【摘要】：波里馬細胞質(zhì)雄性不育(pol CMS)被稱為“第一個有實用價值的油菜雄性不育類型”,目前我國利用三系配套育成的雜交品種60%來自于pol CMS。溫敏型的pol TCMS在低溫下部分恢復育性,高溫下穩(wěn)定不育還可以實現(xiàn)兩系制種。為了解析恢復基因的調(diào)控機制和pol TCMS溫度敏感的分子機理,使pol CMS系統(tǒng)更好的應(yīng)用于油菜雜種優(yōu)勢,本研究用酵母單雜交的方法篩選恢復基因Rfp的調(diào)控因子,用全基因組關(guān)聯(lián)分析的方法對溫敏基因初定位,主要研究成果如下:1.對Rfp基因上游600 bp的啟動子序列進行順式作用元件分析,結(jié)果表明該段啟動子序列含有CAAT box和TATA box、光調(diào)控元件、激素響應(yīng)元件等順式作用元件。構(gòu)建5個Rfp啟動子5?端截短載體,轉(zhuǎn)化擬南芥發(fā)現(xiàn)ATG上游151 bp就能驅(qū)動GUS基因在花蕾中的表達,而ATG上游118 bp不能驅(qū)動GUS基因的表達,根據(jù)實驗結(jié)果我們確定Rfp基因的啟動子核心順式作用元件在ATG上游151 bp內(nèi)。我們用甘藍型油菜恢復系的幼蕾(0.5-1mm)構(gòu)建了cDNA文庫,用ATG上游394 bp構(gòu)建酵母誘餌載體進行篩選,通過點對點驗證獲得一個與誘餌片段結(jié)合的蛋白,經(jīng)過生物信息學分析發(fā)現(xiàn)該基因可能參與生物學脅迫響應(yīng)和細胞間信號傳導的功能。2.從530份甘藍型油菜自然群體中挑選出86份保持系與6330A做雜交構(gòu)建F1群體,將雜交群體秋播于武漢,通過觀察群體的表型發(fā)現(xiàn):在3月初開花時溫度較低(低于16℃),不同材料之間呈現(xiàn)不同的育性等級;4月初溫度升高,群體全部呈現(xiàn)不育表型,說明群體中可能存在一種和溫度響應(yīng)相關(guān)的調(diào)控因子。利用89份甘藍型油菜保持系的60K SNP芯片信息,用Q模型進行全基因組關(guān)聯(lián)分析,共檢測到在A9、A5、A2、A8染色體上有多個標記位點可能與溫敏基因相關(guān)。
[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，

本文編號：2375102