为编号LOC-Os06g40550的候选基因,并预测其功能是参与脂肪酸的运输,基因命名为ABCG15。本实验进行了水稻ABCG15基因的互补表达载体的构建研究,以来源于四川隐性核不育H2S的突变体材料为受体,利用农杆菌介导的遗传转化法获得有价值的转基因植株。获得的互补材料为水稻ABCG15基因的深入研究奠定了基础。实验研究的主要结果如下：1对四川隐性核不育H2S的突变体材料进行观察及鉴定,发现其花药瘦小,呈无色水浸状,其他花器官正常,碘染表现为无花粉不育。2利用扫描电镜(SEM)和半薄切片对四川隐性核不育H2S的突变体及其野生型的花药细胞进行细胞学观察。电镜结果发现,从小孢子时期开始到花药成熟期,野生型材料的花药表皮细胞表面逐渐出现粗糙的网状结构,而突变体材料整个过程却基本上是光滑的。切片结果发现,四分体时期之后小孢子发育异常,绒毡层降解延迟,导致最终表现为无花粉型雄性不育。3根据Rice Tigr上预测的ABCG15基因的CDS序列设计引物,进行PCR反应,最终扩增得到了约2100bp的全长cDNA片段。4将克隆得到的ABCG15基因全长cDNA片段连接到表达载体pOsAct2-1-nos上,得到互补表达载体pOsAct-ABCG15。该载体受Actin2启动子调控。5将构建好的互补表达载体pOsAct-ABCG15利用农杆菌介导的遗传转化法导入到四川隐性核不育H2S的幼穗诱导的胚性愈伤组织中,经过组织培养,抗性筛选,分化,生根,获得再生植株。6利用PCR扩增ABCG15基因对转基因再生植株进行鉴定,证实了ABCG15基因全长cDNA片段正确的插入到受体水稻基因组中,并成功地获得了5株阳性转基因水稻。7用碘化钾染料对ABCG15的转基因阳性植株的育性进行鉴定,碘染结果表明ABCG15基因转基因植株的育性恢复正常。水稻ABCG15基因互补转基因植株的获得为后面进一步研究ABCG15基因的功能提供了保证。更多还原

【Abstract】 Rice male sterility is a widespread phenomenon in plants, receives much concerned because of the successful application of hybrid rice.Breeders get sterile materials through various methods, study the mechanism of infertility, to get control of male sterile trait genes, by cloning of the gene, transforming and combining ability of use to get wide CMS, used for cross breeding, is of great value.Rice male sterility genetic mechanism is complex, anther development, the synthesis of substance metabolism are involved. This study used to Sichuan H2S recessive sterile material of fertility control it by cloning the gene on the chromosome of the sixth, a45kb band,between markers Inde140520and RM20366. Analysis of candidate genes for7of the band’s final preliminary locking it for the number of LOC-Os06g40550candidate genes, and their function is involved in the transport of fatty acids, gene named ABCG15.The experiment succeeded in building the complementarity of rice ABCG15gene expression vector pOsAct2-ABCG15, recessive nuclear male sterile mutant of H2S from Sichuan material for the receptor, using Agrobacterium tumefaciens-mediated genetic transformation of valuable transgenic plants. Get complementary materials laid the Foundation for in-depth study of rice ABCG15gene. Experimental study on the main results are as follows:1Sichuan recessive genic male-sterile mutant material for observation and identification of H2S, its anther small, colorless flood, other floral organs are normal, after iodine dye performance of pollen sterility.2using scanning electron microscopy (SEM) and semi-thin sectioning technique to observe Sichuan recessive nuclear male sterile mutant of H2S anther separation of materials and their wild-type material for cytological observation of epidermal cells. It turns out, from small spores begin to anther mature wild type epidermal cells in anthers of material gradually appeared on the surface roughness of the reticular formation, and mutant materials is basically the whole process was smooth. Biopsy results, abnormalities of the small spores after tetrad, tapetum degradation delay, ultimately resulting in no pollen sterility.3according to the prediction of ABCG15on rice TIGR gene sequences of CDS designed primers, PCR reaction, respectively, final amplification full length cDNA fragment was about2100bp.4clones are ABCG15on the gene expression of full length cDNA fragment attached to carrier pOsAct2-1-nos, expression vector pOsAct-ABCG15are complementary.5Transfer the complementary expression vector of pOsAct-ABCG15imported by using Agrobacterium-mediated genetic transformation of recessive genic male-sterile mutant of H2S to Sichuan material for spike induction of embryogenic callus, through tissue culture, resistance selection, differentiation and rooting and plant regeneration.6Using PCR amplification of the ABCG15gene to identify regeneration of transgenic plants is confirmed by full-length cDNA ABCG15gene fragment properly inserted into a receptor in the rice genome, and successfully got5positive transgenic rice strains.7Potassium iodide dyes for the fertility of ABCG15gene transgenic plants have positive identification, iodine dye showed ABCG15gene transgenic plants’s fertility is restored. Behind the access to complementary rice ABCG15gene transgenic plants ABCG15gene functions provide the assurance of further research.更多还原