牛HSL基因的克隆和生物信息学分析
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摘要
旨在克隆牛HSL基因的CDS序列,并对该基因进行生物信息学、组织表达谱分析。根据GenBank登录的牛HSL基因序列(NM_001080220)设计1对引物,对牛组织样品中的HSL基因CDS序列进行RT-PCR扩增及序列测定;利用半定量RT-PCR方法检测HSL基因在牛各组织中的表达情况;利用生物信息学软件对其所编码的牛HSL蛋白进行分析。结果显示,获得的牛HSL基因CDS全长为2 271 bp,编码756个氨基酸,与GenBank登录的牛HSL基因同源性最高,为99.9%。HSL蛋白含有一个HSL-N superfamily保守结构域,无信号肽结构,具有疏水性。半定量RT-PCR显示,HSL基因在检测的心脏、脾脏、肺脏、肾脏、肝脏、大网膜、皮下脂肪、肌肉8种组织中均有表达,其中在大网膜和皮下脂肪中表达量较高,在肾脏、脾脏、肝脏、肺脏、肌肉和心脏中度表达。该试验可为研究牛HSL蛋白的结构和功能提供参考。
In this study, the CDS sequence of the bovine HSL gene was cloned and bioinformatically analyzed, and its tissue expression profile was characterized. One set of primers was designed based on the bovine HSL gene sequence(NM_001080220)registered in the GenBank, and the CDS sequence of HSL gene in bovine tissue samples was amplified using RT-PCR assay and was determined by sequencing; the expression level of HSL gene in bovine tissues was detected by semi-quantitative RT-PCR;the HSL protein encoded by HSL gene was bioinformatically analyzed. The results showed that the obtained CDS sequence of bovine HSL gene was 2 271 bp in length encoding 756 amino acids, and it shared the highest nucleotide homology(99.9%) with the bovine HSL gene registered in the GenBank; the HSL protein was hydrophobic and contained a HSL-N superfamily conserved domain with no signal peptide structure; semi-quantitative RT-PCR revealed that the HSL gene was expressed in 8 tissues including heart, spleen, lung, kidney, liver, greater omentum, subcutaneous fat and muscle; the highest expression level was observed in the greater omentum and subcutaneous fat, and the moderate expression level was found in kidney, spleen, liver,lung, muscle and heart. This study may provide a reference for assessing the structure and function of bovine HSL proteins.
In this study, the CDS sequence of the bovine HSL gene was cloned and bioinformatically analyzed, and its tissue expression profile was characterized. One set of primers was designed based on the bovine HSL gene sequence(NM_001080220)registered in the GenBank, and the CDS sequence of HSL gene in bovine tissue samples was amplified using RT-PCR assay and was determined by sequencing; the expression level of HSL gene in bovine tissues was detected by semi-quantitative RT-PCR;the HSL protein encoded by HSL gene was bioinformatically analyzed. The results showed that the obtained CDS sequence of bovine HSL gene was 2 271 bp in length encoding 756 amino acids, and it shared the highest nucleotide homology(99.9%) with the bovine HSL gene registered in the GenBank; the HSL protein was hydrophobic and contained a HSL-N superfamily conserved domain with no signal peptide structure; semi-quantitative RT-PCR revealed that the HSL gene was expressed in 8 tissues including heart, spleen, lung, kidney, liver, greater omentum, subcutaneous fat and muscle; the highest expression level was observed in the greater omentum and subcutaneous fat, and the moderate expression level was found in kidney, spleen, liver,lung, muscle and heart. This study may provide a reference for assessing the structure and function of bovine HSL proteins.
引文
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[4]苗志国,李国旺,谢红兵,等.金华猪与长白猪脂肪组织中HSL mRNA丰度的差异研究[J].广东农业科学,2011,43(4):128-129.
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[7]常怀普,钟金城,陈智城,等.大额牛HSL基因外显子I部分片段的序列测定及分析[J].畜牧与兽医,2007,39(3):11-15.
