甘蓝型油菜波里马细胞质雄性不育恢复基因的图位克隆和功能研究

发布时间：2018-06-05 23:07

本文选题：甘蓝型油菜 + 细胞质雄性不育　；参考：《华中农业大学》2016年博士论文

【摘要】：波里马细胞质雄性不育(pol CMS)是甘蓝型油菜杂种优势利用的主要途径之一,同时也是研究核质互作的良好载体。本研究通过图位克隆的方法分离了波里马细胞质雄性不育的育性恢复基因Rfp,并利用细胞学和分子生物学方法对其进行了相关研究,阐述了Pol CMS的败育机理及Rfp基因在甘蓝型油菜中的恢复功能。主要研究结果如下:1.利用不育系6330A和恢复系Bing409构建的回交群体通过图位克隆的方法将波里马细胞质雄性不育恢复基因(Rfp)定位到甘蓝型油菜A9染色体上一段长约32kb的区间。对定位区间中的orf进行序列分析发现其中一个为PPR家族成员,将之作为候选基因构建载体。遗传转化证明该orf确实是波里马细胞质雄性不育恢复基因(Rfp)。2.对该基因的全长(包括启动子区,CDS和3,UTR)在不育系1141A和恢复系Bing409中进行比较测序,比较测序结果表明Rfp与rfp启动子区和3,UTR区分别个有一个SNP,而在CDS上存在45个SNP,导致30个氨基酸的改变,其中12个氨基酸的极性发生变化。亚细胞定位的结果显示Rfp和rfp均定位于线粒体。这表明恢复基因与其等位基因功能的差异主要是由于CDS区的变异导致,但这些变异并未引起蛋白定位的改变。基于上述的测序结果,开发了针对Rfp的功能标记,可以高效地用于波里马细胞质雄性不育恢复材料的筛选和恢复系的回交选育。3.半薄切片结果表明不育单株花药在发育至第5期不能形成四个正常的角隅,向后发育也不能形成药室或者形成1-2个形态异常的药室,而形成的药室中小孢子的发育异常,最终不能形成花粉或者产生干瘪的花粉。透射电镜结果表明不育单株在花药发育的第3期线粒体开始出现异常和崩解,可能引发孢原细胞的PCD,最终导致L2层细胞不能分化出正常的药室内壁,中层,绒毡层以及小孢子母细胞。4.对Rfp基因进行RT-PCR和qPCR分析,结果表明Rfp油菜的根、茎、叶、花和幼蕾各个组织中广泛表达,幼蕾中表达量最高,GUS分析结果与之类似,说明恢复基因可能主要在花药发育早期起重要功能。在花药中GUS染色结果表明Rfp表达的主要部位在L2层细胞特化出的药室内壁,中层,绒毡层以及小孢子母细胞(小孢子)中,这与不育表型相对应。5.为了探究Rfp可能的功能,以pol胞质(不育系)和nap胞质(保持系)线粒体组差异的orf作为探针进行Northern blot的检测。其中以orf224作为探针检测时发现在与不育系相比遗传转化复育系和恢复系中orf224-atp6转录本发生明显减少,而保持系中基本检测不到信号。这表明Rfp可能通过转录后调控的方式参与orf224转录本的加工从而恢复育性。6.对Rfp进行预测发现其具有15个PPR结构域,可能与RNA识别相关,但它本身不具有RNA剪切或者其他加工的结构域,据此推测可能还存在其他未知蛋白参与该过程。通过酵母双杂,在甘蓝型油菜花蕾cDNA文库中共筛选到了4个可能与Rfp互作的基因,通过BiFC的检测证实这些互作在甘蓝型油菜的线粒体中真实存在。对这些互作的基因进行了干涉,它们的具体功能需进一步研究。
[Abstract]:The cytoplasmic male sterility (pol CMS) is one of the main ways to utilize the Heterosis of Brassica napus, and it is also a good carrier for the study of nuclear interaction. In this study, the fertility restorer gene Rfp of the cytoplasmic male sterility of bolma cytoplasmic male sterility was isolated by the method of graph cloning, and was carried out by cytological and molecular biological methods. The mechanism of abortion of Pol CMS and the recovery function of Rfp gene in Brassica napus were discussed. The main results were as follows: 1. the backcross group constructed by the sterile line 6330A and the restorer line Bing409 could locate the BMS cytoplasmic male sterility restorer gene (Rfp) on the A9 chromosome of Brassica napus by using the method of mapping the clone of the cytoplasmic male sterile (Rfp). A section of a length of about 32KB. The sequence analysis of orf in the positioning interval found that one of them was a member of the PPR family as a candidate gene construction carrier. Genetic transformation proved that the ORF was indeed the full length of the ORF cytoplasmic male sterility restorer gene (Rfp).2. (including the promoter region, CDS and 3, UTR) in the sterile line 1141A and The comparative sequencing in the restorer line Bing409 showed that there was a SNP in the promoter region of Rfp and RFP and 3 and UTR, and 45 SNP in CDS, which resulted in the change of 30 amino acids, and the polarity of 12 amino acids changed. The result of subcellular localization showed that both Rfp and RFP were located in the mitochondria. The difference in the function of the allele is mainly due to the variation in the CDS region, but these variations do not cause the change in the protein location. Based on the above sequencing results, a functional marker for Rfp is developed, which can be used efficiently for the screening of the restorer material of the cytoplasmic male sterility and the backcross breeding of the.3. semi thin section. The results showed that the anther of sterile single plant could not form four normal corners in the period of development to fifth stage, and the backward development could not form the medicine chamber or form the 1-2 morphologically abnormal pharmacy, and the development of the medium and small spores in the medicine chamber was abnormal and eventually could not form pollen or produce shrivelled flower powder. Transmission electron microscopy showed that the sterile single plant was in anther hair. The third stage mitochondria began to appear abnormal and disintegrating, which could lead to the PCD of the sporospora cells, which eventually led to the failure of the L2 layer cells to differentiate into the normal chamber wall, the middle layer, the tapetum and the microspore mother cell.4. to RT-PCR and qPCR analysis of the Rfp gene. The results showed that the roots, stems, leaves, flowers and young buds of the Rfp rape were widely expressed. The expression of the young buds was the highest, and the results of GUS analysis were similar, indicating that the restorer gene might play an important role in the early stage of anther development. In the anther, the results of GUS staining showed that the main parts of the Rfp expression were in the inner wall of the chamber, the middle layer, the tapetum and the microspore cells (microspore), which were corresponding to the sterile phenotype. 5. in order to explore the possible function of Rfp, the Northern blot was detected by ORF as a probe with the difference between the pol cytoplasmic (sterile line) and the nap cytoplasmic (maintainer) mitochondrial group. The orf224 as a probe detected a significant decrease in the orf224-atp6 transcript in the genetic transformation lines and the restorer lines compared with the male sterile lines, and in the maintainer line. The signal is not detected basically. This indicates that Rfp may be involved in the processing of orf224 transcriptional transcript by post transcriptional regulation to restore the fertility.6. to predict Rfp and find that it has 15 PPR domains, which may be associated with RNA recognition, but it itself does not have a RNA shear or other processing domain, thus speculates that there may still be other failure. In this process, 4 genes interacting with Rfp were screened in the cDNA Library of Brassica napus by yeast double heterozygosity. The interaction of these interactions in the mitochondria of Brassica napus was confirmed by BiFC detection. The genes of these interacted genes were interfered, and their specific functions needed further study.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

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