Selection and Application of Reference Genes for Quantitative Real-time PCR in Exserohilum turcicum
DAI Yu-Li1, LIU Xiao-Fei1, GAN Lin1, LAN Cheng-Zhong1, TENG Zhen-Yong2, YANG Xiu-Juan1*
1 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; 2 Fujian Seed Station, Fuzhou 350001, China
Abstract：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) and β-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 significantly increased over time under difenoconazole stress, with the highest expression at 24 h, while CYP51 was significantly down expressed at 36 h (P<0.05). When a more stabile gene of ACT was used as the internal reference gene, the expression of CYP51 gene of E. turcicum was significantly up-regulated under difenoconazole stress compared with those of the non-stress group (P<0.05). However, an opposite result of the CYP51 gene expression level of the 2 isolates was obtained when the unstable TUBB gene was served as the reference gene. This study affords an effective correction tool for quantitative analysis of gene expression in E. turcicum, ensures the accuracy of quantitative analysis of gene expression, and provides a reliable reference for further study on the overexpression of fungicide resistant genes in E. turcicum.
代玉立, 刘晓菲, 甘林, 兰成忠, 滕振勇, 杨秀娟. 玉米大斑病菌实时荧光定量PCR内参基因的筛选与应用[J]. 农业生物技术学报, 2023, 31(4): 867-882.
DAI Yu-Li, LIU Xiao-Fei, GAN Lin, LAN Cheng-Zhong, TENG Zhen-Yong, YANG Xiu-Juan. Selection and Application of Reference Genes for Quantitative Real-time PCR in Exserohilum turcicum. 农业生物技术学报, 2023, 31(4): 867-882.
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