SMAD1基因的沉默和过表达及对秦川牛原代成肌细胞生肌的影响  

Silencing and Overexpressing SMAD Family Member 1(SMAD1)Gene and Its Effect on Myogenesis in Primary Myoblast of Qinchuan Cattle(Bos taurus)

作　　者：宁越[1] 米雪 陈星伊 邵建航 昝林森[1,2] NING Yue;MI Xue;CHEN XingYi;SHAO JianHang;ZAN LinSen(College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shanxi;National Beef Cattle Improvement Center, Yangling 712100, Shanxi)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]国家肉牛改良中心,陕西杨凌712100

出　　处：《中国农业科学》2019年第10期1818-1829,共12页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2018YFD0501700);国家肉牛牦牛产业技术体系(CARS-37);陕西省科技统筹创新工程计划(2016KTCL02-15)

摘　　要：【目的】为了探索秦川牛(Bos taurus)SMAD1基因在成肌细胞分化过程中的分子作用机制,通过研究沉默、过表达该基因后对牛原代成肌细胞分化及成肌相关基因表达量的影响,以期为BMP信号通路在牛肌肉组织生长发育过程中的作用机制提供理论依据。【方法】根据Genbank牛SMAD1(NM_001077107.2)已知序列设计并合成靶向沉默SMAD1基因的干扰序列si SMAD1和阴性对照siRNA-NC。以秦川牛原代成肌细胞cDNA为模板,利用PCR技术克隆获得SMAD1基因CDS序列,构建腺病毒过表达穿梭载体pDC316-mCMV-EGFP-b SMAD1。将腺病毒基因组质粒和腺病毒载体穿梭质粒共转染到HEK 293细胞中,通过Cre-loxp重组酶获得重组腺病毒。获得的携带目的基因的腺病毒标记为AD-b SMAD1,重组质粒pDC316-EGFP标记为AD-NC,用做对照组病毒,利用LaSRT法测定腺病毒的滴度。将获得的干扰序列si SMAD1和过表达腺病毒AD-b SMAD1分别侵染秦川牛原代成肌细胞,采用实时荧光定量PCR检测SMAD1基因和成肌标志基因MyoD、Myf5、MyoG的mRNA水平,并观察对细胞分化融合情况的影响。【结果】成功获得了1条能有效沉默SMAD1基因siRNA,将其转染秦川牛原代成肌细胞后进行人工诱导分化,相比对照组,SMAD1基因分别下调了75.4%(诱导1d,P<0.01)、66.7%(诱导3d,P<0.01)、60.0%(诱导6d,P<0.01)、54.7%(诱导9d,P<0.01)。此外,还成功构建了秦川牛SMAD1基因的腺病毒过表达穿梭载体,获得了滴度为1×10^(10)pfu/mL的过表达重组腺病毒AD-b SMAD1。与对照组相比,高滴度过表达病毒AD-b SMAD1侵染秦川牛原代成肌细胞0、1、3、6、9d后,与对照组相比,SMAD1基因的表达水平分别上调了2.10倍(诱导1d)、3.19倍(诱导3d)、105.3倍(诱导3d),P<0.01)和144倍(诱导9d,P<0.01);结合肌管形成过程的变化和实时荧光定量结果证明,SMAD1基因促进原代成肌细胞分化和肌管形成,并显著上调成肌标志基因MyoD、Myf5、MyoG的表达。【结论】【Objective】The aims of the present study were to investigate molecular function of SMAD1 gene in the bovine myoblast cell differentiation and to explore its role in the growth and development of Qinchuan cattle for beef production.【Method】 SMAD1 gene was silenced and overexpressed through RNA interference and adenovirus recombinant over expression vector containing SMAD1. The negative control siRNA-NC and RNA interference specific targeted SMAD1 gene (siSMAD1) were designed and synthesized against the bovine SMAD1 mRNA sequence (CDS), which was obtained by RT-PCR using cDNA of Qinchuan cattle myoblast as template, and then, which was inserted into a shuttle vector pDC316-mCMV-EGFP to construct the over expression of adenovirus shuttle vector pDC316-mCMV-EGFP-bSMAD1. Adenovirus genome plasmid and adenovirus shuttle vectors were cotransfected into HEK293 cell line to pack and obtain recombinant adenovirus. Adenovirus containing target gene was named as AD-bSMAD1, while pDC316-EGFP was used as a reference vector and was considered as control group "AD-NC". Their titers were tested by using LaSRT method. Bovine myoblast were transfected with siSMAD1 and AD-bSMAD1, the mRNA level of SMAD1 gene and myogenesis-related genes, such as MyoD, Myf5 and MyoG, were detected by real-time quantitative PCR, and the impact on myotube formation was observed.【Result】 The mRNA level of SMAD1 gene transfected with siRNA was reduced 75.4%(induced differentiation for 1 day, P<0.01), 66.7%(induced differentiation for 3 days, P<0.01), 60.0%(induced differentiation for 6 days, P<0.01), 54.7%(induced differentiation for 9 days, P<0.01), respectively, compared with the siRNA-NC. The optimum titer of AD-bSMAD1 infectious was found at 1×10^10 pfu/mL. The expression levels of SMAD1 were rapidly increased 2.10 (induced differentiation for 1 day), 3.19 (induced differentiation for 3 days), 105.3 (induced differentiation for 6 days, P<0.01) and 144 (induced differentiation for 9 days, P<0.01) times of the control group after infected

关 键 词：SMAD1基因 RNAI 腺病毒载体 原代成肌细胞 肌管形成 秦川牛 

分 类 号：S823[农业科学—畜牧学]