【作者】 杨路明；

【导师】 张天真；

【作者基本信息】 南京农业大学 ， 遗传学， 2009， 博士

【摘要】 植物细胞质雄性不育(Cytoplasmic male sterility, CMS).是一种不能产生有活力花粉的遗传现象,属母性遗传,在植物杂种优势利用中具有重要价值.利用胞质雄性不育系及恢复系和保持系的三系配套制种方式具有省工、方便、种子纯度高、成本较低等优点,将成为杂种棉商业化制种最有效的途径之一.棉花不仅是世界上最重要的天然纤维和重要的油料作物,而且也是一种重要的生物能源.棉花杂种优势十分明显,可有效提高产量、纤维品质与抗病、抗逆性.棉花由于“三系”中的恢复系存在恢复力不强的缺陷使得棉花CMS的应用受到极大的限制,“三系法”制种仍未实现产业化.因此,克隆棉花细胞质雄性不育育性恢复基因对于研究细胞质雄性不育机理,培育优良恢复系,更好的利用杂种优势具有重要的理论和实践意义.目前已克隆的恢复基因中,除了玉米Rf2编码乙醛脱氢酶外,矮牵牛Rf,萝卜Rf0及水稻的Rf1a和Rf1b都编码的是由PPR基序串联组成的PPR蛋白.PPR基因家族是近年来新发现的一个家族,同时也是植物中最大的家族之一.它编码一种以35个简氨基酸为重复单位串连排列组成的蛋白质,这些重复单元能形成一个超螺旋从而特异的与单链RNA结合.PPR蛋白在细胞器形成和植物发育的许多方面起着重要的调控作用。目前,在许多真核生物中都已经发现了PPR基因,但棉花上还没有关于PPR基因的报道.本研究选用本室根据GenBank棉花EST数据库中的序列开发设计的2803对EST-SSR引物采用基因型代表群分析法对亲本不育系104-7A和恢复系0-613-2R及22株小群体进行筛选,将选中的6对引物对613株BCl群体进行扩增,共筛选到6个与Rf1基因紧密连锁的分子标记.通过整合本室先前构建的助遗传图谱上的原有标记,利用Joinmap3.0作图软件对恢复基因进行精细定位,整合后的图谱含有18个标记(2个RAPD、3个STS标记和13个SSR标记),总遗传距离为0.65cM.本研究所获得的6个与Rf1紧密连锁的EST-SSR标记(NAU2650、NAU2924、NAU3205、NAU3652、NAU3938和NAU4040)表现为共分离,均与Rf1基因相距0.327cM,其中NAU3205为显性标记,其它5个都是共显性标记.利用6.个与助紧密连锁的EST-SSR标记对本室构建的0-613-2R恢复系BAC文库进行PCR筛选。先对二级混合克隆文库进行PCR筛选,筛选到阳性混合克隆后,再对一级混合克隆库进行筛选,最终得到阳性单克隆.6个EST-SSR标记共筛选到7个阳性BAC单克隆,其中NAU4040未筛选到阳性克隆,NAU3938筛选到3个阳性克隆,其它标记各筛选到1个阳性克隆,NotⅠ酶切检测其插入片段大小范围为105-130kb.将本室已获得的与Rf1相关的克隆整合后在助遗传图谱上的18对引物共获得56个阳性BAC克隆.选取助图谱上两侧最近的13个标记所筛选到的31个阳性BAC进行HindⅢ酶切指纹分析,利用Image3.10b和FPC V7.2软件对HindⅢ酶切图谱进行分析后构建Rf1区域的精细物理图谱.利用萝卜、矮牵牛和水稻恢复基因编码的蛋白序列分别作为查询探针对GenBank中棉花ESTs数据库进行tblastn扫描,对获得的所有ESTs利用CAP3软件组装并通过blastx预测每个contig的功能,获得三个与萝卜、矮牵牛和水稻恢复基因同源性较高的contigs。根据这三个contigs的序列设计特异引物对物理图谱上的所有BAC进行筛选,其中根据contig2设计的引物筛选到一个克隆Y43。综合物理图谱与同源序列分析的结果,将棉花Rf1基因定位在BACY43上并对该BAC全长测序.对获得的BAC序列进行基因预测,共获得24个ORF,其中4个ORF(ORF2、ORF3、ORF7和ORF18)编码PPR蛋白并含有线粒体定位信号。通过4个ORF的序列比对及对恢复系和不育系的测序比较,推断ORF3为助基因候选ORF.为了了解陆地棉中PPR基因的分布和数量,本研究从147个陆地棉BAC中找到6个编码PPR蛋白的ORF,同时利用其它植物中已克隆的18个PPR基因编码的蛋白作为tblastn比对的查询探针,对GenBank中陆地棉EST数据库进行扫描,通过CAP3组装拼接鉴定出309个PPR unigene。为进一步研究陆地棉中PPR基因编码的蛋白结构和表达特点,以陆地棉0-613-2R为材料通过RT-PCR的方法克隆了5个PPR基因的全长cDNA序列,分别将它们命名为GhPPR1-GhPPR5,对其基因组结构分析发现它们都不含有内含子,ORF长度从867-1779 bp。结构域分析显示这5个PPR基因编码的氨基酸分别包含5-10个PPR重复单元,其中GhPPR1和GhPPR2属于PLS亚家族,GhPPR3-GhPPR5属于P亚家族,同时这5个基因与18个其它植物中的PPR基因的进化分析也显示同一亚家族中的成员聚集在一起。本研究还利用实时荧光定量PCR对这5个PPR基因在陆地棉不同组织和器官中的表达进行了分析,结果显示5个基因在根、茎、叶、花粉以及纤维中都表达,但在不同时期的相对表达量略有不同。育性基因的分离克隆一直是人们关心的课题,它对于认识不育性机理,揭示生命现象以及更有效地利用杂种优势都有着十分重要的意义。本研究利用图位克隆和同源序列克隆两种方法相结合的策略,在构建Rf1精细遗传图谱和物理图谱的基础上,根据恢复基因同源序列设计引物对物理图谱上的BAC克隆进行扫描,鉴定出包含Rf1基因的BAC克隆并对其全长测序,通过基因预测和特征分析及亲本序列差异分析获得了Rf1的候选ORF.棉花CMS育性恢复基因的成功克隆,可以通过转基因方法,培育具有优良性状的新型恢复系,可实现棉花杂种优势利用的重大突破,从而极大地推动我国棉花育种和生产的发展。同时,获得具有自主知识产权的棉花CMS育性恢复基因,可以更好地保护我国的基因资源。本研究还有助于揭示棉花细胞质雄性不育的分子机理,为进一步研究和利用棉花杂种优势奠定基础。更多还原

