亚洲玉米螟pdp1的克隆及功能分析  

Cloning and Functional Analysis of pdp1 in Ostrinia furnacalis(Lepidoptera:Crambidae)

作　　者：程晓娟[1,2] 严善春[1] 黄勇平[2] 谭安江[2] 

机构地区：[1]东北林业大学林木遗传育种国家重点实验室,哈尔滨150040 [2]中国科学院上海生命科学研究院植物生理生态研究所昆虫科学研究中心/中国科学院昆虫发育与进化生物学重点实验室,上海200032

出　　处：《中国农业科学》2013年第20期4272-4283,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31272037)

摘　　要：【目的】克隆获得亚洲玉米螟(Ostrinia furnacalis)蛋白酶激活受体类转录因子pdp1(proteaseactivated receptor-domain protein1)(Ofpdp1)cDNA序列(GenBank登录号:KC857457),明确其基因结构特征,并进一步研究该基因的表达特性及其在生长发育过程中的作用,为亚洲玉米螟的遗传控制提供潜在的靶标基因。【方法】采用RT-PCR和PCR技术,克隆Ofpdp1 cDNA序列,利用相关软件进行生物信息学分析;利用qPCR(quantitative real-time polymerase chain reaction)研究卵期及幼虫期Ofpdp1表达特征;在原胚期利用RNA干扰技术(RNAi)研究其生物学功能。【结果】克隆得到基因Ofpdp1,全长为960 bp,开放阅读框804 bp,编码267个氨基酸,同源比对发现OfPDP1 C-末端与其它昆虫PAR bZIP(basic leucine zipper)转录因子非常相似,含有3个高度保守的结构域,系统发育分析的分类结果与生物学上物种分类相吻合。qPCR结果显示Ofpdp1的mRNA表达量在卵发育76 h左右存在显著的表达高峰。在亚洲玉米螟原胚期,注射Ofpdp1的双链RNA(dsRNA)48 h后,Ofpdp1 mRNA表达量被沉默了71%。【结论】成功地从亚洲玉米螟中克隆得到Ofpdp1,Ofpdp1进化非常保守,属于PAR bZIP亚家族;Ofpdp1可能在幼虫定型时期胚胎生理结构的变化过程中起到重要作用。[ Objective ] The objectives of this study are to clone pdpl （protease-activated receptor-domain protein 1 gene） which named Ofpdpl （GenBank accession number： KC857457） from the Asian corn borer, Ostriniafurnacalis （Guen6e）, and to identify its typical hallmarks, then to analyze the mRNA expression characteristics and physiological function of Ofpdpl. It will provide potential target gene for genetic control of O. furnacalis. [Method] The cDNA sequence of Ofpdpl was isolated using RT-PCR and PCR methods. Based on the sequencing results, the bioinformatics analysis of nucleic acid and putative amino acid was conducted. The developmental expression pattern of Ofpdpl was determined by qPCR. RNAi experiment was performed at proembryo stage to explore the physiological function. [Result] The cDNA sequence （960 bp） of Of PDP1 was obtained, including a complete open reading frame （ORF） of 804 bp, which encoded 267 amino acids. Of PDP1 was a transcription factor of PAR bZIP （basic leucine zipper） subfamily which included several conserved regions in the carboxy terminal region of the protein. The result of phylogenetic analysis was consistent with the classification of the species in biology. The qPCR results showed a high level expression at the 76 hof eggs＇ development process. It could be declined by 71% in the transcript levels of Ofpdpl in the eggs injected with Ofpdpl-specific dsRNA in 48 h compared to those in the control eggs. [ Conclusion] A complete ORF of Ofpdpl was sequenced with clone strategy from Asian corn borer for the first time. Ofpdpl was highly conserved in the process of evolution, and it belonged to PAR bZIP subfamily. The high levels expression of Ofpdpl at the time of 72 h suggested that Ofpdplcould take an important part in the changing process of embryo physiological structure during the larva＇s forming period.

关 键 词：亚洲玉米螟 Ofpdp1 基因克隆 系统发育分析 MRNA表达 RNA干扰 

分 类 号：S435.132[农业科学—农业昆虫与害虫防治]