甘蓝型油菜pol CMS恢复基因调控因子的筛选和温敏基因的GWAS分析
发布时间：2018-12-12 18:39
【摘要】：波里马细胞质雄性不育(pol CMS)被称为“第一个有实用价值的油菜雄性不育类型”,目前我国利用三系配套育成的杂交品种60%来自于pol CMS。温敏型的pol TCMS在低温下部分恢复育性,高温下稳定不育还可以实现两系制种。为了解析恢复基因的调控机制和pol TCMS温度敏感的分子机理,使pol CMS系统更好的应用于油菜杂种优势,本研究用酵母单杂交的方法筛选恢复基因Rfp的调控因子,用全基因组关联分析的方法对温敏基因初定位,主要研究成果如下:1.对Rfp基因上游600 bp的启动子序列进行顺式作用元件分析,结果表明该段启动子序列含有CAAT box和TATA box、光调控元件、激素响应元件等顺式作用元件。构建5个Rfp启动子5?端截短载体,转化拟南芥发现ATG上游151 bp就能驱动GUS基因在花蕾中的表达,而ATG上游118 bp不能驱动GUS基因的表达,根据实验结果我们确定Rfp基因的启动子核心顺式作用元件在ATG上游151 bp内。我们用甘蓝型油菜恢复系的幼蕾(0.5-1mm)构建了cDNA文库,用ATG上游394 bp构建酵母诱饵载体进行筛选,通过点对点验证获得一个与诱饵片段结合的蛋白,经过生物信息学分析发现该基因可能参与生物学胁迫响应和细胞间信号传导的功能。2.从530份甘蓝型油菜自然群体中挑选出86份保持系与6330A做杂交构建F1群体,将杂交群体秋播于武汉,通过观察群体的表型发现:在3月初开花时温度较低(低于16℃),不同材料之间呈现不同的育性等级;4月初温度升高,群体全部呈现不育表型,说明群体中可能存在一种和温度响应相关的调控因子。利用89份甘蓝型油菜保持系的60K SNP芯片信息,用Q模型进行全基因组关联分析,共检测到在A9、A5、A2、A8染色体上有多个标记位点可能与温敏基因相关。
[Abstract]:Polima cytoplasmic male sterility (pol CMS) is called "the first type of male sterility of rape with practical value". At present, 60% of the hybrids bred with three lines in China come from pol CMS.. The thermo-sensitive pol TCMS partially restored fertility at low temperature, and stable sterility at high temperature could also realize two-line seed production. In order to elucidate the regulation mechanism of restorer gene and the molecular mechanism of pol TCMS temperature sensitivity and make pol CMS system more suitable for rapeseed heterosis, the regulation factors of restoring gene Rfp were screened by yeast single hybrid method. The main results of this study are as follows: 1. The promoter sequence of 600 bp upstream of Rfp gene was analyzed by cis-acting elements. The results showed that the promoter sequence contained CAAT box and TATA box, photoregulatory elements, hormone response elements and other cis-acting elements. Construct 5 Rfp promoters? The truncated vector was transformed into Arabidopsis thaliana and it was found that ATG upstream 151 bp could drive the expression of GUS gene in flower buds, while ATG upstream 118 bp could not drive GUS gene expression. According to the experimental results, we determined that the promoter core of Rfp gene is in the upstream 151 bp of ATG. The cDNA library was constructed from 0.5-1mm of Brassica napus restorer line, and yeast bait vector was constructed with 394 bp upstream of ATG. A protein binding to bait fragment was obtained by point-to-point verification. Bioinformatics analysis revealed that the gene may be involved in biological stress response and intercellular signal transduction. 2. 2. 86 maintainer lines and 6330A were selected from 530 natural populations of Brassica napus to construct F1 population. The hybrid population was seeded in Wuhan in autumn. The phenotypic observation showed that the temperature was lower (lower than 16 鈩，
本文编号：2375102
[Abstract]:Polima cytoplasmic male sterility (pol CMS) is called "the first type of male sterility of rape with practical value". At present, 60% of the hybrids bred with three lines in China come from pol CMS.. The thermo-sensitive pol TCMS partially restored fertility at low temperature, and stable sterility at high temperature could also realize two-line seed production. In order to elucidate the regulation mechanism of restorer gene and the molecular mechanism of pol TCMS temperature sensitivity and make pol CMS system more suitable for rapeseed heterosis, the regulation factors of restoring gene Rfp were screened by yeast single hybrid method. The main results of this study are as follows: 1. The promoter sequence of 600 bp upstream of Rfp gene was analyzed by cis-acting elements. The results showed that the promoter sequence contained CAAT box and TATA box, photoregulatory elements, hormone response elements and other cis-acting elements. Construct 5 Rfp promoters? The truncated vector was transformed into Arabidopsis thaliana and it was found that ATG upstream 151 bp could drive the expression of GUS gene in flower buds, while ATG upstream 118 bp could not drive GUS gene expression. According to the experimental results, we determined that the promoter core of Rfp gene is in the upstream 151 bp of ATG. The cDNA library was constructed from 0.5-1mm of Brassica napus restorer line, and yeast bait vector was constructed with 394 bp upstream of ATG. A protein binding to bait fragment was obtained by point-to-point verification. Bioinformatics analysis revealed that the gene may be involved in biological stress response and intercellular signal transduction. 2. 2. 86 maintainer lines and 6330A were selected from 530 natural populations of Brassica napus to construct F1 population. The hybrid population was seeded in Wuhan in autumn. The phenotypic observation showed that the temperature was lower (lower than 16 鈩，
本文编号：2375102
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