摘要
【目的】基于黄脊竹蝗若虫不同龄、成虫不同性别及不同组织筛选稳定表达的内参基因,为黄脊竹蝗后续转录调控研究提供参考。【方法】依据黄脊竹蝗转录组测序结果,以TUB、RPS27A、RPL10、AK、GAPDH、EF1A、RPS3、Actin和18S rRNA常用的9种昆虫内参基因作为候选,本地Blast后获得碱基序列并设计引物。利用GeNorm、NormFinder和BestKeeper软件分析这9种内参基因的稳定性,筛选出在若虫不同龄、成虫不同性别与不同组织(触角、头、唇须、肌肉组织、精/卵巢)中表达量最为稳定的内参基因。【结果】9种候选内参基因在黄脊竹蝗体内均为首次验证并报道,且与其他昆虫相应基因同源性高(高于70%),差异值小;9种候选内参基因的引物均具有良好的扩增效率(均在90%~120%);在所有样品中,18S rRNA表达水平最高(Ct=10.78±2.58,P<0.05),Actin表达水平差异值最大(Ct=23.55±3.84,P<0.05)。黄脊竹蝗不同龄若虫的内参基因稳定性分析时,3个软件排在前2位的内参基因均为RPS3与RPL10;基于不同组织分析时,GeNorm与BestKeeper软件表明RPL10是最合适内参基因,而NormFinder软件则表明GAPDH为最合适内参基因;在黄脊竹蝗成虫不同性别分析时,3种软件评估结果均表明GAPDH和RPL10为合适内参基因。【结论】筛选出RPL10为在不同条件下于黄脊竹蝗体内表达稳定并具有差异性的基因,可作为黄脊竹蝗转录组学及功能基因组学研究的内参基因。
【Objective】To provide a reference for further transcriptomics studies of the yellow-spined locust,Ceracris kiangsu,the reference genes were screened based on different instars,different genders of adults and different tissues of C.kiangsu.【Method】According to the transcriptome sequencing result,nine internal reference genes commonly used in insects,TUB,RPS27A,RPL10,AK,GAPDH,EF1A,RPS3,Actin,and 18S rRNA were chosen as candidates.The base sequences were obtained,and primers were designed after local Blast.GeNorm,NormFinder and BestKeeper were used to analyze the stability of the nine reference genes,and then the most suitable reference genes were screened according to the expression levels in different instars,genders of aduits,and tissues(antennae,head,labial palp,muscle tissue,testis/ovary)of adults.【Result】Nine candidate reference genes of C.kiangsu were verified and reported for the first time,and they showed high homology(more than 70%)with the corresponding genes of other insects.The primers for the nine candidate reference genes all had good amplification efficiency(within 90%-120%).In all samples,the expression level of 18S rRNA was the highest(Ct value=10.78±2.58,P<0.05),and the difference in Actin expression level was the largest(Ct=23.55±3.84,P<0.05).In analyzing the stability of the reference genes of C.kiangsu at different stages of nymphs,the top two of the three software were RPS3 and RPL10.Based on the analysis of different tissues,RPL10 was the most suitable reference gene suggested by GeNorm and BestKeeper,while GAPDH was most suitable one according the analysis of NormFinder.In terms of the analysis of different genders of the C.kiangsu adults,GAPDH and RPL10 were proved to be the two most suitable reference genes by the three software.【Conclusion】RPL10 with stable and different expression under various conditions in C.kiangsu has bee screened as reference gene,which can be used as an internal reference gene for the research of transcriptomics and functional genomics for C.kiangsu.
作者
方林鑫
李志红
张守科
张威
舒金平
王浩杰
Fang Linxin;Li Zhihong;Zhang Shouke;Zhang Wei;Shu Jinping;Wang Haojie(Research Institute of Subtropical Forestry, CAF Hangzhou 311400)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2022年第1期70-77,共8页
Scientia Silvae Sinicae
基金
国家自然科学基金面上项目(31670660)。
