鲫两种不同生长激素cDNA的分子克隆和分析

MOLECULAR CLONING AND ANALYSIS OF TWO DISTINCT GROWTH HORMONE cDNAs FROMCAR ASSIUS AURATUS AURATUS

从鲫脑垂体组织提取总RNA ,采用 3′RACE PCR的方法 ,从单一垂体总RNA中扩增出编码两种不同类型鲫生长激素的cDNA :生长激素Ⅰ (GrowthhormoneⅠ ,GHⅠ )和生长激素Ⅱ (GrowthhormoneⅡ ,GHⅡ )。将两种类型的鲫GHcDNA分别克隆到pGEM TEasyVector上进行序列测定和分析。克隆的鲫GHⅠ和GHⅡcDNA均包括编码 188个氨基酸残基的GH成熟肽序列和 3′端的非翻译区 ,但不含信号肽序列和 5′端非编码区。序列分析结果表明 ,鲫GHⅠ的碱基序列和推测的氨基酸序列与国外已经发表的金鱼GHⅠ的同源性分别为 98 7%和 97 9% ;GHⅡ的碱基序列和推测的氨基酸序列与金鱼GHⅡ的同源性分别为 99 1%和 99 5% ,虽然同源性较高 。

Growth hormone, a 22-kDa single-chain polypeptid e hormone synthesized and secreted by the anterior pituitary gland, plays an impo rtant role in the regulation of growth, development,metabolism and appetite.It i s one of the best-studied pituitary hormones that have been used as models for studying of physiology, nutritional composition, regulating of gene expression, structure-function relationships and gene evolution. In mammals,growth hormone variants arising from differential processing of a single primary RNA transcript have been reported, whereas the growth hormone variants found in teleost appear to be the products of different genes.In previous studies, a few teleost fishes h ave two distinct growth hormones, such as chum salmon(Oncorhynchus keta), ch inook salmon (Oncorhynchus tshawytscha), coho salmon(Oncorhynchus kisutch),Atlantic salmon(Salmo salar), rainbow trout (Oncorhynchus mykiss),At lantic cod(Gadus morhua),Japanese eel(Anguilla japonica),goldfish(Car assius auratus),etc.Growth hormone gene duplication was believed to be the re sult of polyploidization among ancestral aquatic vertebrates. There is a close r elationship betweenCarassius auratus auratusand goldfish, belonging to the same family of Cyprinidae.Carassius auratus auratuswere reported to be pol yploid, so it was possible thatCarassius auratus auratusalso had two disti nct growth hormone genes in a single body.Gene duplication was a rare event. Whe ther there were two formsCarassius auratus auratusgrowth hormone cDNAs enc oding distinct growth hormone was still unknown,it was significative to study th e growth hormone polypeptides ofCarassius auratus auratus.\;The experimental fish (Carassius auratus auratus)came from the farm of Pear l River Fisheries Research Institute. Total RNA was isolated from the pituitary ofCarassius auratus auratususing High Pure RNA Isolation Kit of Roche Ltd. The cDNA encoding growth hormone Ⅰ(GHⅠ) and growth hormone Ⅱ(GHⅡ) mature peptid es together with 3′ end noncoding regions were amplified by 3′RACE-PCR (Rapid Amplification of cDNA Ends-Polymerase Chain Reaction) strategy using the total isolated RNA as template. The first strand of cDNA was synthesized from poly (A ) using oligo dT-adapter primer provided by the kit.The 5′-PCR primer with a EcoR Ⅰ recognising sequence was designed based on the growth hormone sequences of goldfish and common carp.The 3′-PCR primer is M13 primer M4 matching to the 3′-end of oligo dT-adapter primer.The amplified cDNA fragments were electrophoresed on 1.0% low-melt agarose gel,purified with the Agarose Gel Purification Kit,and ligated with pGEM-T Easy Vector by T 4 DN A ligase overnight.E. coliDH5α was transformed with the ligation product . Luria-Bertani (LB) agar plates containing 100μg/mL ampicillin was used to sc reen the recombinant colonies. Insertion of the PCR product was verified by rest riction enzyme digestion, agarose gel electrophoresis and sequencing.The cDNA s equences and deduced amino acid sequences were compared with other growth hormon e sequences in the GenBank database. The PCR product was about 1100 base pairs. There were no signal peptide sequence in the cloned cDNAs. Both GHⅠ and GHⅡ mature peptides cDNAs ofCarassius a uratus auratus were composed of 564 base pairs, encoding 188 amino acid residu es, but the length of 3′ noncoding region of GHⅠ and GHⅡ is different. The 3 ′ noncoding region of GHⅠ has 480 base pairs, whereas GHⅡ has 537 base pairs. T he homology of the nucleotide sequences of two forms growth hormone mature pepti de and deduced amino acid sequence were 92.6% and 91.5%, respectively. There wer e 45 different nucleotides in the coding region between GHⅠ and GHⅡ,resulting in a difference of 16 amino acid residues between the two deduced growth hormone amino acid sequences. Further analysis of sequences indicated thatCarassius auratus auratus GHⅠ was similar to that of Cyprinid,such as goldfish and comm on carp GH which contain five cysteine residues (Cys-49,Cys-123,Cys-161,Cys- 178,and Cys-186). However,Carassius auratus