大白菜polCMS恢复基因BrRfp1的图位克隆和不同恢复基因位点恢复机理的研究

发布时间：2018-07-24 20:32

【摘要】：波里马细胞质雄性不育(pol CMS)是在甘蓝型油菜中发现的第一个有实用价值的胞质雄性不育类型,后被转育大白菜等十字花科作物中,在十字花科作物的杂交育种中有重要的应用价值。Pol胞质被转育到大白菜后,在大白菜中有天然的恢复系,成为大白菜杂交育种应用最广泛的不育源之一。大白菜pol CMS恢复基因的图位克隆和恢复机理的研究,对于pol CMS在大白菜杂交育种中的应用,加快育种进程有重要意义,另外对细胞质遗传和核质互作研究也有重要价值。本研究以大白菜pol CMS不育系94C9和4个恢复系92S105、01S325、00S109和88S148为材料,对4个恢复系的恢复基因进行了等位性分析,并图位克隆了恢复基因BrRfp1,找出了非恢复等位基因的关键突变位点,还研究了2个恢复基因的恢复机理。这些研究为深入理解pol CMS/Rf系统的分子机理奠定基础。本研究主要取得了如下结果:1.在4个大白菜pol CMS的恢复系中鉴定出2个BrRfp位点。恢复系92S105、01S325、00S109的恢复基因位于同一基因位点,恢复系88S148的恢复基因位于另外的基因位点,将这两个恢复基因位点命名为BrRfp1和BrRfp2。2.利用恢复系92S105和不育系94C9构建的BC1F1和BC1F2定位群体,图位克隆了Br Rfp1基因。BrRfp1基因是位于大白菜A09染色体上的一个编码PPR蛋白的基因。3.通过比较4个BrRfp1基因和6个非恢复等位基因的推导氨基酸序列,发现BrRfp1基因第11个PPR基序中的3个氨基酸突变在非恢复等位基因的推导氨基酸序列中都存在,可能是导致这些基因丧失恢复功能的关键突变。4.根据BrRfp1基因和非恢复等位基因的核苷酸序列多态性,开发了与BrRfp1基因共分离的显性标记SC718,该标记在不同群体中均可检测出BrRfp1基因是否存在,可用于分子标记辅助育种和大白菜种质资源中新恢复系的筛选。5.全不育、半恢复和全恢复花蕾的花药在组织形态学上有明显的差异。全不育花蕾在1 mm时花药已经停止发育,半恢复花蕾的花药只能发育出2-3个药室,全恢复花蕾的花药则发育出完整的4个药室。6.采用环化RT-PCR的方法,研究了orf224-atp6转录本在全不育、半恢复和全恢复花蕾中的差异。结果表明,全不育和半恢复花蕾中的表达相同,都能检测出2个orf224-atp6转录本,但在全恢复的花蕾中能检测出3个orf224-atp6转录本。7.采用RT-qPCR的方法,对orf224和atp6基因在全不育、半恢复和全恢复花蕾中的表达进行了定量。结果发现,与全不育花蕾相比,半恢复和全恢复花蕾中orf224基因mRNA的量都显著降低,且二者之间差异不显著。半恢复和全恢复花蕾中atp6基因mRNA的量也比不育花蕾显著降低,但在花蕾小于4 mm时,半恢复花蕾中atp6基因mRNA的量比全恢复花蕾中明显减少,因此推测BrRfp2基因引起育性半恢复的原因可能与atp6基因mRNA的量显著减少有关。8.采用western杂交的方法,研究了ORF224蛋白在不育、半恢复和全恢复花蕾中积累量的差异。结果表明,与不育花蕾相比,半恢复和全恢复花蕾中ORF224蛋白的积累量都显著降低,几乎检测不到。
[Abstract]:Pol CMS is the first useful type of cytoplasmic male sterility found in Brassica napus, and then transferred to cruciferous crops such as cabbage and cabbage. It has important application value in cross breeding of Cruciferae crops,.Pol cytoplasm is transferred to Chinese cabbage, and it has natural restorability in Chinese cabbage. It has become one of the most widely used sterile sources of Chinese cabbage hybrid breeding. The study on the mapping and recovery mechanism of the pol CMS restorer gene of Chinese cabbage is of great significance for the application of Pol CMS in Chinese cabbage breeding and the accelerated breeding process. In addition, it is also of great value to the study of cytoplasmic inheritance and nuclear cytoplasm interaction. The pol CMS sterile line 94C9 of Chinese cabbage and 4 restorer lines 92S105,01S325,00S109 and 88S148 were used as materials to analyze the allele of the restorer genes of the 4 restorer lines. The restorer gene BrRfp1 was cloned and the key mutation sites of the non recovery alleles were found, and the recovery mechanism of 2 restorer genes was also studied. These studies are to understand Po in depth. The molecular mechanism of L CMS/Rf system lays the foundation. The main results are as follows: 1. 2 BrRfp loci were identified in the restorer lines of 4 Chinese cabbage, pol CMS. The recovery gene of the restorer line 92S105,01S325,00S109 was located at the same gene site, and the restorer gene of the restorer line 88S148 was located at the other gene site, and these two restorer genes were located. The point is named BrRfp1 and BrRfp2.2. using the BC1F1 and BC1F2 locative population constructed by the restorer line 92S105 and the sterile line 94C9. The Br Rfp1 gene.BrRfp1 gene is cloned as a encoding PPR protein on the A09 chromosome of Chinese cabbage, and the gene.3. is found by comparing the amino acid sequences of 4 genes and 6 non restorative alleles. The mutation of 3 amino acids in the Eleventh PPR sequences of the BrRfp1 gene exists in the deduced amino acid sequence of the non recovery allele. It may be the key mutation that leads to the loss of the recovery function of these genes,.4., based on the nucleotide sequence polymorphism of the BrRfp1 gene and the non recovery allele, opens the dominant marker SC7 that is co separated from the BrRfp1 gene. 18, the marker can detect the existence of BrRfp1 gene in different populations. It can be used in molecular marker assisted breeding and the New Restorer Line in Chinese Cabbage Germplasm to screen.5. all sterile. The anther of semi recovery and full recovery is distinctly different in morphology. The anther has stopped developing at 1 mm and half recovery. The anther of flower bud can only develop 2-3 medicine chambers, and the anther of the full recovery of bud's anther develops a complete 4 pharmacy.6. using the method of cyclization RT-PCR. The difference of the orf224-atp6 transcript in full sterility, semi recovery and full recovery is studied. The results show that 2 orf224-atp can be detected by the same expression in the full and semi recovery buds. 6 transcriptional transcripts, but 3 orf224-atp6 transcripts were detected in the full recovery buds by RT-qPCR, and the expression of orf224 and Atp6 genes in full sterile, semi restored and fully restored buds was quantified. The results showed that the amount of orf224 gene mRNA in the semi and full buds was significantly lower than that in the full sterile buds. There was no significant difference between the two. The amount of Atp6 gene mRNA in the semi recovery and full buds was also significantly lower than that of the sterile buds, but when the bud was less than 4 mm, the amount of Atp6 gene mRNA in the semi restored bud was significantly lower than that in the full bud. Therefore, the reason that the BrRfp2 gene caused the fertility half recovery may be significantly reduced by the quantity of the Atp6 gene mRNA. The difference in the accumulation of ORF224 protein in the sterility, semi recovery and full recovery of buds was studied by the method of Western hybridization with.8.. The results showed that the accumulation of ORF224 protein in the semi recovery and full recovery buds was significantly lower than that of the sterile buds, and it was almost not detected.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S634.1

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