【作者】 张晓；

【导师】 郭三堆；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 棉花细胞质雄性不育(CMS)是母性遗传性状,在杂交棉制种中有重要的应用价值。通过不育系、保持系和恢复系的“三系”配套,以及“三系法”制种,可实现杂交棉种子规模化生产。目前,国内外已育成哈克尼西棉、三裂棉、陆地棉、海岛棉等胞质的不育系,并实现三系配套,其中陆地棉胞质不育系P30A(来源于104-7A)为核心的三系配套已应用于生产实践。导致植物细胞质雄性不育的基因被认为是位于线粒体基因组上,但是关于棉花CMS相关基因的研究报道很少。本文以P30A及其保持系P30B、恢复系Y18R为主要实验材料,对其线粒体基因组进行了Southern blot分析,克隆了不育胞质和可育胞质之间的差异序列,获得了胞质特异的RFLP、SCAR、SSR分子标记,并对差异序列进行了表达分析,为进一步克隆不育基因奠定了基础。本研究通过同源克隆的方法,获得了棉花31个线粒体基因的保守序列,从中选取20个基因做探针对不育系P30A、保持系P30B、恢复系Y18R的线粒体基因组(mtDNA)进行Southern blot分析,发现atpA、atp9、ccmB、nad1bc、nad6、nad7c、rrn18等7个基因在不育胞质和可育胞质间存在RFLP多态性。其中atpA基因的差异最明显,它在可育胞质和不育胞质中的两个RFLP片段均不相同。利用反向PCR(IPCR)方法,克隆到了可育胞质和不育胞质中atpA基因的所有EcoRI限制片段。在此基础上,应用TAIL-PCR方法,获得了可育胞质和不育胞质中atpA基因的所有HindIII限制片段。在可育胞质中,EcoRI限制片段长度分别是2225 bp和5083 bp,HindIII限制片段的长度分别是11689 bp和8501 bp;不育胞质中,EcoRI限制片段长度分别是2194 bp和3297 bp,HindIII限制片段的长度分别是11658 bp和9139 bp。序列分析发现,棉花atpA基因在可育胞质和不育胞质中各有两个拷贝:一个完整拷贝和一个3’截短型拷贝。完整atpA基因全长1524 bp;而可育胞质和不育胞质中的截短型atpA编码序列分别在1352 bp和1336 bp处截断。在完整atpA拷贝RFLP片段的3’端,即atpA基因下游第161-212 bp处,可育胞质(P30B)和不育胞质(P30A)存在差异,是一个SSR位点,该位点在P30B可育胞质中是(TAA)7(TA)6,在哈克尼西棉CMS系(CMS-D2)、P30A、晋A、湘远A不育胞质中是(TAA)3(TA)2,而在三裂棉CMS系(CMS-D8)胞质中是(TAA)4(TA)3,我们将其开发成SSR标记:SSR160。在atpA截短型拷贝RFLP片段上,即atpA编码序列截断位点(“断点”)后,可育胞质和不育胞质中分别存在一段515 bp(“N515”)和555 bp(“S555”)的差异序列,这两条差异序列完全不同,我们将其开发成SCAR515和SCAR555标记。这三个标记可用于鉴定棉花可育胞质和不育胞质。根据以上结果,对国内外的几种不同CMS系棉花的mtDNA进行了RFLP分析和差异片段的序列分析,发现在atpA基因位点,CMS-D2、P30A、晋A、湘远A这四种CMS系的RFLP图谱相同,而CMS-D8与CMS-D2、P30A、晋A、湘远A、P30B都不同。通过TAIL-PCR方法,克隆到了CMS-D8的差异EcoRI限制片段(4540 bp),发现其中的序列与P30A相比存在5处SNP位点,根据SNP位点,开发出CMS-D8特异分子标记:SCARD8,该标记可将CMS-D8与其他CMS系进行区分。应用RT-PCR方法对atpA进行表达分析,发现棉花可育胞质和不育胞质中全长atpA基因和截短型atpA基因均转录。分析atpA基因的RNA编辑情况时发现,棉花atpA基因全长拷贝(1524bp)中存在6处RNA编辑位点,且不育胞质与可育胞质的RNA编辑率基本相同;在截短型拷贝中存在4处RNA编辑位点,不育胞质的RNA编辑率明显高于可育胞质,说明不育胞质截短型拷贝可能仍承受较高选择压,很可能具有新功能。同时,利用环化RT-PCR (cRT-PCR)法克隆到不育胞质atpA全长拷贝和截短型拷贝的转录本全长,发现atpA全长拷贝转录本中包含完整SSR160位点;atpA截短型拷贝转录本中包含完整“S555”序列。在atpA基因3’侧翼区,存在一些短重复序列,这些序列来自于nad6基因3’部分编码序列。在不育胞质中,这些短重复序列位于关键的“断点”位置,推测其在不育胞质线粒体基因组重排中发挥重要的介导作用。根据短重复序列在两种胞质中的位置和拷贝数不同开发出一个SCAR标记:SCARN6,可用于鉴别棉花可育胞质和不育胞质。Northern blot分析显示,nad6基因在棉花“三系”及F1代中都只有一个转录本(850 bp),且丰度基本一致。利用cRT-PCR法克隆到nad6基因的转录本全长,发现其mRNA在基因组终止密码子之前-14和-15 bp处提前终止,即该基因无终止密码子。在atpA基因5’上游存在rrn18-rrn5基因簇,并且不育胞质比可育胞质缺失一个rrn18拷贝,推测rrn18基因与棉花CMS可能相关。本研究首次克隆到了棉花细胞质雄性不育胞质和可育胞质线粒体基因组中atpA基因的所有EcoRI和HindIII限制片段,获得两种胞质在atpA基因3’下游存在的差异序列,开发出用于鉴定可育胞质和不育胞质的SCAR和SSR标记,确定了CMS-D8胞质与其它CMS系之间的差异序列,开发出CMS-D8胞质特异SCAR分子标记。同时发现nad6基因3’部分编码序列成为短重复序列,并在CMS系线粒体基因组重排中发挥重要介导作用;发现不育胞质比可育胞质缺失一个rrn18基因拷贝;获得不育胞质中atpA全长拷贝和截短型拷贝以及nad6基因的转录本全长,并发现截短型atpA基因的编辑率在两种胞质间存在明显差异。这些结果为进一步克隆棉花细胞质雄性不育基因奠定了基础。更多还原

