【摘要】：波里馬細(xì)胞質(zhì)雄性不育(pol CMS)是在甘藍(lán)型油菜中發(fā)現(xiàn)的第一個(gè)有實(shí)用價(jià)值的胞質(zhì)雄性不育類(lèi)型,后被轉(zhuǎn)育大白菜等十字花科作物中,在十字花科作物的雜交育種中有重要的應(yīng)用價(jià)值。Pol胞質(zhì)被轉(zhuǎn)育到大白菜后,在大白菜中有天然的恢復(fù)系,成為大白菜雜交育種應(yīng)用最廣泛的不育源之一。大白菜pol CMS恢復(fù)基因的圖位克隆和恢復(fù)機(jī)理的研究,對(duì)于pol CMS在大白菜雜交育種中的應(yīng)用,加快育種進(jìn)程有重要意義,另外對(duì)細(xì)胞質(zhì)遺傳和核質(zhì)互作研究也有重要價(jià)值。本研究以大白菜pol CMS不育系94C9和4個(gè)恢復(fù)系92S105、01S325、00S109和88S148為材料,對(duì)4個(gè)恢復(fù)系的恢復(fù)基因進(jìn)行了等位性分析,并圖位克隆了恢復(fù)基因BrRfp1,找出了非恢復(fù)等位基因的關(guān)鍵突變位點(diǎn),還研究了2個(gè)恢復(fù)基因的恢復(fù)機(jī)理。這些研究為深入理解pol CMS/Rf系統(tǒng)的分子機(jī)理奠定基礎(chǔ)。本研究主要取得了如下結(jié)果:1.在4個(gè)大白菜pol CMS的恢復(fù)系中鑒定出2個(gè)BrRfp位點(diǎn)。恢復(fù)系92S105、01S325、00S109的恢復(fù)基因位于同一基因位點(diǎn),恢復(fù)系88S148的恢復(fù)基因位于另外的基因位點(diǎn),將這兩個(gè)恢復(fù)基因位點(diǎn)命名為BrRfp1和BrRfp2。2.利用恢復(fù)系92S105和不育系94C9構(gòu)建的BC1F1和BC1F2定位群體,圖位克隆了Br Rfp1基因。BrRfp1基因是位于大白菜A09染色體上的一個(gè)編碼PPR蛋白的基因。3.通過(guò)比較4個(gè)BrRfp1基因和6個(gè)非恢復(fù)等位基因的推導(dǎo)氨基酸序列,發(fā)現(xiàn)BrRfp1基因第11個(gè)PPR基序中的3個(gè)氨基酸突變?cè)诜腔謴?fù)等位基因的推導(dǎo)氨基酸序列中都存在,可能是導(dǎo)致這些基因喪失恢復(fù)功能的關(guān)鍵突變。4.根據(jù)BrRfp1基因和非恢復(fù)等位基因的核苷酸序列多態(tài)性,開(kāi)發(fā)了與BrRfp1基因共分離的顯性標(biāo)記SC718,該標(biāo)記在不同群體中均可檢測(cè)出BrRfp1基因是否存在,可用于分子標(biāo)記輔助育種和大白菜種質(zhì)資源中新恢復(fù)系的篩選。5.全不育、半恢復(fù)和全恢復(fù)花蕾的花藥在組織形態(tài)學(xué)上有明顯的差異。全不育花蕾在1 mm時(shí)花藥已經(jīng)停止發(fā)育,半恢復(fù)花蕾的花藥只能發(fā)育出2-3個(gè)藥室,全恢復(fù)花蕾的花藥則發(fā)育出完整的4個(gè)藥室。6.采用環(huán)化RT-PCR的方法,研究了orf224-atp6轉(zhuǎn)錄本在全不育、半恢復(fù)和全恢復(fù)花蕾中的差異。結(jié)果表明,全不育和半恢復(fù)花蕾中的表達(dá)相同,都能檢測(cè)出2個(gè)orf224-atp6轉(zhuǎn)錄本,但在全恢復(fù)的花蕾中能檢測(cè)出3個(gè)orf224-atp6轉(zhuǎn)錄本。7.采用RT-qPCR的方法,對(duì)orf224和atp6基因在全不育、半恢復(fù)和全恢復(fù)花蕾中的表達(dá)進(jìn)行了定量。結(jié)果發(fā)現(xiàn),與全不育花蕾相比,半恢復(fù)和全恢復(fù)花蕾中orf224基因mRNA的量都顯著降低,且二者之間差異不顯著。半恢復(fù)和全恢復(fù)花蕾中atp6基因mRNA的量也比不育花蕾顯著降低,但在花蕾小于4 mm時(shí),半恢復(fù)花蕾中atp6基因mRNA的量比全恢復(fù)花蕾中明顯減少,因此推測(cè)BrRfp2基因引起育性半恢復(fù)的原因可能與atp6基因mRNA的量顯著減少有關(guān)。8.采用western雜交的方法,研究了ORF224蛋白在不育、半恢復(fù)和全恢復(fù)花蕾中積累量的差異。結(jié)果表明,與不育花蕾相比,半恢復(fù)和全恢復(fù)花蕾中ORF224蛋白的積累量都顯著降低,幾乎檢測(cè)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S634.1

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