【Abstract】 Plant cytoplasmic male sterility (CMS) is a maternally inherited trait that is unable to produce viable pollens, which plays an important role in utilization of hybrid vigor. Comparing with hand-emasculation and pollination, and genetic male sterile lines, utilization of CMS lines is much more effective and economical in producing commercially hybrid seeds. Cotton is not only the world’s most important natural textile fiber and a significant oilseed crop, but also a crop that is of significance for foil energy and bioengergy production. The heterosis in cotton hybrid is much significant, especially in improving yields, fiber qualities, disease-resistances and stress tolerance. Some hybrid strains of cotton with CMS have been applied in production, but the utilization of CMS is rather limited because of the low yield of restoring lines and the noxiousness of the exotic cytoplasm in the male sterile lines. Therefore, the cloning of restoring gene plays an important value on studying the mechanism of CMS and heterosis utilization.With the exception of Rf2 from maize, all cloned Rf genes are members of the pentatricopeptide repeat gene family (PPR).The PPR protein family is one of the largest families which was found recently. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. Up to now, PPR proteins have been identified in many eukaryotes, but there is still no report on PPR gene in cotton.A total of 2803 pairs of EST-SSR primers designed from cotton ESTs in Genbank were screened the restoring line 0-613-2R and the CMS line 104-7A with 22 small populations by genotypic difference analysis. The selected primers were amplified by using 613 individuals BC1 mapping populations, and 6 new EST-SSR primers were found to closely link to the Rf1 of cotton. Integrating other markers linked with the Rf1 gene identified by Yin et al (2006), fine map was made with Joinmap3.0 software. The genetic mapping contained 19 makers that were 13 for SSR,2 for RAPD, and 3 for STS and the total genetic distance was 0.65cM. The 6 EST-SSR primers (NAU2650、NAU292、NAU3205、NAU3652、NAU3938 and NAU4040)added to the genetic map of Rf1 in this study showed co-segregation, and all the 6 primers had a genetic distance of 0.327cM from Rf1.Among them, only NAU3205 was a dominant marker and other five markers showed co-dominance.In order to construct a fine physical map of Rf1, the 6 EST-SSR primers were used to screen the restoring line 0-613-2R BAC library. Finally 7 single positive clones were attained by PCR method. The 7 clones were digested with Not I enzyme indicating insert size of them with a range of 105 to 130 kb. After integrating with previous results of our laboratory, there are totally 56 single positive clones availably.31 clones selected from 13 markers tightly most linked to the Rf1 were used to digested with HindⅢ.The fingerprintings of these clones were used by Image 3.10b and FPC V7.2 software to construct physical map.The Rf genes of rice, radish and petunia were used as query probes for tBLASTn searches of the cotton EST database. All