【Abstract】 Cytoplasmic male sterility (CMS) is a maternally inherited trait. It has been widely used in breeding programs to produce F1 hybrid seed in crops. The“three-line system”(involving a CMS line, maintainer line, and restorer line) can allow for large-scale cultivation of cotton hybrids. In cotton, there are several different types of CMS lines: CMS-D2 (G. Harknessii), CMS-D8 (G. trilobum), 104-7A, JinA, XiangyuanA and so on. CMS line P30A (cytoplasm originated from 104-7A) has been used in practical production.CMS-determining factors are believed to be located at the mitochondrial genomes. But CMS-associated genes in cotton are almost unknown. In this study, CMS line P30A, maintainer line P30B, restorer line Y18R, were used as the main materials. Mitochondrial gene probes were used to identify the differences between the P30B normal fertile (N-) and the P30A male sterile (S-) cytoplasm with Southern blot method. The differential sequences between N- and S-cytoplasm were cloned, and cytoplasm-specific RFLP, SCAR and SSR markers were developed. Further expression analysis was performed on the differential sequences. These work made foundation for cloning CMS-determining genes in cotton.Conserved sequences of 31 mitochondrial genes were obtained from mitochondrial genome of cotton by homolog cloning strategy. 20 mitochondrial genes were selected to perform Southern blot analysis on mtDNA of CMS line P30A, maintainer line P30B, and restorer line Y18R. Among the 20 genes, atpA, atp9, ccmB, nad1bc, nad6, nad7c and rrn18 revealed restriction fragment length polymorphisms (RFLPs) between N- and S-cytoplasm. The differences on atpA locus were most obvious.All of the EcoRI restriction fragments of atpA were cloned by inverse PCR (IPCR) technique for the first time. And further, all of the HindIII fragments were cloned with TAIL-PCR method. In N-cytoplasm, the EcoRI restriction fragments were 2225 bp and 5083 bp in length, the HindIII restriction fragments were 11689 bp and 8501 bp in length. In S-cytoplasm, the EcoRI restriction fragments were 2194 bp and 3297 bp in length, the HindIII restriction fragments were 11658 bp and 9139 bp in length. Both N- and S-cytoplasm had an intact atpA copy and a 3’truncated copy. The full length of atpA was 1524 bp.The truncated copies in N- and S-cytoplasm were truncated at 1352 bp and 1336 bp of atpA coding region, respectively.At the 3’flanking, and 161-212 bp downstream of, the intact atpA gene, a simple sequence repeat (SSR) locus was found between N- and S-cytoplasm. In N-cytoplasm, the locus was (TAA)7(TA)6; in P30A, JinA, XiangyuanA and CMS-D2, the locus was (TAA)3(TA)2; in CMS-D8, the locus was (TAA)4(TA)3. And the SSR locus was developed to be a molecular marker: SSR160.As far as the 3’truncated atpA copy was concerned, the truncating site (breakpoint) was different in N- and S-cytoplasm. Following the breakpoints, chimerical fragments of 515 bp (“N515”) and 555 bp (“S555”) were found in N- and S-cytoplasm, respectively. According to the chimerical sequences, two cytoplasm-specific sequence characterized amplified region (SCAR) markers were successfully developed. Three of the PCR markers can be used to distinguish N- and S-cytoplsam in cotton.Additionally, based on SSR polymorphisms and SNPs in truncated region, five CMS lines can be classified into two major