玉米芽再生能力调控基因的全基因组关联分析

发布时间：2018-07-28 09:54

【摘要】：植物芽再生能力是遗传转化的基础。幼胚是玉米(Zea mays L.)常用的遗传转化外植体,不同基因型玉米的芽再生能力差别很大。目前关于调控玉米芽再生能力的基因鲜有报道,因此挖掘调控玉米芽再生能力的重要基因不仅有利于提高玉米遗传转化频率而且还有助于加深人们对芽再生能力遗传调控分子基础的理解。本研究选取367份来自全球不同地区的玉米自交系,先后在2013年和2014年夏天种植,取授粉后11d的幼胚进行组织培养实验。在培养过程中,对诱导培养0d、诱导培养15d、诱导培养30d、分化培养0d、分化培养30d 5个时期的愈伤组织状态及愈伤组织芽分化情况进行观察,并对初级愈伤组织诱导率、成熟愈伤组织诱导率、愈伤组织状态、愈伤组织增殖能力、愈伤组织芽再生率及单个愈伤组织芽再生数6个性状进行量化分析。继而通过全基因组关联分析方法找到组织培养能力相关的基因。主要研究结果如下:(1)通过对授粉后11d的幼胚进行组织培养,得到了367份玉米自交系幼胚的组织培养表型数据。结果表明,不同基因型的玉米自交系之间表型差异很大,如CIMBL3、CML426、CML323等玉米自交系愈伤组织状态较好,芽再生频率分别能达到75%、65.5%、64.5%,而有些自交系形成的愈伤组织不能分化形成芽。(2)结合RNA测序获得的高质量的SNP数据,通过全基因组关联分析,共得到了132个标记-性状关联,并通过功能注释得到了58个候选基因,这些候选基因分别与6个性状相关。(3)对玉米芽再生过程6个性状间的相关性分析发现,初级愈伤组织诱导率与成熟愈伤组织诱导率存在相关性,而愈伤组织诱导率与愈伤组织芽再生率之间并不存在相关性,愈伤组织增殖能力与愈伤组织芽再生率之间也不存在相关性,愈伤组织诱导、愈伤组织的生长速度和愈伤组织芽分化过程是相互独立的,高愈伤组织诱导率并不一定伴随高的愈伤组织芽分化率,因此,愈伤组织的诱导和愈伤组织芽分化过程是受不同遗传因子调控的。(4)选择4个玉米愈伤组织芽再生相关的基因进行了初步功能分析。GRMZM2G074850和GRMZM2G045404基因的过表达能够提高芽的再生频率,而过表达GRMZM2G168393基因后则降低了愈伤组织芽再生率,过表达GRMZM2G138689基因后对愈伤组织芽再生率几乎没有影响。说明GRMZM2G168393、GRMZM2G074850和GRMZM2G045404可能在调控玉米愈伤组织芽再生率中发挥重要作用。
[Abstract]:The ability of plant bud regeneration is the basis of genetic transformation. Immature embryos are corn (Zea mays L.) The shoot regeneration ability of different maize genotypes is very different from that of common genetic transformation explants. At present, there are few reports on genes regulating the regeneration ability of maize buds. Therefore, digging the important genes to regulate the regeneration ability of maize bud is not only helpful to improve the transformation frequency of maize, but also to deepen the understanding of the molecular basis of genetic regulation of shoot regeneration ability. In this study 367 maize inbred lines from different regions of the world were planted in the summer of 2013 and 2014 respectively. The immature embryos were pollinated 11 days after pollination for tissue culture. In the course of culture, the callus status and callus bud differentiation were observed at 0 d, 15 d, 30 d, 0 d, 30 d and 30 d, respectively, and the rate of callus induction was also studied. The induction rate of mature callus, the status of callus, the ability of callus proliferation, the rate of callus bud regeneration and the number of callus regeneration in single callus were analyzed quantitatively. Then the gene related to tissue culture ability was found by whole genome association analysis. The main results were as follows: (1) the phenotypic data of 367 maize inbred lines were obtained by tissue culture of immature embryos at 11 days after pollination. The results showed that the phenotypes of maize inbred lines with different genotypes varied greatly, such as CIMBL3, CML426CML323 and other maize inbred lines, and the callus status of maize inbred lines such as CIMBL3 and CML426CML323 was better. The frequency of bud regeneration could reach 75% 65.5% and 64.5%, respectively, while some inbred lines could not differentiate and form buds. (2) combined with the high quality SNP data obtained by RNA sequencing, 132 marker-trait associations were obtained by whole genome association analysis. Through functional annotation, 58 candidate genes were obtained, which were related to 6 characters respectively. (3) the correlation analysis of 6 characters in the regeneration process of maize bud was found. The primary callus induction rate was correlated with mature callus induction rate, but there was no correlation between callus induction rate and callus bud regeneration rate. There was no correlation between callus proliferation ability and callus bud regeneration rate. Callus induction, callus growth rate and callus bud differentiation were independent of each other. High callus induction rate is not always accompanied by high callus bud differentiation rate. Callus induction and callus bud differentiation were regulated by different genetic factors. (4) four genes related to callus regeneration in maize were selected for preliminary functional analysis. The overexpression of GRMZM2G074850 and GRMZM2G045404 genes could increase the regeneration frequency of maize callus. Overexpression of GRMZM2G168393 gene decreased callus bud regeneration rate, and overexpression of GRMZM2G138689 gene had little effect on callus bud regeneration rate. It was suggested that GRMZM2G168393GRMZM2G074850 and GRMZM2G045404 might play an important role in regulating the shoot regeneration rate of maize callus.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513

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