摘要
特定条件下稳定表达内参基因的筛选是利用实时荧光定量PCR研究基因表达的关键基础。本研究以不同温度胁迫后后的瓜实蝇Bactrocera cucurbitae 2龄幼虫、雌虫和雄虫为试验材料,对其9个候选内参基因表达进行了RT-qPCR分析,并利用ge Norm、Norm Finder、Best Keeper和Ref Finder 4款软件分析各候选内参基因在3种虫态中的表达稳定性。结果表明,18S rRNA基因Ct值(≤5)较小,不适合作为内参基因使用,其它8个内参基因在瓜实蝇2龄幼虫中的稳定性排序为Rp L32> RPS13> TBP>β-tubulin> TUBE> Actin2> G6PDH> Actin3。雌虫表达稳定性由高到低为:RPS13> Rp L32>β-tubulin> TBP> TUBE> Actin2> Actin3> G6PDH。雄虫表达稳定性为Rp L32> RPS13>β-tubulin> TBP> TUBE> Actin2> G6PDH> Actin3。ge Norm软件分析各个虫态内参基因最佳组合均为2个。RPS13和Rp L32组合可作为瓜实蝇不同虫态下温度胁迫后的内参基因。
It was a critical step of selecting some suitable reference genes under specific conditionsin gene expression by qRT-PCR analysis.We used the 2-instar larvae,females and males of Bactrocera cucurbitae(Coquillett)as the experimental material after the different temperature stress,and analyzed the expressions of 9 candidate reference genes.The candidate internal stability of gene expression in 3 different stages had been indentified using geNorm,NormFinder,BestKeeper and RefFinder.The results showed that 18S rRNA was exclude,because its Ct value was less than five.Through the comprehensive analysis of above four software,the stability rank of eight candidate reference gene in 2-instar larva was RpL32>RPS13>TBP>β-tubulin>TUBE>Actin2>G6PDH>Actin3;The expression stability order were RPS13>RpL32>β-tubulin>TBP>TUBE>Actin2>Actin3>G6PDH in female and RpL32>RPS13>β-tubulin>TBP>TUBE>Actin2>G6PDH>Actin3 in male.It showed that the best combination of reference genes was 2 in 3 different stages of B.cucurbitae by the geNorm software.The RpL32 and RPS13 can be use as the reference genes for 2-instar larva,female and male of B.cucurbitae(Coquillett)after the different temperature stress.
作者
王凤英
杨朗
黎柳锋
廖世纯
廖仁昭
姜建军
WANG Feng-Ying;YANG Lang;LI Liu-Feng;LIAO Shi-Chun;LIAO Ren-Zhao;JIANG Jian-Jun(Institute of Plant Protection,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pest,Nanning 530007,China)
出处
《环境昆虫学报》
CSCD
北大核心
2018年第5期1097-1105,共9页
Journal of Environmental Entomology
基金
广西自然科学基金(2015GXNSFBA139072
2018GXNSFAA138068)
广西作物病虫害生物学重点实验室项目(2016-ST-4)
国家自然科学基金(31360438)
