鸡肌肉组织实时荧光定量PCR及Western杂交内参基因的筛选

Identification of Reference Genes by Real-Time Quantitative PCR and Western Blot in Chicken Muscle Tissues

本研究对5种常用"持家基因"作为鸡肌肉组织发育早期基因和蛋白检测内参基因的有效性进行了评价,为鸡肌肉发育相关分子检测中内参基因的选择提供科学依据。应用实时荧光定量PCR(RT-qPCR)和Western blot技术,分别检测了鸡12胚龄、17胚龄、1日龄和14日龄胸肌中β肌动蛋白(ACTB)、β微管蛋白(TUBB)、TATA结合蛋白(TBP)、甘油醛-3-磷酸脱氢酶(GAPDH)和热休克蛋白70(HSP70)的mRNA和蛋白表达水平。结果表明,在鸡发育早期胸肌组织中,仅HSP70mRNA和蛋白在各阶段间表达水平差异不显著,GAPDH、TUBB、TBP和ACTB的mRNA和蛋白表达水平在检测的各个时间点间均差异显著或极显著(P<0.05或P<0.01)。HSP70更适合作为鸡肌肉发育早期基因和蛋白检测的内参基因。本结果为研究发育期鸡肌肉组织相关功能基因和蛋白提供了必要的方法学基础,对其他物种的相关研究也有重要借鉴意义。

This research evaluated the availability of 5'housekeeping genes'as reference genes for gene expression detection in different early developmental stages of muscle tissues in chicken,which would provide scientific evidences for selecting reference genes in the molecules detection related to muscle development of chicken.Real-Time quantitative PCR(RT-qPCR)and Western blot were used to test the mRNA and protein levels ofβ-actin(ACTB),β-tubulin(TUBB),TATA-binding-protein(TBP),Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and Heat shock 70 kDa protein(HSP70)in breast muscle of chicken at embryonic day 12 and 17,day 1and day 14post-hatch,respectively.The results showed that,only mRNA and protein levels of HSP70didn't significantly change in the early developmental stages of breast muscle in chicken,while that of other genes significantly altered(P<0.05 or P<0.01).HSP70 was more suitably used as reference gene for gene or protein expression detection in the early developmental stages of muscle tissues in chicken.This study provides methodological basis for functional genes and proteins studies of muscle development in chicken.It also could play a referential role in other species.