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【目的】褐飞虱Nilaparvata lugens(Stl)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-actin2,β-actin3,28S rRNA,18S rRNA和α-2-tubulin 6个内参基因的表达量,用geN orm和BestK eeper软件分析确定最稳定表达的内参基因,并检测热胁迫后hsp70和hsp90基因在处理褐飞虱成虫体内的表达模式。【结果】geN orm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-actin1=β-actin3>28S rRNA>α-2-tubulin>18S rRNA>β-actin2;在雄虫体内为:β-actin1=β-actin3>α-2-tubulin>28S rRNA>18S rRNA>β-actin2。BestK eeper软件分析结果显示,在热胁迫的雌、雄虫体内β-actin1均最稳定,18S rRNA次之,β-actin2最不稳定。两种软件分析结果基本一致。以β-actin1为校正内参基因,荧光定量PCR分析hsp70和hsp90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hsp90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-actin1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。
【Objective】 Nilaparvata lugens (Stl) is one of the major pests that damage rice. Temperature is one of the major environmental factors affecting its outbreak and migration. The aim of this study was to investigate the regulation of heat shock protein gene expression in N. lugens adapted to high temperature stress. 【Method】 The female and male N. lugens were treated with high temperature (30 ℃ -40 ℃) for 1 h and 2 h, respectively. The levels of β-actin 1, β-actin2, β-actin3 and 28S rRNA, 18S rRNA and α-2-tubulin, and the most stable expression of the reference genes was determined by geNorm and BestK eeper software. The expression of hsp70 and hsp90 genes in adult BPH adults after heat stress mode. 【Result】 The results of geN orm software analysis showed that the stability of the internal reference genes of BPH in heat stress was β-actin1 = β-actin3> 28S rRNA> α-2-tubulin> 18S rRNA> β-actin2. The body of the worm was: β-actin1 = β-actin3> α-2-tubulin> 28S rRNA> 18S rRNA> β-actin2. The results of BestK eeper software showed that β-actin1 was the most stable, 18S rRNA second, and β-actin2 the most unstable in both heat-stressed female and male. The two software analysis results are basically the same. The expression of hsp70 and hsp90 under different heat stress conditions was analyzed by using β-actin1 as the internal control gene. The results showed that there was no significant difference between hsp70 expression and the control at 26 ℃. The expression pattern of hsp90 gene was up-regulated by high temperature, and the highest expression of hsp90 in female and male was treated at 40 ℃ and 38 ℃ for 2 h, respectively. 【Conclusion】 The β-actin1 gene can be used as a corrective reference gene for the gene expression pattern analysis of brown planthopper (BPH) in heat stress. The hsp90 gene of BPH can be induced by high temperature, which may play an important role in the process of BPH stress adaptation.