黑皮冬瓜LINE逆转座子RT序列的克隆与特征分析  

Cloning and Sequence Analysis of Reverse Transcriptase of LINE-Retrotransposons in Black Wax Gourd

作　　者：赵芹[1] 谢大森[1] 江彪[1] 何晓明[1] 罗少波[1] 李明珠[1] 

机构地区：[1]广东省农业科学院蔬菜研究所,广东广州510640

出　　处：《热带作物学报》2015年第3期466-473,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学青年基金(No.31311643);广东省自然科学基金(No.10451064001006063);广州市珠江科技新星项目(No.2013086)

摘　　要：根据LINE逆转座子逆转录酶保守序列设计简并引物,PCR扩增黑皮冬瓜“B98K”基因组DNA,获得580 bp左右目的条带。将PCR产物回收、克隆并测序,获得23条逆转酶序列,利用生物信息学软件分析其长度变异、碱基变化、相似性及系统进化关系。结果表明：这些序列长度在557~593 bp区间变异,同源比对核苷酸序列相似性为39.0%~99.3%,存在高度异质性,主要表现为缺失突变、移码突变与终止密码子突变,核苷酸序列聚类分析分为4个家族,家族1与家族2分别包含15和5个成员,占总序列数的65.22%和21.74%。对推导的氨基酸序列分析发现,第12位氨基酸残基处存在一保守的苯丙氨酸（Phe）,多处位置存在半保守氨基酸残基;氨基酸序列相似性在20.0%~99.5%之间;其中8条序列可能具有转录活性,8条与15条序列分别发生移码与终止密码子突变。与已知物种构建系统发育进化树,发现冬瓜LINE逆转座子RT序列较保守,且与拟南芥、李、油菜等有较近的亲缘关系。研究结果为后续利用分子标记研究冬瓜种质遗传变异及基因组进化奠定基础。Degenerate primers were designed according to the RT conserved motifs of LINE retrotransposons to amplify genomic DNA of black wax gourd ＂ B98K＂ for investigating and understanding genetic relationship,characteristics and evolution of LINE retrotransposon families from black wax gourd. The PCR products of 580 bp were obtained and cloned to p MD18-T vector, and the positive clones were screened and sequenced. The full-length variation, base composition, homology and phylogenetic relationship of twenty-three different sequences were analyzed by bio-informatics softwares. The results indicated that length of RT sequences concentrated on the region of 557-593 bp, and sequence similarities varied from 39.0%-99.3%. These sequences showed high heterogeneity which was mainly characterized by deletion, frameshift and termination codon mutation. Four families were divided by alignment analyses of nucleotide sequences, and family1 and family2 contained most members of black wax gourd LINE retrotransposons and accounted for 65.22% and 21.74%, respectively. The analysis results of deduced amino acid sequences showed that there is a conservative phenylalanine（ phe） in the 12 th amino acid residue position of the translated sequences and lots of semi-conservative points appeared in many positions. Eight sequences were found to have transcriptional activity, moreover eight and fifteen sequences presented termination codon and frameshift mutations respectively, and amino acid sequence similarities ranged from 20.0% to 99.5%.The phylogenic tree constructed by these sequences and other RT sequences from Gen Bank showed that RT sequences of LINE retrotransposons in black wax gourd were conservative, and shared high homology with other species like Arabidopsis, plum and rape, etc. This work offers the basis for developing molecular markers to analyze the genetic variability and research the origin and evolution pathways of black wax gourd.

关 键 词：黑皮冬瓜 LINE逆转座子 逆转录酶 序列分析 

分 类 号：S642.3[农业科学—蔬菜学]