【作者】 许小勇；

【导师】 方智远；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2013， 博士

【摘要】 细胞质雄性不育（CMS）是高等植物中广泛存在的生物现象。对CMS及其恢复系的研究利用，一方面可省去繁琐的手工去雄，在农作物制种中拥有巨大的应用潜力；另一方面也为揭示细胞核和细胞质间遗传互作交流提供一个很有价值的研究模型。油菜polima CMS是已经在十字花科植物中广泛应用的不育源，但是到目前为止其细胞质雄性不育的发生机制仍不清楚，育性恢复基因也没被克隆。本课题组在上世纪末期已经成功将pol-like CMS导入到大白菜中，育成大白菜CMS7311，并且发现白菜基因组中存在其育性恢复基因，开展了一系列相关研究。本试验在已有的试验研究基础之上，主要进行了大白菜CMS7311不育发生相关基因筛选鉴定，以及大白菜育性恢复基因BcRfp的分子标记定位等研究，取得了以下研究结果：（1）筛选鉴定了白菜花蕾发育过程中稳定表达的内参基因采用三种统计软件分别比较了8个候选基因在白菜不同大小花蕾发育过程中的表达稳定性。GeNom软件计算结果表明这些基因的相对稳定性顺序为：Tub/GAPDH> Cyp> EF1a> U34559>BcTip41> Apr>18S rRNA，其中，Tub和GAPDH是最稳定的内参基因（M值为0.614）。NormFinder和BestKeeper软件的计算结果也表明Tub依然是最稳定的内参基因。综合三种软件的计算结果，确定Tub作为最佳内参基因，可用于白菜花蕾发育过程相关基因表达分析。（2）进行了大白菜CMS及其可育株花蕾发育相关基因差异表达分析通过cDNA-AFLP比较分析CMS和恢复系杂交分离后代中不育和可育株花蕾发育相关的基因表达变化，通过回收测序及Blast分析，共发现32个差异条带在数据库中找到同源基因，其中有10条来自于不育池，其余都来自可育池。这些基因涉及到逆境胁迫反应、合成与代谢，细胞重建以及转录调控，脂肪合成代谢等。（3）大白菜雄性不育发生相关基因表达定量对cDNA-AFLP分析所获得的部分差异片段进行表达定量，发现6个基因BcMS2, BcGRP17, BcPME31, BcROPGEF8, BcTNL3和BcUP的表达量在不育和可育株花蕾之间表现出显著性差异。其中，前三个基因已经被证明其功能缺失可导致雄性不育的发生，在该不育材料的表达量很低甚至是不表达，预示了CMS相关不育基因可能抑制着这些花蕾尤其是雄蕊正常发育所必需功能基因的表达，从而导致雄性不育的发生；首次发现后两个基因BcROPGEF8和BcTNL3在不育花蕾中的高表达，有可能参与了雄性不育的诱导或应激反应，具体的功能尚不清楚。最后一个基因BcUP功能未知，只在花蕾发育早中期表达，且在不育材料中表达显著低于可育材料。（4）克隆了大白菜雄性不育发生相关的未知功能基因BcUP基于cDNA-AFLP差异表达结果，通过RACE等技术克隆了一个BcUP，全长1407bp，完整的开发阅读框1212bp，编码403氨基酸残基的蛋白，Blast比对发现该基因与拟南芥的未知功能基因AT3G56750同源，相似性达90%。生物信息学预测其具有糖基转移酶结构域，推测该基因可能具有岩藻糖基转移酶功能。（5）大白菜育性恢复基因BcRfp分子标记筛选及遗传定位利用AFLP、SRAP、SSR等分子标记技术对不育系和恢复系杂交后代分离群体进行分子标记筛选鉴定，获得一系列与BcRfp连锁的分子标记，连锁遗传计算分析发现这些标记分布于BcRfp的两边，最近的两个标记26920和SSR03的遗传距离分别为0.44cM和0.7cM。这些标记将BcRfp基因定位于A09染色体上约380kb的范围内，为进一步精细定位克隆奠定基础，也为快速进行新品种的选育提供分子标记辅助筛选。更多还原

【Abstract】 Cytoplasmic male sterility (CMS) is widely existence in higher plants. Study andapplication of CMS and its restoration (Rf), not only excuse us from artificial emasculationand contain potential capacity in crop seed production, but also provide a valuable researchmode for revealing the interactions between nuclei and cytoplasm. The polima (pol) CMS wasfound in Brassica napus, and has been widely used in Cruciferae, but the mechanism of malesterility was still unknown. In our team, the pol-like CMS has been successfully transferredinto heading Chinese cabbage, the restorer gene of which was also found in Chinese cabbage.In this research, we separated and identified six specific expressed genes between male sterileand fertile plants; screened the molecular markers and located the restorer gene BcRfp. Thedetail results were as follows.First, screening and identification of the reference gene that expressed most stable duringflower bud development in heading Chinese cabbage. Three statistical softwares were used tocompare the expression stablity of eight candidate genes in different sizes of buds. The resultsby GeNom software showed the order of expression stability was: Tub/GAPDH> Cyp>EF1a> U34559> BcTip41> Apr>18S rRNA, Tub and GAPDH were the most stablereference gene with the M value0.614. As the results of NormFinder and BestKeepersoftware, Tub was also the most stable reference gene. Comprehense three softwarecalculation results, Tub was regarded as the best reference gene, and could be used for thegene expression analysis during flower bud development in heading Chinese cabbage.Second, screening and identification of CMS and restorer related gene. CDNA-AFLPanalysis was used to identify genes differentially expressed during flower bud developmentbetween cytoplasmic male sterile plant (Ms) and male fertile plant (Mf) derived from thehybrid of CMS7311and a restorer (Rf). Thirty-two transcripts of different fragments (TDFs)of over80bp in length were identified by cDNA-AFLP anslysis. Among them, ten TDFswere found expressing in Ms bud, and the others expressing in Mf bud. Gene ontologyanalysis revealed that these genes were involved in stress response, synthesis and metabolism of fatty acid, cell reconstruction etc.Third, six genes closely related to male sterile were selected and their expression duringthe flower bud development of Ms and Mf line were investigated by qRT-PCR. Three genes,BcGRP17, BcMS2, and BcPME31, which were necessary for normal male organ development,showed significantly lower expressions in Ms bud; two genes, BcROPGEF8and BcTNL3,were induced significantly higher expression in Ms bud by unknown stress and involved inthe formation of male sterility. Another function unknown gene BcUP was only expressed atthe early and middle stage during the flower bud development,and the expression in Ms budwas obviuously lower that that in Mf bud.Fourth, clone of unknown gene BcUP. Based on the results of cDNA-AFLP analysis, afull length cDNA BcUP with1407bp was cloned by RACE method, which containing acomplete development reading frame1212bp and encoding a protein with403amino acidresidues. Blast result showed that this gene was homology with an unknown function geneAT3G56750from Arabidopsis, with the similarity about90%. A glycosyltransferase domainwas predicted by bioinformatics suggesting that this gene may have a fucosyl transferasefunction.Fifth, screening of molecular marker linked to the restorer gene BcRfp of CMS andmapping. By molecular marker techniques such as AFLP, SRAP, SSR etc, a series of linkedmolecular markers were identified. Genetic linkage analysis showed that these markersdistributed in both sides of BcRfp, of which two markers26920and SSR03with the nearestgenetic distances0.44cM and0.7cM, respectively. These markers located the BcRfp gene onchromosome A09with about380kb of Brassica rapa database, which made a basis forfurther positional cloning of the BcRfp gene and for rapid selection of new varieties.更多还原