从江香猪lncGHR1基因的克隆、组织表达及超表达研究  

The Cloning,Tissue Expression and Over-expression Study of lncGHR1 Gene in Cong Jiang Xiang Pig(Sus scrofa)

作　　者：崔小芝 吴巧群 吴小敏 朱晓锋 谌颖莲 谢玲玲 赵佳福[1,2] 陈伟[1,2] 许厚强[1,2,3] CUI Xiao-Zhi;WU Qiao-Qun;WU Xiao-Min;ZHU Xiao-Feng;CHEN Ying-Lian;XIE Ling-Ling;ZHAO Jia-Fu;CHENWei;XU Hou-Qiang(Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region of Ministry of Education,Guizhou University/Key Laboratory of Animal Genetics,Breeding and Reproduce in Guizhou Province,Guiyang 550025,China;College of Animal Sciences,Guizhou University,Guiyang 550025,China;School of Medicine,Guizhou University,Guiyang 550025,China;Livestock and Poultry Germplasm Testing Center of Guizhou Province,Guiyang 550025,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [2]贵州大学动物科学学院,贵阳550025 [3]贵州大学医学院,贵阳550025 [4]贵州省种畜禽种质测定中心,贵阳550025

出　　处：《农业生物技术学报》2020年第12期2165-2175,共11页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划(2015BAD03B02-3);贵州省农业重大产业科学研究攻关项目(黔教合KY字[2019]011);贵州省科技计划项目(黔科合LH字[2017]7274号);贵州大学2017年度学术新苗培养及创新探索专项(黔科合平台人才[2017]5788号)。

摘　　要：生长激素受体(growth hormone receptor,GHR)在动物生长发育过程中具有重要作用。为研究GHR基因相关长链非编码RNAs(long noncoding RNAs,lncRNAs)在从江香猪(Sus scrofa)不同组织中的表达规律,本研究以贵州从江香猪和大白猪为研究对象,以生长发育相关的miR-146b-3p为关联点,通过SWChen 2.3程序筛选GHR相关lncRNAs,对筛选出来的lncRNAs进行克隆验证。以候选lncRNA NONSUST017828.1(GenBank No.TCONS_00020564,命名为lncGHR1)为研究对象,对其进行生物信息学及组织表达谱分析,并构建lncGHR1-CMV超表达载体,转染至人源胚胎肾细胞(human embryonic kidney cells,HEK-293T)和从江香猪原代肌细胞,检测细胞增殖相关基因YAP(Yes-associated protein)和TAZ(transcription activator)的表达情况。结果显示,lncGHR1二级结构最小自由能为-131.20 kcal/mol,含3个ORF;局部比对结果发现,lncGHR1与猪GHR 3'UTR相似程度最高;lncGHR1与miRNA结合的抑制系数为4.96%,其中miR-146-3p与lncGHR1亲和评分最高。lncGHR1在从江香猪脾脏和肺脏中表达量最高,心脏中最低;在大白猪肝脏中表达量最高,心脏中最低;lncGHR1在从江香猪脾脏、肺脏、背最长肌和小肠中的表达量均极显著高于大白猪(P<0.01),但肝脏中的表达量极显著低于大白猪(P<0.01),其他组织表达差异不显著。lncGHR1在HEK-293T细胞和从江香猪原代肌细胞中的超表达,显著提高TAZ基因的表达(P<0.05),但对YAP影响不大(P>0.05)。以上结果表明lncGHR1在生长发育过程中发挥了重要作用,对解析从江香猪生长缓慢、个体矮小的成因机制及猪的遗传改良育种具有重要意义。Growth hormone receptor(GHR)plays an important role in animal growth and development.In order to study the expression of long noncoding RNAs(lncRNAs)related to GHR gene in Cong Jiang Xiang pig(Sus scrofa),this paper selected Guizhou Cong Jiang Xiang pig and Yorkshire pig as the research objects,used the growth and development related miR-146 b-3 p as correlation point,screened out the GHR gene related lncRNAs by SWchen 2.3 program,and verified the lncRNAs by cloning and sequencing.The candidate lncRNA NONSUST017828.1(GenBank No.TCONS00020564,named lncGHR1)was used as the further research object.Bioinformatics and tissue expression spectrum analysis were performed,the lncGHR1-CMV over-expression vector was constructed and transfected to human embryonic kidney cells(HEK-293 T)and primary myocytes of Cong Jiang Xiang pig,effects of lncGHR1 on the expression of Yes-associated protein(YAP)and transcription activator(TAZ)were detected by qRT-PCR.The results showed that the minimum free energy of the secondary structure of lncGHR1 was-131.20 kcal/mol and contained 3 ORF.The results of local comparison showed that lncGHR1 was the most similar with GHR 3’UTR of pig.The inhibition coefficient of lncGHR1 binding to miRNA was 4.96%,and the score of miR-146-3 p with lncGHR1 was the highest.The expression of lncGHR1 was the highest in the spleen and lung of Cong Jiang Xiang pig,which was significantly higher than that in other tissues(P<0.01),the lowest in the heart;the highest expression of Yorkshire was found in liver,followed by lung;the expression of lncGHR1 in heart,spleen,lung,kidney,large intestine,small intestine and longissimus dorsi muscle of Cong Jiang Xiang pig was significantly higher than that of Yorkshire pig(P<0.01),but the expression of lncGHR1 in liver was significantly lower(P<0.01).Over-expression of lncGHR1 in 293 T cells and primary myocytes of Cong Jiang Xiang pig significantly increased the expression of TAZ gene(P<0.05),but the expression of YAP gene was not significantly different(P>0.05).These resu

关 键 词：从江香猪 生长激素受体(GHR) 长链非编码RNAs(lncRNAs) 差异表达 

分 类 号：S828[农业科学—畜牧学]