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[9]柴志欣,王永,罗晓林,等.麦洼牦牛H-FABP、HSL基因多态性及与生长性状的相关分析[J].中国农业科学,2013,46(14):3022-3031.
[10]ZECHNER R,KIENESBERGER P C,H AEMMERLE G,et al.Adipose trig lyceride lipase and the lipolytic catabolismof cellular fat stores[J].J Lipid Res,2009,50:3-21.
[11]KAZALA E C,JENNIFER L P,FRED J L,et al.Hormone-sensitive lipase activity in relation to fat content of muscle in Wagyu hybrid cattle[J].Lives Prod Sci,2003,79:87-96.
[12]田万年.延边黄牛背最长肌差异表达基因的克隆鉴定及特征分析[D].延吉:延边大学,2011.
[13]杨家大,陈祥,龙威海,等.放牧条件下山羊激素敏感性甘油三酯脂肪酶基因的表达[J].西南农业学报,2015,28(5):2263-2267.
[14]王琦,张利红,张立杰,等.猪激素敏感脂酶和甘油三酯水解酶基因组织表达特性研究[J].广东农业科学,2011,43(4):128-129.
[15]刘俊霞,罗军,滕炎玲,等.西农萨能奶山羊激素敏感脂酶基因CDS区的克隆与生物信息学分析[J].农业生物技术学报,2010,18(3):539-544.
[2]KRAEMER F B,SHEN W J.Hormone-sensitive lipase knockouts[J].Nutr Metab,2006,3(1):12-19.
[3]屈博文,金朝,李永辉,等.淮南猪皮下与肌内脂类代谢相关基因表达差异的研究[J].河南科技学院学报,2015,43(5):40-44.
[4]苗志国,李国旺,谢红兵,等.金华猪与长白猪脂肪组织中HSL mRNA丰度的差异研究[J].广东农业科学,2011,43(4):128-129.
[5]强巴央宗,商鹏,杨涛,等.藏猪激素敏感脂肪酶(HSL)基因遗传多样性分析[J].西南农业学报,2012,25(5):1904-1906.
[6]黄艳娜,何凤愿.陆川猪激素敏感性脂肪酶基因的克隆及序列分析[J].黑龙江畜牧兽医,2017(1):98-101.
[7]常怀普,钟金城,陈智城,等.大额牛HSL基因外显子I部分片段的序列测定及分析[J].畜牧与兽医,2007,39(3):11-15.
[8]马志杰,钟金城,陈智华,等.牛科动物HSL基因序列分析及其分子进化研究[J].遗传学报,2007,34(1):26-34.
[9]柴志欣,王永,罗晓林,等.麦洼牦牛H-FABP、HSL基因多态性及与生长性状的相关分析[J].中国农业科学,2013,46(14):3022-3031.
[10]ZECHNER R,KIENESBERGER P C,H AEMMERLE G,et al.Adipose trig lyceride lipase and the lipolytic catabolismof cellular fat stores[J].J Lipid Res,2009,50:3-21.
[11]KAZALA E C,JENNIFER L P,FRED J L,et al.Hormone-sensitive lipase activity in relation to fat content of muscle in Wagyu hybrid cattle[J].Lives Prod Sci,2003,79:87-96.
[12]田万年.延边黄牛背最长肌差异表达基因的克隆鉴定及特征分析[D].延吉:延边大学,2011.
[13]杨家大,陈祥,龙威海,等.放牧条件下山羊激素敏感性甘油三酯脂肪酶基因的表达[J].西南农业学报,2015,28(5):2263-2267.
[14]王琦,张利红,张立杰,等.猪激素敏感脂酶和甘油三酯水解酶基因组织表达特性研究[J].广东农业科学,2011,43(4):128-129.
[15]刘俊霞,罗军,滕炎玲,等.西农萨能奶山羊激素敏感脂酶基因CDS区的克隆与生物信息学分析[J].农业生物技术学报,2010,18(3):539-544.