四倍体野生种花生Ty3-gypsy类反转录转座子RT基因的分离及序列分析

Isolation and Sequence Analysis of Reverse Transcriptase of Ty3-gypsy-like Retrotransposons from Arachis monticola

本研究从四倍体野生种花生中分离了Ty3-gypsy类反转录转座子的反转录酶(RT)基因序列,分析了其序列特点和差异,为研究其转录活性和功能奠定基础。根据Ty3-gypsy类反转录转座子RT基因的保守区设计简并引物,PCR扩增四倍体野生种花生(Arachis monticola)(PI468199)的DNA,目的条带经回收、克隆和测序后,进行生物信息学分析。结果显示,目的条带大小约为430 bp,共有29条AmRT2-X基因序列被分离出,这些序列长度变化范围为397~433 bp,AT所占比例范围为55.66%~65.97%,核苷酸序列间相似性在45.1%~97.2%之间,存在较高异质性;29条AmRT2-X基因序列被聚类划分为8个家族,家族A是包含序列最多的家族;翻译成氨基酸后,有10条AmRT2-X基因序列发生了无义突变;氨基酸序列间相似性在22.3%~98.6%之间,呈现高度异质性;绝大部分AmRT2-X基因序列的保守基序一致;系统进化树显示,所有RT基因序列被分为6类,其中,Ⅰ类包含20条AmRT2-X基因序列,Ⅱ类中的4条Am RT2-X基因序列与来自拟南芥、多花黑麦草、火龙果、牡丹、果梅、枣、山桑、白皮松等物种的序列之间亲缘关系较近;通过比对花生EST数据库,发现2条具有转录活性的四倍体野生种花生Ty3-gypsy类反转录转座子。本研究为Ty3-gypsy类反转录转座子全长序列的分离及其功能的研究提供一定基础。

The reverse transcriptase sequences of Ty3-gypsy-like retrotransposons were isolated from tetraploid wild peanut species,and their sequences characteristics and differences were analyzed,which will lay a foundation for studying their transcriptional activity and function.The genomic DNA of tetraploid wild peanut species(Ara-chis monticola)(PI468199)was amplified using a pair of degenerate primers designed according to the conservative region of reverse transcriptase of Ty3-gypsy-like retrotransposons.Then the target band was recovered,cloned and sequenced.Finally,the obtained sequences were analyzed through bioinformatics analysis.The results showed that the size of the target band was approximately 430 bp.Twenty-nine AmRT2-X sequences were obtained successfully.The length of sequences ranged from 397 bp to 433 bp.The proportion of AT content ranged from 55.66%to 65.97%.The similarity between nucleotide sequences ranged from 45.1%to 97.2%.These indicated that the nucleotide sequences of reverse transcriptase existed higher heterogeneity.The twenty-nine AmRT2-X sequences were clustered into eight families.Family A was the main component of Ty3-gypsy-like retrotransposons.After being translated into amino acids,ten AmRT2-X sequences had nonsense mutations.The similarity between amino acids ranged from 22.3%to 98.6%.These indicated that the amino acids sequences of reverse transcriptase existed high heterogeneity.The conservative motifs of most AmRT2-X sequences were identical.The evolutionary tree showed that all RT gene sequences were classified into six categories.Among them,classⅠcontained twenty AmRT2-X sequences.ClassⅡcontained four AmRT2-X sequences,which had close relationship with Arabidopsis,ryegrass,pitaya,peony,mume,jujube,mulberry and pine.Two Ty3-gypsy-like retrotransposons from tetraploid wild peanut species with transcriptional activity were found by aligning with peanut EST database.The reverse transcriptase sequences obtained in this study will provide a basis for further isolating full length sequences and studying their function.