红麻UG93细胞质雄性不育相关基因的筛

发布时间：2018-03-31 03:25

本文选题：红麻　切入点：线粒体基因　出处：《广西大学》2016年硕士论文

【摘要】：植物细胞质雄性不育(cytoplasmic male sterility, CMS)普遍存在于植物界,植物CMS不育机理的阐明对于人工创制新的细胞质雄性不育资源、调控CMS的育性具有重要意义。前人对植物CMS机理的研究,大多数是以胞质差异较大的不育系和保持系为材料,比较二者线粒体基因组的差异,由此挖掘与植物CMS密切相关的基因。由于细胞质雄性不育系和保持系胞质来源不同细胞质基因组遗传信息差别很大,容易造成CMS以外的累赘遗传信息的干扰。因此本研究以红麻细胞质近等基因系UG93A、UG93B及F1 (UG93A/福红992)为研究材料,对红麻线粒体基因nad3、nad9、nad4L、 nad6、nad9、sdh4、cob、 atpl、atp4等15个基因进行了克隆和表达分析,以此挖掘与红麻CMS密切相关的基因,获得的结果如下：1.获得不育系UG93A、保持系UG93B和F1代15个基因编码区,这些基因在UG93A、UG93B和F1中编码区序列差异仅为单碱基突变。利用荧光定量PCR方法研究CMS相关基因在花药发育双核期的表达量,结果表明不育系中CMS相关基因的表达量总体上要低于保持系。2.通过比较分析红麻线粒体基因RNA编辑与CMS的关系,结果表明15个基因在不育系和保持系中RNA编辑差别不大,推测这些基因的RNA编辑可能与红麻UG93细胞质雄性不育无关。3.红麻CMS系和保持系atp4基因的克隆和序列对比发现,细胞质雄性不育系UG93A中atp4基因起始密码子上游5’端存在4 bp碱基缺失,1 bp碱基插入和4个核苷酸序列差异,Northern Blot技术分析表明其转录本条带并无差异,但在不育系、保持系和F1代双核期花药中表达量存在显著差异,不育系UG93A atp4的表达量是保持系的0.31倍,F1代是保持系的1.32倍,且双核期不育系UG93A复合体V的活性是保持系的0.90倍,推测atp4基因可能与红麻细胞质雄性不育相关,atp4基因在红麻花粉发育能量代谢过程中有着重要的作用。4.红麻不育系、保持系和恢复系花药不同发育时期线粒体复合体V活性和ATP含量测定结果显示,在花药发育的单核期和双核期线粒体复合体V的活性均表现出不育系低于保持系和恢复系,呈递增趋势,而且单核期复合体V活性低于双核期。红麻不育系、保持系和恢复系在花药发育的单核期ATP含量没有显著差异,而双核期呈现出递增趋势,结果表明花药发育的双核期能量供给不足可能与雄性不育密切相关。
[Abstract]:Cytoplasmic male sterility (CMSs) exists widely in the botanical world. The elucidation of the mechanism of plant CMS sterility is of great significance in creating new cytoplasmic male sterility resources and regulating the fertility of CMS. Most of them were male sterile lines and maintainer lines with different cytoplasm, and the difference of mitochondrial genome between them was compared. Therefore, the genes closely related to plant CMS were excavated. The genetic information of cytoplasmic genomes of cytoplasmic male sterile lines and maintainer lines varied greatly from different cytoplasmic sources. It is easy to interfere with the unwanted genetic information other than CMS. Therefore, 15 genes of kenaf mitochondria, nad9nad9nad4L, nad6nad9sdh4cob and atplatp4, were cloned and expressed in kenaf cytoplasm, UG93A / Fuhong 992 and UG93A / FUG93B / F1 UG93A / Fuhong 992, respectively. The results were as follows: 1. The male sterile line UG93A, the maintainer line UG93B and F1 had 15 coding regions. The sequence difference of these genes in UG93AnUG93B and F1 was only a single base mutation. The expression of CMS related genes in anther developmental binucleate stage was studied by fluorescence quantitative PCR. The results showed that the expression of CMS related genes in male sterile lines was generally lower than that in maintainer lines. By comparing and analyzing the relationship between RNA editing and CMS of kenaf mitochondrial gene, the results showed that there was no significant difference between RNA editing of 15 genes in male sterile lines and maintainer lines. It is speculated that the RNA editing of these genes may not be related to the cytoplasmic male sterility of kenaf UG93. The cloning and sequence comparison of atp4 gene of kenaf CMS line and maintainer line were found. In cytoplasmic male sterile line UG93A, there were 4 BP deletion 1 BP insertion and 4 nucleotide sequence difference at the 5 'end of the initiation codon of atp4 gene. Northern Blot analysis showed that there was no difference in the transcriptional band, but there was no difference in the transcriptional band in the male sterile line. There was significant difference in the expression of UG93A atp4 between the maintainer line and the F1 generation. The expression of UG93A atp4 was 1.32 times of that of the maintainer line, and the activity of UG93A complex V was 0.90 times higher than that of the maintainer line. It is speculated that atp4 gene may play an important role in the energy metabolism of kenaf pollen development, which may be related to the cytoplasmic male sterility of kenaf. The activity of mitochondrial complex V and the content of ATP at different stages of anther development of maintainer and restorer line showed that the activity of mitochondrial complex V in anther development stage was lower than that in maintainer line and restorer line. The activity of complex V in mononuclear stage was lower than that in binuclear stage. There was no significant difference in ATP content between maintainer line and restorer line in anther development stage, but there was an increasing trend in binuclear stage in kenaf male sterile line, maintainer line and restorer line. The results showed that the lack of energy supply in the binuclear stage of anther development may be closely related to male sterility.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S563.5

【相似文献】