辣椒Ty1-copia类逆转座子逆转录酶序列的克隆及分析

Cloning and Sequence Analysis of Reverse Transcriptase of Ty1-copia-like Retrotransposons in Capsicum annuum

以辣椒属5个栽培种叶片为试材,利用Ty1-copia类逆转座子逆转录酶的保守序列设计简并引物,PCR扩增后均获得了260bp左右的目的条带。对C.annuum的扩增产物进行纯化、克隆和测序,经Mega4和DNAstar软件分析后,获得25条逆转录酶序列,核苷酸序列长度变化范围为225～272bp,这些序列存在高度的异质性,主要表现为缺失突变,同源性范围为23.1%～93.2%,核苷酸聚类分为7个组。翻译成氨基酸后,有9条序列存在1～3个不同程度的终止密码子突变,4条序列存在移框突变。将这25条逆转录酶的氨基酸序列与已发表的不同物种同一类型逆转座子的逆转酶序列进行聚类分析,表明辣椒的Ty1-copia型逆转座子与烟草、矮牵牛、马铃薯有很近的关系。

Five cultivars belonging to five Capsiucm species were employed as experimental materials and the degenerate oligonucleotide primers were designed based on the conserved domains of the Ty1-copia retrotrans-poson reverse transcriptase in this research.The 260 bp fragments were amplified by PCR from five tested species of Capsiucm.One of the amplicons were purified from C.annuum to be cloned and sequenced.Twenty-five different sequences of reverse transcriptase were obtained from C.annuum after analysis by Mega4 and DNAstar softwares.These nucleotide sequences showed high heterogeneity mainly characterized by deletion mutation.The length of the nucleotide sequences varied from 225 bp to 272 bp,and homology ranged from 23.1% to 93.2%.Seven groups were clustered by alignment analyses of these nucleotide sequences.The deduction of amino acids revealed that nine sequences presented stop codon mutation with one base to three bases and four sequences presented frame shift mutation.In this research we confirmed that the Ty1-copia-like retrotransposon reverse transcriptase in Capsicum annuum have a closer relationship with those in Nicotiana tabacum,Petunia hybrida and Solanum tuberosum based on cluster and alignment analyses of the reverse transcriptase among the twenty-five sequences and other sequences from other species.