美国白蛾HcGST-E1的克隆及基于RNAi的功能探究
Cloning and functional analysis of the Hyphantria cunea glutathione-S-transferase gene, HcGST-E1
高琳娜，陈麒羽，孟 香，陈 敏
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DOI：10.7679/j.issn.2095-1353.2023.003
作者单位：北京林业大学林木有害生物防治北京市重点实验室，北京 100083
中文关键词：美国白蛾；谷胱甘肽-S-转移酶；HcGST-E1 基因；RNA 干扰；槲皮素
英文关键词：Hyphantria cunea; glutathione-S-transferase; HcGST-E1; RNAi; quercetin
中文摘要：
【目的】 谷胱甘肽-S-转移酶（GSTs）是昆虫体内一种重要的解毒酶。基于美国白蛾Hyphantria cunea中肠转录组数据筛选鉴定了 HcGST-E1 为显著相应槲皮素诱导的差异表达基因。为进一步探究该基因的功能，我们克隆获得HcGST-E1全长序列，并通过RNA干扰（RNA interference，RNAi）技术验证了HcGST-E1在美国白蛾幼虫响应槲皮素过程中的作用，为明确美国白蛾对寄主植物中的次生物质槲皮素的解毒适应机制提供科学依据。【方法】 基于HcGST-E1的核酸序列设计引物，克隆HcGST-E1的全长序列并合成 dsHcGST-E1，通过显微注射法用 dsHcGST-E1注射美国白蛾4龄幼虫沉默HcGST-E1基因，进而分析了该基因的沉默效率以及沉默后的美国白蛾4龄幼虫取食槲皮素对体重、存活率等生理指标的影响。【结果】 dsHcGST-E1可有效抑制 HcGST-E1基因表达，相同处理时间内，注射1 000 ng的dsHcGST-E1 比注射 500 ng的dsHcGST-E1沉默效率高，沉默效率最高为62.3%。用0.5%和1.0%槲皮素人工饲料分别饲喂 HcGST-E1沉默的美国白蛾幼虫，10 d后其存活率分别为40.00%和33.33%，显著低于对照组的73.33%和66.67%（P < 0.05）和空白组的86.67%和80.00%（P < 0.05）。注射dsHcGST-E1后的美国幼虫体重在处理时间内无明显波动，体重增长量均低于注射无菌水的对照组和未经注射的空白组。其中，0.5%槲皮素饲养的实验组美国白蛾幼虫的体重与对照组幼虫体重差异不显著，与空白组在第 6 天差异显著（P < 0.01）；1.0%槲皮素饲养的实验组幼虫的体重低于对照组幼虫的体重但差异不显著，与空白组在第4天和第6天差异显著（P < 0.05）。【结论】 HcGST-E1 沉默后，美国白蛾幼虫对槲皮素的适应能力下降，生长发育受到影响，表明 HcGST-E1 参与了美国白蛾对槲皮素解毒代谢过程。研究结果为阐明美国白蛾对含槲皮素的寄主植物的适应机制奠定基础。
英文摘要：
[Objectives] To clone the Hyphantria cunea glutathione S-transferases (GST) gene (HcGST-E1 ) and verify the role of this gene in the response of H. cunea to quercetin, a common secondary substance in host plants. [Methods] HcGST-E1 was cloned based on the quercetin-induced midgut transcriptome of H. cunea and real-time, quantitative PCR results. The gene was then silenced using RNAi technology and the effect of this on the weight and survival of quercetin-treated 4th instar larvae investigated. [Results] The HcGST-E1 was successfully cloned and silenced. dsHcGST-E1 effectively inhibited the expression of HcGST-E1 with a maximum silencing efficiency of 62.3%. Over the same period of time, 1 000 ng of dsHcGST-E1 was more effective than 500 ng dsHcGST-E1. The survival rates of HcGST-E1 silenced larvae that had been fed an artificial diet containing either 0.5% or 1.0% quercetin were 40.00% and 33.33%, respectively, significantly lower than their respective control groups（73.33% and 66.67%; P < 0.05）or blank control groups（86.67% and 80.00%; P < 0.05）. The body weight of HcGST-E1 silenced H. cunea larvae did not fluctuate significantly, and their weight was lower than that of larvae in both respective, and blank, control groups. Larvae in the 5% quercetin treatment group were not significantly lighter than those in the respective control group, but were significantly lighter than those in the blank control groups on day 6 (P < 0.01). Similarly, larvae in the 1.0% quercetin treatment group were not significantly lighter than those in the respective control group, but were significantly lighter than those in the blank control groups on days 4 and 6 (P <0.05). [Conclusion] Silencing the HcGST-E1 gene increased the susceptibility of H. cunea larvae to quercetin, retarding their growth and development. This indicates that HcGST-E1 plays a role in the detoxification and metabolization of quercetin. These results provide a basis for further research on the role of GST genes in the resistance of H. cunea to plant secondary compounds.