AA野生种花生Ty1-copia类反转座子RT基因的克隆与序列分析

Cloning and sequence analysis of reverse transcriptase genes of Ty1-copia-like retrotransposons in wild peanut species with AA genome

关键词:  花生, Ty1-copia类反转录转座子, 反转录酶, 野生种, 异质性

Key words:  peanut, Ty1-copia-like retrotransposons, reverse transcriptase(RT), wild species, heterogeneity

摘要:

该研究旨在克隆Ty1-copia类反转录转座子RT(reverse transcriptase)基因,为分离花生属Ty1-copia类反转录转座子全长序列和研究其功能提供序列基础。根据RT基因的保守区设计简并引物,以两份AA染色体组野生种花生Arachis duranensis为试材,利用PCR扩增其基因组DNA,回收、克隆和测序目的条带后,对所获得序列进行生物信息学分析。结果表明:(1)目的条带大小约为260 bp,分别从两份野生种花生材料中克隆到41条和27条RT基因序列,68条序列的长度变化范围为256 ～270 bp,AT所占比例范围为55.86%～68.42%,AT与GC比例范围为1.27～2.17,核苷酸序列间相似性范围为49.8%～99.2%,存在较高异质性。(2)68条序列被分为6个家族,家族Ⅰ和Ⅳ为主要家庭。(3)68条序列中的19条发生了无义突变,A. duranensis(PI219823)要比A. duranensis(PI262133)的无义突变率高。(4)氨基酸序列间相似性为4.7%～100%,呈现高度异质性。(5)各家族中代表序列的蛋白质三级结构在整体构型上一致,但在螺旋结构数、折叠结构数、转角数和氢键数上存在较大差别。(6)序列间保守基序总体一致,但也存在一定变异,呈现一定异质性; 系统进化树将68条序列分为10类,大部分序列都聚在A和B两大类中。(7)另有部分AA染色体组野生种花生的RT基因序列与其他物种植物的RT基因序列亲缘关系较近,推测不同物种植物间可能发生过Ty1-copia类反转录转座子的横向传递。该研究为花生属基于Ty1-copia类反转录转座子的新分子标记开发和应用奠定基础。

Abstract:

The purpose of this study was to clone the reverse transcriptase(RT)genes of Ty1-copia-like retrotransposons, and to provide sequences basis for isolating the full-length sequences of Ty1-copia-like retrotransposons and studying their function in genus Arachis. Degenerate primers were designed according to the conserved region of RT genes, the genomic DNA of wild peanut species Arachis duranensis with AA genome was amplified by PCR using the degenerated primers. The amplified targeted bands were recovered, cloned and sequenced, and then the sequences were analyzed through bioinformatics analysis. The results were as follows:(1)The amplified targeted bands were all about 260 bp in size. Forty-one and twenty-seven RT genes sequences were cloned from the two wild peanut species respectively. The length of sixty-eight sequences varied from 256 to 270 bp. The proportion of AT, AT/GC and the similarity between nucleotide sequences ranged from 55.86% to 68.42%, 1.27 to 2.17, and 49.8% to 99.2% respectively, showing a higher heterogeneity.(2)The sixty-eight sequences were divided into six families, Family I and Family IV were the main families.(3)Nineteen of sixty-eight sequences had nonsense mutations, and Arachis duranensis (PI219823)had a higher nonsense mutations rate than Arachis duranensis (PI262133).(4)The similarity between amino acid sequences ranged from 4.7% to 100%, showing a high heterogeneity.(5)The tertiary structures of proteins representing sequences in each family were basically similar in overall configuration, but there were great differences in the number of helix structures, folding structures, turns and hydrogen bonds.(6)The conserved motifs among sequences were generally consistent, but there were also some variations, showing a certain degree of heterogeneity. The phylogenetic tree divided sixty-eight sequences into ten classes. Most of sequences were clustered in A and B classes.(7)Some of RT genes sequences from two wild peanut species with AA genome were closely related to RT genes sequences from other plant species, which indicated that there might be transposon horizontal transmission between them. This study provides reference for the development and application of new molecular markers based on Ty1-copia-like retrotransposons in genus Arachis.