不同地理种群二化螟Ty3/gypsy反转座子天冬氨酰蛋白酶(AP)基因序列的克隆与分析  

Molecular cloning and analysis of aspartic protease( AP) gene in Ty3 /gypsy retrotransposon in different geographical populations of Chilo suppressalis( Lepidoptera: Pyralidae) in China

作　　者：李晓欢[1,2] 罗光华[1] 韩召军[2] 方继朝[1,2] 

机构地区：[1]江苏省农业科学院植物保护研究所,南京210014 [2]南京农业大学植物保护学院,南京210095

出　　处：《昆虫学报》2014年第5期530-537,共8页Acta Entomologica Sinica

基　　金：国家水稻产业技术体系项目(Cars-001-25);国家自然科学基金项目(31201505);江苏省农业科技自主创新资金项目CX(13)5025

摘　　要：【目的】Ty3/gypsy反转座子是广泛存在于生物体内的一类反转座子。反转座子上的天冬氨酰蛋白酶(aspartic protease,AP)基因是反转座子发生转座所需的一个重要基因。但由于该基因家族成员间变异较大,较难利用简并引物克隆得到该基因,所以对该基因家族成员的研究很少。【方法】本研究采用PCR方法克隆了二化螟Ty3/gypsy反转座子的AP基因序列,并对其序列特征和地理种群变异进行了分析。【结果】克隆获得的二化螟Chilo suppressalis(Walker)Ty3/gypsy反转座子中的AP基因具有独立的开放阅读框(open reading frame,ORF),长528 bp,编码的蛋白含175个氨基酸残基(GenBank登录号:KF886014)。Conserved Domain Search在线工具分析显示,该蛋白中含有一个特异的Asp_protease_2保守功能域。从7个二化螟不同地理种群中共克隆获得70份AP基因拷贝。对同一基因座位上的AP序列多重比对分析,发现共存在46处碱基替换,其中碱基转换(transition)有31处,碱基颠换(transversion)有15处,70份拷贝中有69份拷贝是完整的ORF,能编码完整的蛋白。从碱基替换形式看,A→G的变异形式出现最多,有15处;其次是T→C的变异形式,有11处;其余的变异形式都很少。对比这7个不同地理种群,没有发现碱基的替换存在明显的地理区划差异。【结论】碱基的替换形式与二化螟所处的地理区域无明显相关性。本研究对于认识反转座子序列的变异特点有所帮助。[ Aim] Ty3/gypsy is a retrotransposon that is widespread in many organisms. Aspartic protease （AP） gene is important for the retrotransposon transposition. Since the AP gene is difficult to be clonedbased on degenerate primers because the members of AP family have low similarities, there are few researches about AP genes. [ Methods] The AP gene in Ty3/gypsy retrotransposon was cloned from Chilosuppressalis （Walker） with PCR method, and its sequence characteristics and variations in the Chinese populations were analyzed. [ Results ] The AP gene has a single open reading frame （ORF） of 528 bpencoding a protease of 175 amino acids （GenBank accession no. ： KF886014）. The Conserved Domain Search analysis showed that there is a conserved Asp_protease_2 domain in this AP protein. Seventy copies of APgene were cloned from 7 field populations. Multiple sequences alignment revealed that there are 46 single-base substitutions including 31 transitions and 15 transversions. Sixty-nine of 70 copies have intact ORF. The basechanges of A→G （15） and T→C （11） occur more frequently than the others. [ Conclusion] There is no significant relationship between the patterns of base substitutions and the distribution area of C. suppressals.This study will be helpful to investigate the variation features of retrotransposon.

关 键 词：二化螟 反转座子 Ty3 GYPSY 天冬氨酰蛋白酶 基因克隆 序列分析 

分 类 号：Q966[生物学—昆虫学]