快速冷驯化条件下稻水象甲内参基因的筛选及LoTPS基因表达  

Screening of Reference Genes and Analysis of LoTPS Gene Expression in Lissorhoptrus oryzophilus under Rapid Cold Hardening

作　　者：祁立中 王晓 王庆泰 王尚[1] 张炬红[1] QI Lizhong;WANG Xiao;WANG Qingtai;WANG Shang;ZHANG Juhong(College of Plant Science,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学植物科学学院,吉林长春130062

出　　处：《华北农学报》2023年第3期176-183,共8页Acta Agriculturae Boreali-Sinica

基　　金：吉林省自然科学基金项目(20200201128JC)。

摘　　要：为筛选出适合快速冷驯化条件下稻水象甲成虫荧光定量PCR的内参基因,并分析快速冷驯化低温胁迫下海藻糖合成酶基因(LoTPS)的表达模式,从稻水象甲转录组数据库中筛选出10个具有完整开放阅读框的待选内参基因,以稻水象甲成虫为试验材料,通过实时荧光定量PCR测定内参基因在不同快速冷驯化条件下的表达水平,通过常用的内参基因稳定性分析方法ΔCt法和geNorm、NormFinder和BestKeeper等软件综合评价待选的10个内参基因表达的稳定性,测定稻水象甲海藻糖合成酶基因在不同快速冷驯化条件下的表达变化,分析其与低温的相关性。试验结果表明,在ΔCt法分析中RPS18表达丰度最高,geNorm软件分析中较稳定表达的内参基因是RPS18和G6DPH,G6DPH在NormFinder软件分析中是表达最稳定的内参基因,BestKeeper软件分析表明,RPL13和α-tubulin表达较稳定,综合以上各分析方法得出RPS18基因在快速冷驯化条件下的稳定性较高。以RPS18为内参基因进一步分析LoTPS基因在快速冷驯化条件下的表达模式发现,LoTPS基因表达量在快速冷驯化条件下有所增加,各冷驯化处理均高于对照。因此,内参基因RPS18在稻水象甲成虫体内表达相对稳定,可以作为稻水象甲不同快速冷驯化条件下的内参基因,低温胁迫促进了在快速冷驯化条件下海藻糖合成酶基因的上调表达,且随着冷驯化时间的延长而表达量增加。To screen out the stable reference gene for Real-time quantitative PCR under rapid cold hardening conditions,ten candidate reference genes from the transcriptome sequencing of Lissorhoptrus oryzophilus Kuschel were selected.Real-time quantitative PCR was used to obtain the expression levels of the selected candidate reference genes in adults under different rapid cold hardening conditions.The expression stability of the selected candidate reference genes was analyzed byΔCt method,geNorm,NormFinder and BestKeeper softwares.ΔCt predicted the most stable candidate reference gene was RPS18.It was revealed that RPS18 and G6DPH were the most stable reference genes based on the geNorm analysis,while G6DPH,RPL13 andα-tubulin were the most stable reference genes based on the NormFinder analysis and BestKeeper analysis,respectively.The stability of RPS18 gene was relatively high based on comprehensive analysis.It was found that the expression level of LoTPS gene increased under rapid cold acclimation when RPS18 was used as an internal reference gene,and it was higher in all the cold acclimation treatments than control.Therefore,RPS18 was relatively stable and could be used as an internal reference gene under different rapid cold acclimation conditions.Low temperature stress promoted the up-regulation of LoTPS gene under rapid cold acclimation,and the expression level increased with the prolongation of cold acclimation time.

关 键 词：稻水象甲 快速冷驯化 内参基因 海藻糖合成酶 基因表达 

分 类 号：S435.11[农业科学—农业昆虫与害虫防治]