中国农业科学2016，Vol.49Issue(10)：1998-2007,10.DOI:10.3864/j.issn.0578-1752.2016.10.015
山羊 IGFBP-2基因的克隆、序列分析及其组织表达
Cloning, Sequence Analysis and Tissue Expression of IGFBP-2 Gene in Goat (Capra hirus)
摘要
Abstract
Objective]The objectives of this study were to clone goat IGFBP-2 (Insulin-like growth factor binding protein -2) coding sequence (CDS), to analyze its sequence characteristics, mRNA and protein expression profiles. It could provide data for further investigation on the function and expression regulation of IGFBP-2 gene in the growth and development of postnatal goat.[Method]The IGFBP-2 mRNA sequences of Ovis aries (NM_001009436) and Bos taurus (NM_174555) were downloaded from GenBank database, and the primers were designed according to the conserved regions by using Primer Premier 6.0 software after sequences alignment, then PCR amplification and the positive clones were obtained using TA clone technology. A complete coding sequence of IGFBP-2 gene was obtained from sequencing. The CDS and coding amino acid sequences were analyzed by using EditSeq, DNAMAN 6.0, MEGA 6.0 and ExPASy online software. The real-time quantitative PCR (Q-PCR) primers were designed using the acquired CDS sequence of the IGFBP-2 gene. The expression level of the IGFBP-2 gene and protein in postnatal Nanjiang brown goat’s different tissues (heart, liver, lung, longissimus dorsi muscle, semimembranosus muscle and triceps brachii muscle) and development stages (3 d,30 d,60 d,90 d and 120 d) were detected using Q-PCR and western blotting.[Result]A full-length CDS of 954bp was cloned for goat IGFBP-2, and encodes a protein of 217 amino acid. The GC and AT content is 69.39% and 30.61%. The molecular weight of goat IGFBP-2 protein is 33.8808 kD, and its theoretical isoelectric point is 7.82. The secondary structure of the protein contains random coil (68.14%), α-helix (18.30%) and extended strand (13.56%). Protein structure domain analysis showed that its amino acid sequence contains conserved IB (IGFBP homologues) and TY (Thyroglobulin type Ι repeats) domains, IB domain in the 37-125 amino acids and TY domain in the 250-302 amino acids. Phosphorylation sites prediction indicated that there are five Ser phosphorylation sites and seven Thr phosphorylation sites exists in IGFBP-2 protein sequence. Glycosylation sites prediction found that there are ten N-glycosylation sites and two O-glycosylation sites exists in IGFBP-2 protein sequence. Sequence analysis indicated that the IGFBP-2 CDS sequence of the Nanjiang brown goat is similar with those of the sheep (98.99%), cattle (97.73%), pig (87.12%), human (78.33%) and mouse (76.26%). Sequence analysis revealed that the IGFBP-2 amino acid sequence of the Nanjiang brown goat is similar with those of the sheep (99.24%), cattle (98.10%), pig (87.07%), human (70.27%) and mouse (73.00%). Furthermore, goat IGFBP-2 has the closest phylogenetic relationship to the sheep and cattle IGFBP-2 in amino acids sequences. The mRNA and protein levels of IGFBP-2 in the liver were significantly higher than that in other tissues (P<0.01), and LD were the second (P<0.01); in the liver, the mRNA and protein levels of IGFBP-2 were upregulated from 3 day to 120 day. In addition,the mRNA level of IGFBP-2 showed an “up-down-up” expression pattern during postnatal longissimus dorsi muscle development.[Conclusion]IGFBP-2 was cloned and its tissue expression patterns were investigated. The sequence characteristics of IGFBP-2 is conserved in species, and liver is the main expression tissue of goat IGFBP-2 mRNA and protein. The expression of IGFBP-2 mRNA and protein occurred with some regularity and indicated that IGFBP-2 may play an important role in early growth and development of postnatal goat.关键词
山羊/IGFBP-2/基因克隆/荧光定量 PCR/蛋白免疫印迹杂交Key words
goat/IGFBP-2/gene cloning/real-time PCR/Western blotting引用本文复制引用
占思远,董尧,李利,仲涛,王林杰,张红平..山羊 IGFBP-2基因的克隆、序列分析及其组织表达[J].中国农业科学,2016,49(10):1998-2007,10.基金项目
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