辣椒不育系和保持系线粒体差异基因获得及SNP分析

Acquisition of different gene of pepper Cytoplasmic Male Sterile and maintainer line and Single Nucleotide Polymorphism analysis

通过对辣椒细胞质雄性不育(CMS)系及其相应保持系育性相关基因的分析,检寻SNP位点,旨在探索其与辣椒细胞质雄性不育性的关系。以高盐-蛋白酶K法提取线粒体DNA,采用AFLP技术分析了辣椒不育系和保持系的mtDNA的差异,从128对引物扩增产物中发现了不育系和保持系的差异片段,经回收、克隆获得不育系特异片段CanLE长度140 bp,保持系特异片段CanLB长度126 bp。利用Blast对其进行序列比对分析,其中CanLE与细胞色素P450家族蛋白同源,CanLB和赖氨酰-tRNA合成酶同源。进一步分别克隆并获得了不育系和保持系辣椒细胞色素P450全长,序列分析发现,两者存在单核苷酸多态性(SNP),其中5个碱基差异,3个为同义突变,2个氨基酸发生改变。

To explore the relationship between Pepper CMS and its parent line,the fertility and SNP sites for Pepper CMS and its maintained line were investigated.Capsicum mitochondrial DNA（mtDNA） was extracted from leaves of cytoplasmic male sterile line and its maintainer line based on high salt proteinase K method in the study.Amplified Fragment Length Polymorphsim（AFLP） technique was applied to compare the mtDNA difference between CMS line and maintainer of pepper.The specific fragments were detected in the products amplified by 128 primers.The size of the specific fragments was around 126 and 140 bp.Sequencing analysis by using BLAST program showed that the fragment CanLE is similar to cytochrome P450 and the fragment CanLB is related to lysyl-tRNA synthetase.The full length of cytochrome P450 gene from pepper was cloned.Five SNP mutations was discovered by comparing the sequences of the CMS and its maintainer line.Three nucleotide variations didn＇t cause the changes of amino acids,thus they were synonymy mutations.However the rest of the two nucleotide variations resulted in the changes of their corresponding amino acids.