朱红毛斑蛾实时荧光定量PCR内参基因的筛选

Screening of reference genes for quantitative real-time PCR in Phauda flammans(Walker)(Lepidoptera:Phaudidae)

筛选朱红毛斑蛾Phauda flammans(Walker)在不同成虫组织、性别及发育阶段处理条件下稳定表达的内参基因,为进一步开展朱红毛斑蛾相关基因的定量研究提供参考。本研究以不同成虫组织(头、胸、腹、足、翅和触角)、不同成虫性别和不同发育阶段(卵、幼虫、蛹和成虫)为实验材料,对10个候选内参基因进行实时荧光定量PCR(qRT-PCR),并使用GeNorm、NormFinder和BestKeeper软件及RefFinder网站对候选内参基因的表达稳定性进行评价和综合分析。结果表明:在朱红毛斑蛾不同成虫组织基因定量研究中,TUB2>GAPDH>TUB1>AK>EF1α>ACTIN3>TBP>TUB3>ACTIN2>RPL32,建议以TUB2和GAPDH作为内参基因;在不同成虫性别基因定量研究中,TUB1>EF1α>ACTIN3>RPL32>ACTIN2>TUB2>AK>GAPDH>TUB3>TBP,建议以TUB1和EF1α作为内参基因;在不同发育阶段基因定量研究中,ACTIN3>TBP>TUB1>EF1α>TUB3>ACTIN2>GAPDH>RPL32>TUB2>AK,建议以ACTIN3和TBP作为内参基因。基于GeNorm分析,最佳内参基因使用数目为2个。

This study aims to screen and verify stably expressed genes under given conditions as reference genes for quantitative real-time PCR(qRT-PCR)in Phauda flammans(Walker),which can provide the basis for following experiments on gene quantification of this moth.Based on transcriptomics sequencing results in P.flammans and reference genes reported in other lepidopteran species,10 genes were selected as candidate reference genes.Then the primers of qRT-PCR were respectively designed,and their expression levels in different adult tissues(head,thoraxe,abdomen,leg,wing and antenna),sexes,and different developmental stages(egg,larva,pupa and adult)were tested by qRT-PCR.The expression stability of candidate genes were evaluated by GeNorm,NormFinder and BestKeeper softwares and RefFinder website.According to the comprehensive rank of RefFinder,the expression stability of above ten candidate reference genes in different adult tissues were TUB2>GAPDH>TUB1>AK>EF1α>ACTIN3>TBP>TUB3>ACTIN2>RPL32;the expression stability of above candidate reference genes in different adult sexes were TUB1>EF1α>ACTIN3>RPL32>ACTIN2>TUB2>AK>GAPDH>TUB3>TBP;the expression stability of above candidate reference genes in different developmental stages were ACTIN3>TBP>TUB1>EF1α>TUB3>ACTIN2>GAPDH>RPL32>TUB2>AK.Three pairs of genes(TUB2 and GAPDH,TUB1 and EF1α,ACTIN3 and TBP),were recommended as reference genes to study on the gene quantification of different adult tissues,sexes and developmental stages in P.flammans,respectively.