梨小食心虫实时荧光定量PCR内参基因的筛选

Screening of reference genes for real-time quantitative PCR in Grapholita molesta(Lepidoptera:Tortricidae)

【目的】本研究旨在探明梨小食心虫Grapholita molesta在不同发育阶段、成虫不同组织以及不同浓度的3种杀虫剂处理后成虫中稳定表达的内参基因,为后续对梨小食心虫目的基因表达的研究奠定基础。【方法】基于梨小食心虫转录组数据筛选10个候选内参基因(β-actin,18S rRNA,β-tubulin,EF-1α,RPL13,RPL32,RSPL40,UBC7,α-tubulin和RPS20);通过实时荧光定量PCR(qRT-PCR)测定候选内参基因在梨小食心虫不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、成虫不同组织(头、前肠、中肠、后肠、脂肪体、马氏管、精巢和卵巢)以及不同浓度的3种杀虫剂(阿维菌素:19.819,72.897和179.663μg/mL;吡虫啉:17.638,163.323和762.986μg/mL以及高效氯氟氰菊酯:33.791,96.123和198.282μg/mL)通过玻璃管药膜法处理后成虫中的表达量;利用geNorm,NormFinder,ΔCt,BestKeeper和RefFinder对10个候选内参基因的表达稳定性进行评价。选择梨小食心虫细胞色素P450基因CYP354A 32进行验证。【结果】结合qRT-PCR结果和软件评价结果表明,梨小食心虫不同发育阶段内参基因表达稳定性从高到低依次为β-tubulin,18S rRNA,EF-1α,RPL13,β-actin,RPS20,UBC7,RPL32,α-tubulin和RSPL40;成虫不同组织内参基因表达稳定性从高到低依次为UBC7,β-tubulin,β-actin,18S rRNA,RSPL40,EF-1α,RPS20,RPL13,RPL32和α-tubulin;不同浓度阿维菌素、吡虫啉和高效氯氟氰菊酯处理后成虫中内参基因表达稳定性从高到低依次为RPS20,RPL13,β-tubulin,β-actin,RPL32,RSPL40,EF-1α,UBC7,α-tubulin和18S rRNA。用所获得的内参基因组合对CYP354 A2表达特性进行的分析结果表明,用β-tubulin,18S rRNA和EF-1α组合作为内参基因时CYP354 A2在高龄幼虫及成虫中表达量较高,用UBC7,β-tubulin和β-actin组合作为内参基因时在成虫精巢和卵巢中有较高表达,且用RPS20,RPL13和β-tubulin组合作为内参基因时不同浓度杀虫剂处理后仅有19.819μg/mL阿维菌素处理时CYP354 A2表达量高于对照,其余浓度杀虫剂处理时CYP354 A2表达量均低于对照。【结论】梨小食心虫不同发育阶段目的基因表达的研究推荐使用β-tubulin,18S rRNA和EF-1α组合作为内参基因;梨小食心虫成虫不同组织目的基因表达的研究推荐使用UBC7,β-tubulin和β-actin组合作为内参基因;不同浓度阿维菌素、吡虫啉和高效氯氟氰菊酯处理梨小食心虫成虫后目的基因表达的研究推荐使用RPS20,RPL13和β-tubulin组合作为内参基因。

【Aim】The aim of this study is to find out the reference genes stably expressed in different developmental stages and adult tissues of Grapolita molesta and in its adults after treatment with different concentrations of three insecticides,so as to lay a foundation for the subsequent study on target gene expression in G.molesta.【Methods】Ten candidate reference genes(β-actin,18S rRNA,β-tubulin,EF-1α,RPL13,RPL32,RSPL40,UBC7,α-tubulin and RPS20)were selected based on G.molesta transcriptome data.The expression levels of the candidate reference genes in different developmental stages(egg,1st-5th instar larvae,pupa and adult)and different adult tissues(head,foregut,midgut,hindgut,fat body,Malpighian tubules,testis and ovary)of G.molesta and in G.molesta adults treated with three insecticides at different concentrations(avermectin:19.819,72.897 and 179.663μg/mL;imidacloprid:17.638,163.323 and 762.986μg/mL;and lambda-cyhalothrin:33.791,96.123 and 198.282μg/mL)with the method of residual film in glass tube were detected using real-time quantitative PCR(qRT-PCR).The expression stabilities of the 10 candidate reference genes were evaluated by geNorm,NormFinder,ΔCt,BestKeeper and RefFinder.The cytochrome P450 gene CYP354A 32 of G.molesta was selected for validation.【Results】qRT-PCR results combined with software evaluation results revealed that the expression stabilities of the reference genes in different developmental stages of G.molesta were ranked in a descending order ofβ-tubulin,18S rRNA,EF-1α,RPL13,β-actin,RPS20,UBC7,RPL32,α-tubulin and RSPL40,those in different adult tissues were ranked in a descending order of UBC7,β-tubulin,β-actin,18S rRNA,RSPL40,EF-1α,RPS20,RPL13,RPL32 andα-tubulin,and those in adults after exposure to different concentrations of avermectin,imidacloprid and lambda-cyhalothrin were ranked in a descending order of RPS20,RPL13,β-tubulin,β-actin,RPL32,RSPL40,EF-1α,UBC7,α-tubulin and 18S rRNA.The expression characteristics of CYP354 A2 analyzed by using the obtained reference gene combinations showed that CYP354 A2 was highly expressed in the old larvae and adults when using the combination ofβ-tubulin,EF-1αand 18S rRNA as the reference genes,and was highly expressed in the testis and ovary of adults when using the combination of UBC7,β-tubulin andβ-actin as the reference genes.After G.molesta adults were exposed to different concentrations of insecticides,only the expression level of CYP354 A2 in the treatment with 19.819μg/mL avermectin was higher than that in the control,while the expression levels of CYP354 A2 in treatments with other concentrations of insecticides were lower than that in the control when using the combination of RPS20,RPLB andβ-tubulin as the refernece genes.【Conclusion】The combination ofβ-tubulin,18S rRNA and EF-1αis recommended as the reference genes for studying the target gene expression in different developmental stages of G.molesta,that of UBC7,β-tubulin andβ-actin as the reference genes for studying the target gene expression in different adult tissues of G.molesta and that of RPS20,RPL13 andβ-tubulin as the reference genes for studying the target gene expression in adults of G.molesta after treatment with different concentrations of avermectin,imidacloprid and lambda-cyhalothrin.