陆川猪激素敏感性脂肪酶基因的克隆及序列分析
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摘要
为了获得广西陆川猪激素敏感性脂肪酶(HSL)基因编码区序列(CDS),试验从11月龄陆川猪背最长肌组织中提取总RNA,利用反转录-聚合酶链式反应(RT-PCR)方法分段扩增出HSL基因CDS。结果表明:成功分段克隆出陆川猪HSL基因CDS,拼接后长度为2 295 bp,与Gen Bank数据库中公布的国内地方猪种梅山猪的同源性为99.9%,与外来猪种长白猪的同源性为99.7%,并发现其1 044位点存在C→T,其碱基T是陆川猪所特有,尚未导致氨基酸突变。利用生物软件构建系统进化树显示,与广西陆川猪遗传距离最近的是梅山猪,它们聚为同一支,最远的是人。陆川猪HSL基因的二级结构包含α-螺旋、β-折叠。
To obtain the coding sequence( CDS) of hormone- sensitive lipase( HSL) gene from Luchuan pig in Guangxi,total RNA was extracted from the Longissimus dorsi tissue of 11- month- old Luchuan pig,and the CDS fragments of HSL gene were amplified by reverse transcription- polymerase chain reaction( RT- PCR). The results showed that the CDS fragments of HSL gene were successfully cloned,and the length of the CDS was 2 295 bp after splicing. The CDS of HSL gene from Luchuan pig shared 99. 9% and 99. 7% homology with that from the local breed of domestic Meishan pig and the foreign breed Landrace published in Gen Bank,respectively. It was found that there was a mutation from C to T at 1 044 locus,and its base T was unique to Luchuan pig and didn't cause amino acid mutation. A phylogenetic tree constructed by biological software showed that Luchuan pig in Guangxi was genetically closest to Meishan pig,both were clustered into the same branch,while Luchuan pig in Guangxi was genetically farthest to human. The secondary structure of HSL gene from Luchuan pig contained α- helix and β-fold.
To obtain the coding sequence( CDS) of hormone- sensitive lipase( HSL) gene from Luchuan pig in Guangxi,total RNA was extracted from the Longissimus dorsi tissue of 11- month- old Luchuan pig,and the CDS fragments of HSL gene were amplified by reverse transcription- polymerase chain reaction( RT- PCR). The results showed that the CDS fragments of HSL gene were successfully cloned,and the length of the CDS was 2 295 bp after splicing. The CDS of HSL gene from Luchuan pig shared 99. 9% and 99. 7% homology with that from the local breed of domestic Meishan pig and the foreign breed Landrace published in Gen Bank,respectively. It was found that there was a mutation from C to T at 1 044 locus,and its base T was unique to Luchuan pig and didn't cause amino acid mutation. A phylogenetic tree constructed by biological software showed that Luchuan pig in Guangxi was genetically closest to Meishan pig,both were clustered into the same branch,while Luchuan pig in Guangxi was genetically farthest to human. The secondary structure of HSL gene from Luchuan pig contained α- helix and β-fold.
引文
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[2]FRAYN K N,COPPACK S W,FIELDING B A,et al.Coordinated regulation of hormone-sensitive lipase and ipoprotein lipase in the human adipose tissue in vivo:implications for the control of fat storage and fat mobilization[J].Adv Enzyme Regul,1995(35):163-178.
[3]KRAEMER F B,SHEN W J.Hormone-sensitive lipase knockouts[J].Nutr Metab,2006,3(1):12-19.
[4]HOLM C,KIRCHGESSNER T G,SVENSON K L,et al.Hormonesensitive lipase:sequence,Expression and chromosomal localization to 19cent-ql3.3[J].Science,1988,241(4872):1503-1506.
[5]艾庆辉,袁禹惠,麦康森,等.半滑舌鳎稚鱼脂肪分解关键酶基因的克隆及饲料中脂肪水平对其表达的调控[J].中国海洋大学学报,2014,44(10):65-71.
[6]陈启亮.铜对黄颡鱼和矛尾复虾虎鱼脂类代谢的影响及机理研究[D].武汉:华中农业大学,2015.
[7]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.
[8]王燕,汪以真.猪激素敏感酯酶基因克隆与序列[J].中国兽药杂志,2006,40(5):12-14.
[9]张振波.猪脂蛋白脂酶(LPL)和激素敏感脂肪酶(HSL)基因的序列和多态性分析及体外表达的研究[D].武汉:华中农业大学,2005.
[10]张家富,何若钢,江永强,等.陆川猪胴体品质及肉质的研究[J].养猪,2010(6):33-36.
[11]雷明刚.猪脂蛋白脂酶(LPL)和激素敏感脂肪酶(HSL)基因的分子遗传学基础研究[D].武汉:华中农业大学,2003.
[12]曲亮,黄瑞华,李开桢,等.H-FABP和HSL基因多态性对苏淮猪胴体性状的影响[J].南京农业大学学报,2008,31(3):107-112.
[13]王安娜.贵州地方猪、长×约杂交猪HSL和GDF-8基因多态性差异及其与瘦肉率的相关性研究[D].贵阳:贵州大学,2008.
