摘要
为发掘棉花细胞质雄性不育相关基因,采用RFLP分子标记、Northern blot、同源克隆及RT-qPCR的方法,对离核木棉细胞质雄性不育系‘07-113A’及其保持系‘07-113B’进行线粒体差异基因发掘和表达分析。结果表明:1)以12个线粒体基因为探针,结合EcoRⅠ和HindⅢ2种限制性内切酶对不育系和保持系进行RFLP分析,发现atpA/EcoRⅠ及ccmB/EcoRⅠ2个基因/酶组合存在RFLP多态性;2)以atpA和ccmB为探针,对‘07-113A’和‘07-113B’进行Northern blot分析,检测到atpA、ccmB在不育系和保持系中转录本大小均相同;3)获得atpA的CDS全长及其部分侧翼序列,比对结果表明,不育系和保持系的atpA编码序列相同;与保持系相比,不育系5′端侧翼序列有2个碱基的变异,3′端侧翼序列有4个碱基的变异;4)RT-qPCR分析发现,不育系中atpA在败育后的表达量是保持系的0.44倍,极显著低于保持系。推测‘07-113A’花粉败育可能与atpA的异常表达相关。
To explore cotton cytoplasmic male sterility(CMS) genes,the mitochondrial differential genes and their expression levels between CMS line ‘07-113 A’ and its maintainer line ‘07-113 B’ were compared by RFLP,Northern blot,homologous cloning and Real-time quantitative PCR(RT-qPCR) analyses.The results showed that:1) A total of twelve mitochondrial genes were used as probes and digested with EcoR Ⅰ and Hind Ⅲ restriction endonuclease in RFLP analysis.Among which,only atpA/EcoR Ⅰ and ccmB/EcoR Ⅰ combinations displayed polymorphism;2) The Northern blot analysis of atpA and ccmB genes revealed same transcript size between the two lines;3) The CDS sequence of atpA and its partial flanking sequences were cloned from the CMS line and its maintainer line.The results indicated that there was no difference between the sterile line and the maintainer line in the coding sequences,while there were 2 bp variations at the 5′ flanking sequence and 4 bp variation at the 3′ flanking sequence of the sterile line compared with the maintainer line.4) RT-qPCR analysis showed that the expression of the atpA in the CMS line was 0.44,relatively to the control ‘07-113 B’(P<0.01).It is speculated that CMS line(‘07-113 A’) pollen abortion may be associated with abnormal expression of atpA.
作者
李斌
郑杰
孔祥军
李敏
廖小芳
周步进
李枝玲
周瑞阳
LI Bin;ZHENG Jie;KONG Xiangjun;LI Min;LIAO Xiaofang;ZHOU Bujin;LI Zhiling;ZHOU Ruiyang(College of Agriculture,Guangxi University,Nanning 530004,China;Guangxi Colleges and Universities Key Laboratory of Plant Genetics and Breeding,Nanning 530004,China)
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2019年第11期59-69,共11页
Journal of China Agricultural University
基金
国家自然科学基金(31360348)
广西研究生教育创新计划资助项目(YCBZ2017014).