the hit sequences were assembled by CAP3 assembly tool and 63 contigs were attained. Blastx analysis was used for function prediction of these contigs, and 3 contigs were predicted to have a higher similarity with Rf genes of radish, petunia and rice. Specific primers of the 3 contigs were designed to screen the BAC clones in the physical map of the Rf1. We identified a clone Y43 screened by the primers of contig2, and presumed this BAC probably containing the Rf1 gene.24 ORF were acquired by sequencing and gene prediction of this BAC. ORF2、ORF3、ORF7and ORF18 were predicted to encode PPR protein and have a mitochondrially targeted signals in their N-terminal amino acids. Sequences alignment of the four ORF and the restoring line 0-613-2R and the CMS line 104-7A, the ORF3 was identified as a candidate ORF for the Rf1 gene which is almost entirely composed of 16 repeats of the 35-aa PPR motif.In order to study the number and distribution of PPR genes in upland cotton, we identified 6 ORF encoding PPR protein in 147 BAC sequences, and 309 PPR unigenes were found through extensive survey of the EST database of upland cotton. Furthermore, we isolated five full length cDNA of PPR genes from 0-613-2R which were named GhPPRl-GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPRl-5 contain from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPRl-2 belonged to PLS subfamily and GhPPR3-5 belonged to P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All the five GhPPR genes were constitutively expressed in roots, stems, leaves, pollens, and 15dpa fibers based on real-time quantitative RT-PCR, but the relative expression levels were a little different.The isolation and clone of restoring genes is concerned because it pays an important value on studying the mechanism of sterility and heterosis utilization. In our study, the strategy combining map-based cloning and homology cloning was used to isolate the Rf1 of cotton. On the basis of physical map construction of Rf1, specific primers were designed from homology EST of Rf genes to screen those BAC clones on the physical map of Rf1. The BAC clone containing Rf1 gene was identified to be sequenced, and finally a candidate ORF encoding PPR protein for Rf1 was identified through bioinformatics analysis and sequence alignment. This is the basic work for isolation and clone of restoring genes. We can breed new cotton restoring strains with excellent traits, break through the utilization of heterosis and greatly push cotton breeding and development of production through transgenic methods. The fertile restoring gene with property right also can protect our gene resources. The results of the research can be contributive to reveal the molecular machine about cotton CMS and establish a foundation for the study and use with cotton heterosis.更多还原