›› 2015, Vol. 58 ›› Issue (5): 479-486.doi:

• 研究论文 • 上一篇    下一篇

褐飞虱热胁迫下内参基因的筛选及热激蛋白基因表达分析(英文)

姜建军, 黄立飞, 陈红松, 杨朗*   

  1. (广西农业科学院植物保护研究所, 广西作物病虫害生物学重点实验室, 南宁 530007)
  • 出版日期:2015-05-20 发布日期:2015-05-20
  • 作者简介:姜建军, 男, 1980年生, 河南周口人, 博士, 助理研究员, 研究方向为昆虫生态与分子生物学, E-mail: jiangjianjun8008@126.com

Identification of reference genes and expression analysis of  heat shock protein genes in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), after exposure to heat stress(In English

JIANG Jian-Jun, HUANG Li-Feng, CHEN Hong-Song, YANG Lang*   

  1. (Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pest, Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China)
  • Online:2015-05-20 Published:2015-05-20

摘要: 【目的】褐飞虱Nilaparvata lugens (Stål)是为害水稻的重要害虫之一,温度是影响其暴发、迁飞的主要环境因子之一。本研究旨在探讨研究褐飞虱对高温胁迫适应性的热激蛋白基因表达调控模式。【方法】分别以不同的高温(30℃~40℃)处理褐飞虱雌、雄虫1 h和2 h,利用荧光定量PCR技术检测其体内的β-actin 1, β-actin 2, β-actin 3, 28S rRNA, 18S rRNA和α-2-tubulin 6个内参基因的表达量,用geNorm 和BestKeeper软件分析确定最稳定表达的内参基因,并检测热胁迫后hsp 70和hsp 90基因在处理褐飞虱成虫体内的表达模式。【结果】geNorm软件分析结果表明,热胁迫后褐飞虱内参基因稳定性在雌虫体内为:β-actin 1=β-actin 3>28S rRNA>α-2-tubulin>18S rRNA>β-actin2;在雄虫体内为:β-actin1=β-actin 3>α-2-tubulin>28S rRNA>18S rRNA>β-actin 2。BestKeeper软件分析结果显示,在热胁迫的雌、雄虫体内 β-actin 1均最稳定,18S rRNA次之,β-actin 2最不稳定。两种软件分析结果基本一致。以β-actin 1为校正内参基因,荧光定量PCR分析hsp 70和hsp 90在不同热胁迫条件下的表达模式,结果表明,各高温处理下hsp70表达量与对照26℃下的表达量没有显著性差异;而hsp 90基因表达模式表现为被高温诱导上调表达,在雌、雄虫体内表达量达到最高的处理条件分别为40℃和38℃处理2 h。【结论】β-actin 1基因可以作为热胁迫下褐飞虱雌雄虫体内基因表达模式分析的校正内参基因使用。褐飞虱 hsp90基因能被高温诱导表达,该基因可能在褐飞虱适应热胁迫过程中起着重要的作用。

Abstract: 【Aim】 The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a serious rice pest in China and Southeast Asia. The occurrence and migration of N. lugens is thought to be related to temperature. This study was conducted to understand the expression patterns of heat shock protein genes (hsps) in the adaptation to temperature stress in N. lugens. 【Methods】 Female and male N. lugens adults were exposed to high temperature (30℃-40℃) for 1 h and 2 h, respectively. Real-time PCR was used to detect the expression of β-actin 1, β-actin 2, β-actin 3,28S rRNA, 18S rRNA and α-2-tubluin in their bodies. The most stable candidate reference gene was identified using geNorm and BestKeeper software. The expression levels of hsp70 and hsp90 genes in the treated N. lugensadults were measured using RT-qPCR. 【Results】 The most stable reference gene in both female and male adults of N. lugens after exposure to heat stress was β-actin 1. The expression levels of hsp70 after heat stress ranging from 30℃ to 40℃ in both female and male adults were not significantly different compared with those in the control group. The expression level of hsp 90 displayed significant up-regulation and reached the highest levels in female adults and male adults exposed to 40℃ and 38℃ for 2 h, respectively. 【Conclusions】β-actin 1 can be used as the reference gene for normalization of gene expression under high temperature stress in N. lugensadults. The expression of hsp 90 is induced by heat shock and the over-expression of hsp 90 might be involved in the enhancement of thermal tolerance in N. lugens adults.

Key words: Nilaparvata lugens, heat shock protein, expression pattern, reference gene, qRT-PCR