火龙果溃疡病菌实时荧光定量PCR内参基因的筛选  

Screening of Reference Genes for RT-qPCR in Neoscytalidium dimidiatum

作　　者：姚姿婷 曹雪颖 肖雪 李瑞芳[1] 韦小妹 邹承武[2] 朱桂宁[1] YAO Zi-ting;CAO Xue-ying;XIAO Xue;LI Rui-fang;WEI Xiao-mei;ZOU Cheng-wu;ZHU Gui-ning(Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences,Giuangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China Ministry of Agriculture and Rural Affairs,Nanning 530007;College of Agriculture,Guangxi University,Nanning 530004)

机构地区：[1]广西壮族自治区农业科学院植物保护研究所、广西作物病虫害生物学重点实验室、农业农村部华南果蔬绿色防控重点实验室,南宁530007 [2]广西大学农学院,南宁530004

出　　处：《生物技术通报》2023年第5期92-102,共11页Biotechnology Bulletin

基　　金：广西农业科学院科技发展基金资助项目(桂农科2021ZX28);广西农业科学院基本科研业务专项(桂科农2021YT063)。

摘　　要：火龙果溃疡病是由火龙果溃疡病菌引起的重要真菌病害,近年来在世界火龙果产区的爆发严重影响了火龙果产业的发展。为研究火龙果溃疡病菌响应逆境胁迫的基因表达和功能,需要筛选火龙果溃疡病菌在各种条件下表达稳定的内参基因。以火龙果溃疡病菌菌株LJ02为研究对象,以在马铃薯葡萄糖液体培养基(PDW)培养的菌丝体为对照组,以在LB培养基培养的菌丝体和分别在添加过氧化氢、吡唑醚菌酯和苯醚甲环唑的PDW培养的菌丝体为处理组。利用实时荧光定量PCR技术以及内参基因稳定性评估软件(geNorm、NormFinder、Bestkeeper和RefFinder),评估18个候选内参基因(CYT1、SDH2_1、TBCC、SUI1、RPL19、PPH、ATP5B、UBE2_16、RPL13、UBE2_2、PRS17、SUCLA2、ATP5A、TUB1_2、ACT1、EFTU、EF1A和GAPDH)的表达稳定性。结果显示,火龙果溃疡病菌的18个候选基因在上述培养条件下的Ct值介于14.80-24.66之间,表达水平适中;稳定性最高的内参基因是CYT1,其次为SUI1,GAPDH和ACT1的稳定性最差;最少使用2个内参基因组合可以提高定量PCR分析结果的准确性,此时SUCLA2和ATP5A是最稳定的内参基因组合。该结果可为研究火龙果溃疡病菌生物学过程中的基因表达提供理论依据。The pitaya canker disease is a serious fungal disease caused by Neoscytalidium dimidiatum.In recent years,the outbreak of the pitaya canker disease in the world's pitaya producing areas has seriously affected the pitaya industry development.In order to study the expression level and function of genes responding to stress in N.dimidiatum,it is necessary to screen the reference genes that express stably when the pathogen grows under different cultural conditions.The N.dimidiatum strain LJ02 mycelia cultured in potato dextrose liquid medium(PDW)were taken as the control group,and the mycelia cultured in LB medium,and the mycelia cultured in PDW medium supplemented with hydrogen peroxide,pyrazolyl ester and difenoconazole,respectively,were taken as the treatment groups.The expression stabilities of 18 candidate reference genes(CYT1,SDH2_1,TBCC,SUI1,RPL19,PPH,ATP5B,UBE2_16,RPL13,UBE2_2,PRS17,SUCLA2,ATP5A,TUB1_2,ACT1,EFTU,EF1A and GAPDH)were evaluated by real-time quantitative PCR(RT-qPCR)and stability evaluation software(geNorm,NormFinder,Bestkeeper and RefFinder).Based on the results of RT-qPCR and software analysis,it was found that the Ct values of 18 candidate genes were within 14.80-24.66 under different culture conditions,with moderate expression levels.The reference gene with the highest stability was CYT1,followed by SUI1,while GAPDH and ACT1 were with the worst expression stability.Using at least two reference genes may improve analysis accuracy of RT-qPCR.At this time,SUCLA2 and ATP5A is the most stable reference gene combination.The results provide a theoretical basis for gene expression analysis in the biological process of N.dimidiatum.

关 键 词：火龙果溃疡病菌 内参基因 实时荧光定量PCR 菌丝生长 表达分析 非生物胁迫 内参基因分析软件 

分 类 号：S66[农业科学—果树学]