黑尾叶蝉NcPGRP基因克隆及表达模式分析  

Molecular Cloning and Expression Pattern Analysis of Peptidoglycan Recognition Protein Gene in the Green Leafhopper,Nephotettix cincticeps(Hemiptera:Cicadellidae)

作　　者：黄天宇[1,2] 方琦[2] 王桂荣[1] 林克剑[1] 叶恭银[2] 

机构地区：[1]植物病虫害生物学国家重点实验室/中国农业科学院植物保护研究所,北京100193 [2]浙江大学昆虫科学研究所/农业部农业昆虫学重点实验室,杭州310058

出　　处：《中国生物防治学报》2017年第3期304-312,共9页Chinese Journal of Biological Control

基　　金：国家"973计划"(2014CB138404)

摘　　要：为明确黑尾叶蝉肽聚糖识别蛋白(Nephotettix cincticeps peptidoglycan recognition protein,Nc PGRP)的功能,利用RT-PCR克隆长度为552 bp的Nc PGRP开放阅读框,其编码蛋白大小为19.9 k D,无信号肽,含3个锌离子结合/酰胺酶催化位点,1个二氨基庚二酸型(Dap型)肽聚糖识别位点。系统发育分析表明,Nc PGRP与其他昆虫亲缘关系较远。利用显微注射技术对黑尾叶蝉进行病原细菌诱导,定量PCR结果显示Nc PGRP在受大肠杆菌及藤黄微球菌刺激后,其转录水平显著上调。组织分布研究表明,Nc PGRP的m RNA在各组织中均有表达,且头部表达量最高。黑尾叶蝉取食感染水稻普通矮缩病毒RDV的水稻后,Nc PGRP转录水平受抑制。本文研究表明,Nc PGRP是一种诱导型表达的PGRP,黑尾叶蝉感染RDV能显著抑制其转录,进而削弱黑尾叶蝉免疫能力,这可能有利于RDV与黑尾叶蝉共生。To determine the function of peptidoglycan recognition protein in Nephotettix cincticeps （NcPGRP）, we cloned the open reading frame cDNA nucleotide sequence of NcPGRP. The sequence is 552 bp in length, encoding a 19.9 kD protein but without signal peptide. NcPGRP has three Zn2＋-binding and amidase catalyzing sites and one DAP-type peptidoglycan recognition site. Polygenetic tree analysis did not cluster NcPGRP and PGRPs of other species into one group. The real-time PCR results indicated that transcription level of NcPGRP was up regulated after injection of Escherichia coli DH5α and Micrococcus luteus. Expression of NcPGRP mRNA could be detected in all tissues including head, gut, fat body, Malpighian tubes, ovary, testis and cuticle, but was the highest in the head. The transcription level of NcPGRP was down regulated in RDV-infected planthopper. The results suggest that RDV-infected N. cincticeps exhibits a lower resistance to pathogenic bacteria infection, which may be conductive to the symbiosis between N. cincticeps and RDV.

关 键 词：黑尾叶蝉 肽聚糖识别蛋白 水稻普通矮缩病毒 表达模式 免疫 

分 类 号：S43[农业科学—农业昆虫与害虫防治]