Diversity analysis of reverse transcriptase gene (RT) of long terminal repeat retrotransposons in Arachis ipaensis Krapov. et W. C. Greg. with BB genome
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摘要: 利用简并PCR技术从野生花生种（Arachis ipaensis Krapov.et W.C.Greg.）的基因组中扩增分离Ty1-copia类（1类）和Ty3-gypsy类（2类）反转录转座子RT基因，并对其序列特征、多样性、系统进化关系及转录活性进行分析。结果显示：对于1和2类RT基因，目的条带分别约为260和430 bp；分离获得了23和32条序列，长度范围分别为262~266、395~435 bp；AT所占比例分别为61.60%~69.17%和55.79%~61.34%；核苷酸序列间相似性分别为52.5%~98.9%和45.0%~98.8%，氨基酸序列间相似性分别为39.8%~100%和9.0%~97.2%。其中2类基因的异质性高于1类；1类和2类基因分别有3条和15条发生了无义突变，2类的无义突变发生率远高于1类。1类基因的保守基序保守性较高，2类的保守基序呈一定程度的变异。代表序列的蛋白质三级结构基本类似。聚类分析结果显示，1类和2类基因可被分为5个和6个家族。1类和2类基因都有部分序列与其他物种的RT基因序列亲缘关系较近，表明它们之间可能存在两类反转录转座子的横向传递。通过比对花生EST数据库，本研究发现1类有1条以及2类有7条序列为具有转录活性的反转录转座子，且2类基因比1类更具有转录活性。Abstract: The reverse transcriptase gene (RT) sequences of Ty1-copia-like (Type 1) and Ty3-gypsy-like retrotransposons (Type 2) were amplified and isolated from the Arachis ipaensis Krapov. et W. C. Greg. with BB genome by degenerate polymerase chain reaction (PCR). Sequence characteristics, diversity, phylogenetic relationships, and transcriptional activity were then analyzed. Results showed that the target bands for types 1 and 2 were 260 bp and 430 bp in size, respectively. 23 sequences of type 1 retrotransposons and 32 sequences of type 2 retrotransposons were obtained. Sequence lengths of type 1 and type 2 ranged from 262 to 266 bp and from 395 to 435 bp, respectively. The proportion of AT content in type 1 and type 2 ranged from 61.60% to 69.17% and from 55.79% to 61.34%, respectively. Similarity between nucleotide sequences in type 1 and type 2 ranged from 52.5% to 98.9% and from 45.0% to 98.8%, respectively. The similarity between amino acid sequences of type 1 and type 2 ranged from 39.8% to 100% and from 9.0% to 97.2%, respectively. The heterogeneity of type 2 was higher than that of type 1. There were three and 15 nonsense mutations in type 1 and type 2, respectively. The incidence of nonsense mutations in type 2 was much higher than that in type 1. The conserved motifs of type 1 were highly conserved, while the conserved motifs of type 2 showed a certain degree of variation. The protein tertiary structures were similar in overall configuration. Type 2 had more differences in protein structure than type 1. Based on cluster analysis, type 1 and type 2 were divided into five and six families, respectively. The phylogenetic tree showed that type 1 and type 2 were divided into four and 11 classes, respectively, but type 2 sequence categories and diversity were significantly higher than that of type 1. Several type 1 and 2 RT gene sequences were closely related to the RT gene sequences of other species, indicating possible horizontal transmission of retrotransposons. When searching the peanut EST database, one type 1 and seven type 2 sequences from the A.ipaensis BB genome showed transcriptional activity. The type 2 retrotransposons showed greater transcriptional activity than the type 1 retrotransposons. This study not only provides sequences for the isolation of full-length LTR retrotransposons and for studies on their transcriptional activity and function, but also lays the foundation for the development of molecular markers based on LTR retrotransposons in the Arachis genus.
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