玉米大斑病菌实时荧光定量PCR内参基因的筛选与应用  

Selection and Application of Reference Genes for Quantitative Real-time PCR in Exserohilum turcicum

作　　者：代玉立[1] 刘晓菲 甘林[1] 兰成忠[1] 滕振勇[2] 杨秀娟[1] DAI Yu-Li;LIU Xiao-Fei;GAN Lin;LAN Cheng-Zhong;TENG Zhen-Yong;YANG Xiu-Juan(Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests/Fujian Engineering Research Center for Green Pest Management,Institute of Plant Protection,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China;Fujian Seed Station,Fuzhou 350001,China)

机构地区：[1]福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室/福建省作物有害生物绿色防控工程研究中心,福州350013 [2]福建省种子总站,福州350001

出　　处：《农业生物技术学报》2023年第4期867-882,共16页Journal of Agricultural Biotechnology

基　　金：福建省属公益类科研院所专项(2020R1024003);国家重点研发计划项目(2021YFC2600402);福建省农业科学院“5511”协同创新工程(XTCXGC2021011,XTCXGC2021017);福建省农业科学院农作物重大有害生物灾变机制与绿色防控科技创新团队(CXTD2021002-1)。

摘　　要：由异宗配合的子囊菌大斑凸脐蠕孢(Exserohilum turcicum)引起的大斑病(northern leaf blight)是我国玉米(Zea mays)和高粱(Sorghum bicolor)生产的重要真菌病害。为筛选适用于玉米大斑病菌实时荧光定量PCR(quantitative real-time PCR;qRT-PCR)的内参基因,本研究以转录延伸因子1α(elongation factor-1α,EF-1α)、新生多肽相关复合物α多肽(nascent-polypeptide-associated complex alpha polypeptide,NACA)、60S核糖体蛋白L2(60S ribosomal protein L2,RPL2)、60S核糖体蛋白L37a(60S ribosomal protein L37a,RPL37A)、泛素结合酶(ubiquitin-conjugating enzyme,UBC)、α微管蛋白(α-tubulin,TUBA)、肌动蛋白(actin,ACT)和β微管蛋白(β-tubulin,TUBB)为候选内参基因,以玉米大斑病菌4个不同菌株、3个不同发育阶段、体内侵染4个不同时期以及苯醚甲环唑胁迫4个不同时间的样品为实验材料,通过qRT-PCR技术测定玉米大斑病菌各候选内参基因在上述不同条件下的表达量,利用geNorm(v3.5)、NormFinder(v0.953)、BestKeeper(v1.0)、Delta-CT以及RefFinder(http://blooge.cn/RefFinder/)等分析方法综合评价候选内参基因的表达稳定性。同时,分别以筛选的稳定性较好和较差的内参基因,分析玉米大斑病菌羊毛甾醇14α去甲基化酶(lanosterol 14α-demethylase,CYP51)基因在苯醚甲环唑胁迫下的表达规律。结果表明,8个候选内参基因的扩增效率范围为90.3%~97.8%(r>0.99),熔解曲线峰图均为单一峰,符合qRT-PCR的扩增效率要求。TUBA和TUBB适于做玉米大斑病菌不同菌株间基因表达分析的内参基因;RPL2和TUBA是不同发育阶段表达较稳定的内参基因;NACA和TUBA是体内侵染不同时期较理想的内参基因;ACT适于做苯醚甲环唑胁迫不同时间基因表达分析的内参基因。qRT-PCR分析表明,在苯醚甲环唑胁迫下,玉米大斑病菌CYP51基因的表达量随时间的推移显著增加,胁迫24 h时,表达量达到最大,胁迫36 h表达量显著下调(P<0.05)。以稳定性较�Northern leaf blight caused by the heterothallic ascomycete Exserohilum turcicum,it is an important foliar fungal disease that affects corn(Zea mays)and sorghum(Sorghum bicolor)production in China.To select suitable reference genes for quantitative real-time PCR(qRT-PCR)in E.turcicum,the elongation factor-1α(EF-1α),nascent-polypeptide-associated complex alpha polypeptide(NACA),60S ribosomal protein L2(RPL2),60S ribosomal protein L37a(RPL37A),ubiquitin-conjugating enzyme(UBC),α-tubulin(TUBA),actin(ACT)andβ-tubulin(TUBB)genes were served as candidate reference genes,and the samples from 4 different isolates of E.turcicum,three different stages of development,four different periods of infection in vivo,and 4 different times under difenoconazole stress were used as experimental materials in this study.The qRT-PCR technique was used to measure the expressions of candidate reference genes of E.turcicum under different conditions as mentioned above,and the analytical methods of geNorm(v3.5),NormFinder(v0.953),BestKeeper(v1.0),Delta-CT,and RefFinder(http://blooge.cn/RefFinder/)were used for comprehensive assessment the expression stability of candidate reference genes.Meanwhile,the expression pattern of lanosterol 14α-demethylase(CYP51)gene of E.turcicum under difenoconazole stress was analyzed using the screened most stable and least stable reference gene,respectively.The results showed that the amplification efficiencies of the 8 candidate reference genes ranged from 90.3%to 97.8%(r>0.99),with the melting curve peak being a single peak,which met the requirements of amplification efficiency in qRT-PCR.TUBA and TUBB were suitable for reference genes among different isolates of E.turcicum.RPL2 and TUBA were steadier reference genes at different stages of development.NACA and TUBA were more ideal reference genes for different stages of infection in vivo.ACT was appropriate for reference gene at different times under difenoconazole stress.qRT-PCR analysis indicated that the expression of CYP51 gene of E.turcicum signif

关 键 词：大斑凸脐蠕孢 实时荧光定量PCR 内参基因 扩增效率 表达稳定性 

分 类 号：S432.1[农业科学—植物病理学]