亚洲玉米螟pdp1的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2013.20.010

摘要/Abstract

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（protease- activated 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。

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

Abstract: 【Objective】The objectives of this study are to clone pdp1 (protease-activated receptor-domain protein1 gene) which named Ofpdp1 (GenBank accession number: KC857457) from the Asian corn borer, Ostrinia furnacalis (Guenée), and to identify its typical hallmarks, then to analyze the mRNA expression characteristics and physiological function of Ofpdp1. It will provide potential target gene for genetic control of O. furnacalis.【Method】 The cDNA sequence of Ofpdp1 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 Ofpdp1 was determined by qPCR. RNAi experiment was performed at proembryo stage to explore the physiological function. 【Result】The cDNA sequence (960 bp) of OfPDP1 was obtained, including a complete open reading frame (ORF) of 804 bp, which encoded 267 amino acids. OfPDP1 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 h of eggs’ development process. It could be declined by 71% in the transcript levels of Ofpdp1 in the eggs injected with Ofpdp1-specific dsRNA in 48 h compared to those in the control eggs. 【Conclusion】 A complete ORF of Ofpdp1 was sequenced with clone strategy from Asian corn borer for the first time. Ofpdp1 was highly conserved in the process of evolution, and it belonged to PAR bZIP subfamily. The high levels expression of Ofpdp1 at the time of 72 h suggested that Ofpdp1could take an important part in the changing process of embryo physiological structure during the larva’s forming period.

Key words: Ostrinia furnacalis , ofpdp1 , gene cloning , phylogenetic analysis , mRNA expression , RNA interference

程晓娟12, 严善春1, 黄勇平2, 谭安江2. 亚洲玉米螟pdp1的克隆及功能分析[J]. 中国农业科学, 2013, 46(20): 4272-4283.

CHENG Xiao-Juan-12, YAN Shan-Chun-1, HUANG Yong-Ping-2, TAN An-Jiang-2. Cloning and Functional Analysis of pdp1 in Ostrinia furnacalis (Lepidoptera: Crambidae)[J]. Scientia Agricultura Sinica, 2013, 46(20): 4272-4283.

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