[14]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.
[15]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(自然科学版),2015,43(5):40-44.
[16]CHOWDHARY B P,HOMSEN P D,FLLEGREN H,et al.Precise localization of somegenetic markers in pigs using non-radioactive in situ hybridization[J].Anim Genet,1992(23):92.
[17]MELLINK C H,LAHBIB-MANSAIS Y,YERLE M,et al.Localization of four newmarkers to pig chromosomes 1,6 and 14 by radioactive in situ hybridization[J].Cytogenet Cell Genet,1993,64(3/4):256-260.
[18]HARBITZ I,CHOWDHARY B,THOMSEN P D,et al.Assignment of the porcine calcium release channel gene,a candidate for themalignanthy-perthermial locus,to the 6p11-q21 segment of chromosome 6[J].Genomics,1990,8(2):243-248.
[19]LANGIN D,LAURELL H,HOLST L S,et al.Gene organization and primary structure of human hormone-sensitive lipase:possible significance of a sequence homology with a lipase of moraxella TA144,an antarctic bacterium[J].Proc Natl Acad Sci USA,1993,90(11):4897-4901.
[20]LI Z,SUMIDA M,BIRCHBAUER A,et al.Isolation and characterization of the gene for mouse hormone-sensitive lipase[J].Genomics,1994,24(2):259-265.
[21]丁能水,黄路生,任军,等.猪激素敏感脂肪酶(HSL)基因的研究概况[J].遗传,2000,22(5):331-333.
[2]FRAYN K N,COPPACK S W,FIELDING B A,et al.Coordinated regulation of hormone-sensitive lipase and ipoprotein lipase in the human adipose tissue in vivo:implications for the control of fat storage and fat mobilization[J].Adv Enzyme Regul,1995(35):163-178.
[3]KRAEMER F B,SHEN W J.Hormone-sensitive lipase knockouts[J].Nutr Metab,2006,3(1):12-19.
[4]HOLM C,KIRCHGESSNER T G,SVENSON K L,et al.Hormonesensitive lipase:sequence,Expression and chromosomal localization to 19cent-ql3.3[J].Science,1988,241(4872):1503-1506.
[5]艾庆辉,袁禹惠,麦康森,等.半滑舌鳎稚鱼脂肪分解关键酶基因的克隆及饲料中脂肪水平对其表达的调控[J].中国海洋大学学报,2014,44(10):65-71.
[6]陈启亮.铜对黄颡鱼和矛尾复虾虎鱼脂类代谢的影响及机理研究[D].武汉:华中农业大学,2015.
[7]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.
[8]王燕,汪以真.猪激素敏感酯酶基因克隆与序列[J].中国兽药杂志,2006,40(5):12-14.
[9]张振波.猪脂蛋白脂酶(LPL)和激素敏感脂肪酶(HSL)基因的序列和多态性分析及体外表达的研究[D].武汉:华中农业大学,2005.
[10]张家富,何若钢,江永强,等.陆川猪胴体品质及肉质的研究[J].养猪,2010(6):33-36.
[11]雷明刚.猪脂蛋白脂酶(LPL)和激素敏感脂肪酶(HSL)基因的分子遗传学基础研究[D].武汉:华中农业大学,2003.
[12]曲亮,黄瑞华,李开桢,等.H-FABP和HSL基因多态性对苏淮猪胴体性状的影响[J].南京农业大学学报,2008,31(3):107-112.
[13]王安娜.贵州地方猪、长×约杂交猪HSL和GDF-8基因多态性差异及其与瘦肉率的相关性研究[D].贵阳:贵州大学,2008.
[14]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.
[15]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(自然科学版),2015,43(5):40-44.
[16]CHOWDHARY B P,HOMSEN P D,FLLEGREN H,et al.Precise localization of somegenetic markers in pigs using non-radioactive in situ hybridization[J].Anim Genet,1992(23):92.
[17]MELLINK C H,LAHBIB-MANSAIS Y,YERLE M,et al.Localization of four newmarkers to pig chromosomes 1,6 and 14 by radioactive in situ hybridization[J].Cytogenet Cell Genet,1993,64(3/4):256-260.
[18]HARBITZ I,CHOWDHARY B,THOMSEN P D,et al.Assignment of the porcine calcium release channel gene,a candidate for themalignanthy-perthermial locus,to the 6p11-q21 segment of chromosome 6[J].Genomics,1990,8(2):243-248.
[19]LANGIN D,LAURELL H,HOLST L S,et al.Gene organization and primary structure of human hormone-sensitive lipase:possible significance of a sequence homology with a lipase of moraxella TA144,an antarctic bacterium[J].Proc Natl Acad Sci USA,1993,90(11):4897-4901.
[20]LI Z,SUMIDA M,BIRCHBAUER A,et al.Isolation and characterization of the gene for mouse hormone-sensitive lipase[J].Genomics,1994,24(2):259-265.
[21]丁能水,黄路生,任军,等.猪激素敏感脂肪酶(HSL)基因的研究概况[J].遗传,2000,22(5):331-333.