RNAi技术介导大豆蚜hsp70基因对其内源物质及激素表达的影响

Effect of RNAi mediates hsp70 gene on the expression of endogenous substances and hormones in Aphis glycines

通过siRNA hsp70干扰大豆蚜后,分析其体内hsp70基因、热休克蛋白70、内源物质和激素表达变化,寻找siRNA干扰效果最明显时间点。利用RNAi技术介导、Real-time qPCR技术、试剂盒,研究分析siRNA hsp70(0.33μg·μL^-1)干扰2龄大豆蚜后1、2、3、12、24、48、72 h等处理,其体内hsp70基因、热休克蛋白70、内源物质和激素变化情况。结果表明,siRNA hsp70干扰2龄大豆蚜3 h时,hsp70基因表达量极显著低于清水对照组(P<0.01),显著低于阴性对照组(P<0.05),热休克蛋白70表达量、总糖含量、保幼激素含量极显著低于清水对照组和阴性对照组(P<0.01),羧酸酯酶活性、谷胱甘肽S-转移酶活性、蜕皮激素含量极显著高于清水和阴性对照组(P<0.01)。可知,siRNA hsp70(0.33μg·μL^-1)干扰2龄大豆蚜在选测的7个时间处理中,3 h时效果最明显,此时大豆蚜体内各物质含量皆受影响,是开展大豆蚜药剂防治最佳时间点。

The aim of this study was to find the optimal time point of siRNA hsp70 interfering with Aphis glycines by analyzing the expression change of hsp70 gene,heat shock protein 70,endogenous substances and hormones in Aphis glycines during the interfering.This article mainly used RNAi mediated method,real-time q PCR and kits to study the variation of hsp70 gene,heat shock protein 70,endogenous substances and hormones in the 2 nd instar Aphis glycines interfered by siRNA hsp70(0.33μg·μL^-1)with seven durations,that was 1,2,3,12,24,48 and 72 h,respectively.The results showed that when using si RNA hsp70 interfered with Aphis glycines for 3 h,the expression of hsp70 was extremely significantly inhibited compared with the water control(P<0.01)and significantly inhibited compared with the negative control(P<0.05),meanwhile,the expression of the heat shock protein 70,the content of the total sugar and juvenile hormone were extremely significantly inhibited compared with the water control and the negative control(P<0.01),while the carboxylesterase activity,glutathione S-transferase activity and ecdysone content were extremely significantly promoted(P<0.01).The effect of siRNA hsp70(0.33μg·μL^-1)interfered with the 2 nd instar Aphis glycines became the most obvious in 3 h duration treatment among the seven selected treatments.In this duration treatment,the content of various substances in Aphis glycines were affected,so that,it was presumed that 3 h duration was the best time point for Aphis glycines control.