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小菜蛾PGRP基因的克隆及其在昆虫细胞中的表达

发表时间:2012-10-15  浏览量:1341  下载量:625
全部作者: 许小霞,王瑞晓,孙强,李林妙,徐英杰,高钢,黄婉君,王爽,金丰良
作者单位: 华南农业大学资源环境学院
摘 要: 肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是一类可识别肽聚糖和含肽聚糖的细菌模式识别受体,在天然免疫应答中发挥着重要的识别和调节功能。利用同源克隆结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术首次克隆了小菜蛾(Plutella xylostella L.)PGRP的cDNA 全长序列(命名为PxPGRP),GenBank登录号为EU399240. 序列分析表明:PxPGRP基因开放阅读框(open reading frame,ORF)为588 bp,编码195个氨基酸,分子量为21.0 ku,等电点为8.38,成熟肽的分子量为19.0 ku,等电点为8.86. 为了解PxPGRP时空表达模式,利用半定量逆转录PCR(reverse transcription-PCR, RT-PCR)检测PxPGRP在不同龄期的表达情况。结果表明,PxPGRP在小菜蛾不同发育阶段均有表达,但在4龄幼虫中的转录水平较高;利用实时荧光定量(real-time quantitatire PCR, real-time PCR)检测PxPGRP在小菜蛾4龄不同组织的转录情况,结果表明:PxPGRP在小菜蛾4龄幼虫不同组织中的转录水平存在显著差异,表达量从高到低依次为表皮、马氏管、脂肪体、血细胞和中肠。为进一步研究PxPGRP结构和功能的关系,将PxPGRP基因和PEGFP-C3(+)质粒连接,转染Sf9昆虫细胞,利用荧光倒置显微镜观察,结果表明:PxPGRP基因在Sf9细胞中得到了高效融合表达。研究结果为PxPGRP的结构和功能研究奠定了基础,也为小菜蛾的生物防治提供了新的靶标。
关 键 词: 植物保护学;小菜蛾;肽聚糖识别蛋白;克隆;细胞表达�
Title: Cloning and cellular expression of peptidoglycan recognition protein from diamond back moth (Plutella xylostella L.)
Author: XU Xiaoxia, WANG Ruixiao, SUN Qiang, LI Linmiao, XU Yingjie, GAO Gang, HUANG Wanjun, WANG Shuang, JIN Fengliang
Organization: College of Natural Resources and Environment, University of South China Agricultural
Abstract: Peptidoglycan recognition proteins (PGRPs) are one of the pattern recognition receptors which can recognize the peptidoglycan or a class of bacterial containing the peptidoglycan. They play an important role in the regulation and recognition of innate immune response. In this paper, a full length cDNA sequence encoding PGRP was cloned from Plutella xylostella L. by homology and rapid amplification of cDNA ends (RACE) technology (GenBank accession No. EU399240, defined as PxPGRP). The analysis of sequence showed that PxPGRP contains an open reading frame (ORF) of 588 bp, encoding 195 amino acids, with the predicted MW of 21 ku and pI of 8.38, while the mature protein encoding with the predicted MW of 19.04 ku and pI of 8.86. The temporal and partial expression profiles of PxPGRP were investigated by semi-quantitative PCR and real-time quantitative PCR. The analysis of semi-quantitative RT-PCR showed that PxPGRP could be expressed at each instar stage, especially with higher expression in 4th instar and pupae of P. xylostella. The analysis of real time PCR showed that the mRNA transcription level PxPGRP was tissue specific, and the level was from high to low in proper order of epidermis, malpighian tubules, fat body, hemocyte and midgut. In order to obtain recombinant protein and lay a basis of structural and functional study of PxPGRP, the ORF of PxPGRP was ligated into pEGFP-C3(+). The converted fluorescence microscopy confirmed that PGRP had a higher expression in Sf9 cells after transfection. The successful expression of PxPGRP in cells not only lays a foundation for the structural and functional study, but also provides a new target for the biological control of P. xylostella.�
Key words: plant protection; Plutella xylostella; peptidoglycan recognition proteins; clone; cellular expression
发表期数: 2012年10月第19期
引用格式: 许小霞,王瑞晓,孙强,等. 小菜蛾PGRP基因的克隆及其在昆虫细胞中的表达[J]. 中国科技论文在线精品论文,2012,5(19):1850-1857.
 
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