达氏鲟生长激素基因cDNA克隆、表达及免疫荧光定位研究  

c DNA CLONING, EXPRESSION AND IMMUNOLOCALIZATION OF GROWTH HORMONE GENE IN ACIPENSER DABRYANUS

作　　者：单喜双[1,2] 岳华梅[1] 陈细华[1] 叶欢[1] 杨晓鸽[1] 李创举[1] 

机构地区：[1]中国水产科学研究院长江水产研究所,农业部淡水生物多样性保护重点实验室,武汉430223 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水生生物学报》2015年第2期307-314,共8页Acta Hydrobiologica Sinica

基　　金：国家高技术研究发展计划项目课题一(2011AA100401);中央级公益性科研院所基本科研业务费专项资金项目(2013JBFZ01);国家自然科学基金青年科学基金项目(31001104)资助

摘　　要：为研究达氏鲟(Acipenser dabryanus)生长激素(Growth Hormone,GH)基因的功能,合成了达氏鲟垂体SMART c DNA,克隆得到GH全长c DNA序列。达氏鲟GH全长c DNA序列为1008 bp,由52 bp的5′端非编码区(Untranslated region,UTR)、编码214个氨基酸的645 bp开放阅读框(Open reading frame,ORF)和311 bp的3′UTR构成。运用GH氨基酸序列构建进化树分析发现,达氏鲟与两栖类、爬行类和哺乳类的一致性要高于真骨鱼类。实时荧光定量PCR结果表明,达氏鲟GH m RNA主要在垂体和下丘脑中表达,且垂体中GH的表达量约为下丘脑的110倍;Western-blot研究结果与q RT-PCR一致,仅在垂体和下丘脑中检测到生长激素蛋白,且垂体中GH的表达量远高于下丘脑。免疫荧光定位结果显示,GH主要定位于垂体中部,下丘脑中也有少量荧光信号;苏木精-伊红组织切片染色研究表明,GH主要是由嗜酸性的生长激素分泌细胞分泌。研究为深入研究脊椎动物生长激素基因的进化和人工养殖达氏鲟的生长调控提供了基础。The Dabry’s sturgeon （Acipenser dabryanus Dumeril, 1868） mainly distributes in the upper Yangtze River of China. It is currently endangered due to the destruction of their spawning grounds and other anthropogenic interferences. In this study, the full-length cDNA sequence of GH was cloned from SMART cDNAs of pituitary in A. dabryanus. The Dabry’s sturgeon GH cDNA was 1008 bp in length, including a 52 bp 5′-untranslated region （UTR）, a 311 bp 3′ UTR and a 645 bp open reading frame （ORF） encoding a peptide of 143 amino acids. Amino acid sequence alignment revealed that A. dabryanus GH shared extremely high identities （over 95%） with its counterparts in other sturgeons. Moreover, sturgeons showed lower GH sequence identities with other bony fish than that of Amphibia, Reptilia, Aves, and Mammalia. By quantitative real-time PCR, GH mRNAs were detected in the pituitary and hypothalamus but absent in any of other somatic organs examined. Moreover, the expression of GH mRNAs in the pituitary was about 110 times to that of hypothalamus. Western-blot analysis suggested the GH expression in both the pituitary and hypothalamus, with higher expression level in the pituitary, which is consistent with the result of qRT-PCR. Immunofluoresence locali-zation showed strong GH-positive signals in the pars intermedia of the pituitary, together with weak fluorescence signals detected in the hypothalamus. HE staining indicated that GH was mainly secreted by eosinophilic cells in the adenohypophysis. This study will benefit both the evolution study of vertebrate GH genes and the further biological function study of GH in cultured A. dabryanus.

关 键 词：达氏鲟 生长激素 q RT-PCR WESTERN-BLOT 荧光免疫组化 

分 类 号：Q344.1[生物学—遗传学]