groups: one corresponds to CMS-D8 and the other consists of other four CMS lines. Furthermore, a new SCAR marker was identified to distinguish CMS-D8 from other CMS lines.RT-PCR analysis showed that these atpA copies in N- and S-cytoplasm were all transcribed. There were six RNA edit sites in full length atpA coding sequences.The RNA editing rates of the full length atpA gene were the same between the two cytoplasms, but when the truncated copies were conserned, the RNA editing rates in S-cytoplasm were obviously higher than the N-cytoplasm. In S-cytoplasm, the RNA editing rates of truncated copy were close to 100%, while in N-cytoplasm the values were less than 60%. It indicated that the selective pressure upon the truncated copy of S-cytoplasm is heavier than that of N-cytoplasm, and further implied that the truncated copy of N-cytoplasm had lost function, while the truncated copy of S-cytoplasm was likely to possess new function. The full-length cDNAs of the intact and truncated atpA copies in S-cytoplasm were obtained by the cRT-PCR method. The results showed that the transcript of intact atpA gene covered the SSR160 sequence, and the transcript of truncated atpA gene covered the S555 sequence.We found that at the 3’downstream of atpA gene, there were several short repeat sequences which belonged to the 3’coding sequences of nad6 gene. In S-cytoplasm, these short repeat sequences were located at the“breakpoint”loci, this implied that the short repeat sequences played an important role in rearrangement of mitochondrial genome in S-cytoplasm. According to the short repeat sequences, a SCAR marker was developed to distinguish N- and S-cytoplasm of cotton. Northern blot analysis showed that there was an identical transcript of nad6 in“three line”and F1 cultivars. In addition, full length transcript of nad6 gene was obtained with cRT-PCR method, the mRNA of the nad6 gene was found to be processed -14 bp and -15 bp upstream of the inframe stop codons.rrn18-rrn5 gene cluster was found located at the 5’upstream of atpA gene, and an rrn18 copy was found to be deleted in S-cytoplasm when compared with N-cytoplasm. This indicated that rrn18 gene may be associated with CMS in cotton.In this study, all of the EcoRI and HindIII restriction fragments of atpA in N- and S- cytoplasms of cotton were cloned for the first time. SCAR and SSR molecular markers were developed to distinguish N- and S-cytoplasm. And the differential sequences between CMS-D8 from the other four CMS lines were determind. And parts of nad6 coding sequences were found to be participated in the rearrangement of cotton mitochondrial genome, especially in the S-cytoplasm. And one rrn18 copy was found to be deleted in S-cytoplasm when compared with N-cytoplasm. Transcription of atpA and nad6 was analysed. And RNA editing rates of truncated atpA gene were different between N- and S-cytoplasm. The results made foundation for further cloning CMS-determining genes in cotton.更多还原