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

doi:10.3864/j.issn.0578-1752.2019.10.014

摘要/Abstract

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siSMAD1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-bSMAD1。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-bSMAD1,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siSMAD1和过表达腺病毒AD-bSMAD1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 ¹⁰pfu/mL的过表达重组腺病毒AD-bSMAD1。与对照组相比,高滴度过表达病毒AD-bSMAD1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】获得了能有效沉默秦川牛原代成肌细胞SMAD1表达的特异性的干扰序列siSMAD1,以及可成功实现过表达SMAD1基因的腺病毒介导的体外表达载体,可正向调控成肌细胞的分化和肌管形成过程。

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

Abstract:

【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 ¹⁰pfu/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 by AD-bSMAD1, respectively. In addition, both myotube formation and qRT-PCR results proved that SMAD1 gene promoted myoblasts differentiation and myotube formation, as well as the mRNA expression level of MyoD, Myf5, and MyoG. 【Conclusion】 We could conclude from the present study that SMAD1 gene performed its function in the myoblast differentiation and myotube formation. Based upon these findings, SMAD1 gene could be used as potential marker for the breed improvement of Qinchuan cattle through marker assisted selection for beef production.

Key words: mothers against decapentaplegic homolog 1 gene (SMAD1), siRNA, adenovirus vector, primary myoblast, myotube formation, Qinchuan cattle

宁越,米雪,陈星伊,邵建航,昝林森. 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[J]. 中国农业科学, 2019, 52(10): 1818-1829.

NING Yue,MI Xue,CHEN XingYi,SHAO JianHang,ZAN LinSen. Silencing and Overexpressing SMAD Family Member 1 (SMAD1) Gene and Its Effect on Myogenesis in Primary Myoblast of Qinchuan Cattle (Bos taurus)[J]. Scientia Agricultura Sinica, 2019, 52(10): 1818-1829.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.10.014

https://www.chinaagrisci.com/CN/Y2019/V52/I10/1818

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物种 Species	登录号 Accession	物种 Species	登录号 Accession
牛 Bos Taurus	NP_001069691.2	猪 Sus scrofa	NP_999130.1
羊 Capra hircus	XP_017917067.1	小鼠 Mus musculus	NP_032565.2
绵羊 Ovis aries	XP_014956993.1	大鼠 Rattus norvegicu	NP_037262.2
人 Homo sapiens	NP_005891.1	鸡 Gallus gallus	NP_001188384.1
狗 Canis lupus	XP_022259170.1

序列名称 Sequence name	序列（5'→3'） Sequences (5'→3')
siSMAD1	sense：5'-CCAUCUCUGCCUUCAGAAATT-3'
siSMAD1	antisense：5'-UUUCUGAAGGCAGAGAUGGTT-3'
siRNA-NC	sense：5'-UUCUCCGAACGUGUCACGUTT-3'
siRNA-NC	antisense：5'-ACGUGACACGUUCGGAGAATT-3'

基因名称 Gene name	登录号 Accession No.	引物序列（5'→3'） Primer sequences (5'→3')	产物 Size（bp）
CDS-SMAD1	NM_001077107.2	F:CTAGCTAGCCACCATGAATGTGACAAGTTTATTTTCCTTCAC	1452
CDS-SMAD1	NM_001077107.2	R:ATTTGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCC	1452
RT-SMAD1	NM_001077107.2	F:TTCGCATGAGCTTTGTGAAG	98
RT-SMAD1	NM_001077107.2	R:GTGAGCCCATCTGGGTAAGA	98
RT-MyoD	NM_001040478	F:TCCGCGACGTAGACTTGA	84
RT-MyoD	NM_001040478	R:CGAAACACGGGTCATCATAGAA	84
RT-MyoG	NM_001111325	F:GGCGTGTAAGGTGTGTAAG	85
RT-MyoG	NM_001111325	R:CTTCTTGAGTCTGCGCTTCT	85
RT-Myf5	NM_174116.1	F:CCTCTAGTTCCAGGCTCATCTA	90
RT-Myf5	NM_174116.1	R:ACCTCCTTCCTCCTGTGTAATA	90
RT-GAPDH	NM_001034034	F: CCAACGTGTCTGTTGTGGAT	80
RT-GAPDH	NM_001034034	R: CTGCTTCACCACCTTCTTGA	80

稀释倍数 Diluted multiples	上一梯度病毒液 Virus of upper gradient	DMEM完培 DMEM complete medium	上样量 Volume of the sample	滴定量 Volume of the virus
10³	10	90	10	1μL
10⁴	10	90	10	0.1μL
10⁵	10	90	10	0.01μL
10⁶	10	90	10	0.001μL
10⁷	10	90	10	0.0001μL
…	…	…	…	…