Evaluation of Endogenous Reference Genes of Bactrocera (Tetradacus) minax by Gene Expression Profiling under Various Experimental Conditions
Keywords:
Chinese citrus fruit fly, developmental stages, PCR primer sequence reference gene, stable expression, γ-irradiation, temperature stressAbstract
Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is widely used for gene expression analysis in living organisms, and stably expressed endogenous reference genes are needed to obtain accurate results. Some commonly used reference genes varied among different experimental treatments. To obtain suitable reference gene for specific experimental conditions, the stability of 10 reference genes of Bactrocera (Tetradacus) minax (Enderlein) were evaluated in various development stages, and under temperatureand γ-irradiation-stress conditions by semi-quantitative PCR. The present results indicated that the most stable candidate reference gene was RPL32 in eggs, 3rd instars, 1-, 90- and 160-day-pupae and newly emerged female and male adults (< 24 h), and under different stress conditions, i.e., 35°C temperature stress for 0 h, 1 h, 3 h and 5 h, and 4°C temperature stress for 0 h, 12 h, 36 h, 48 h and 60 h. GAPDH, G6PDH and RPL32 were ideal candidate endogenous genes under 35°C temperature stress for 0 h, 1 h, 3 h and 5 h, and under 4°C temperature stress for 0 h, 12 h, 36 h, 48 h and 60 h, and under a Γ irradiation stress of 90 Gy. These results provide basic information for future studies of gene expression in B. minax, and should serve as a resource to screen reference genes for gene expression studies in other insect species.
La transcripcion inversa de la reaccion de la polimerasa en cadena (RT-qPCR) cuantitativa en tiempo real es ampliamente utilizada para el analisis de la expresion de genes en los organismos vivos, y los genes de referencia endogenos expresados de forma estable son necesarios para obtener resultados precisos. Algunos de los genes de referencia utilizados variaron entre los diferentes tratamientos experimentales. Para obtener genes de referencia adecuados para condiciones experimentales especificas, la estabilidad de 10 genes de referencia de Bactrocera (Tetradacus) minax (Enderlein) fueron evaluados en diferentes estadios de desarrollo, y bajo temperaturas y condiciones de estres por irradiacion-γ por medio del metodo de PCR semi - cuantitativo. Los resultados indican que el candidato del gene de referencia mas estable fue RPL32 en los huevos, tercer estadios, pupas de 1, 9 y 160 dias y adultos recien emergidos de ambos sexos (< 24 h) y bajo diferentes condiciones de estres, como el estres por temperatura de 35°C durante 0 h, 1 h, 3 h y 5 h, y el estres de temperatura de 4°C durante 0 h, 12 h, 36 h, 48 h y 60 h. Los GAPDH, G6PDH y RPL32 fueron candidatos ideales de genes endogenos bajo estres de temperatura de 35°C durante 0 h, 1 h, 3 h y 5 h, y bajo 4°C de estres de temperatura por 0 h, 12 h, 36 h, 48 h y 60 h y bajo estres de irradiacion- Γ 90 Gy. Estos resultados proveen informacion basica para estudios futuros de expresion de genes en B. minax y estos serviran como un recurso para detectar genes de referencia para los estudios de expresion de genes en otras especies de insectos.
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