朗德鹅肌肉生成抑制因子基因(MSTN)的克隆及其表达量与日粮能量、血清IGF-I和GH的关系

Cloning of MSTN Gene of Landes Goose and Research of Relationship Between MSTN Gene Expression and Different Energe Diets,or IGF-I and GH in Serum

【目的】采用反转录-聚合酶链反应(RT-PCR)技术,克隆朗德鹅肌肉生成抑制因子基因(MSTN),研究MSTN基因表达和日粮能量及其部分血清激素含量的相互关系,了解MSTN的功能,为水禽分子营养和分子育种提供基础资料。【方法】从朗德鹅(Anser anser)的腿肌中抽提总RNA,用两步法RT-PCR扩增出MSTN基因的cDNA编码序列,以pGEM-TVector为载体,将该片段克隆到大肠杆菌(Escherichia coli)中。通过筛选阳性克隆,双酶切鉴定后测序;以MSTN基因的克隆为基础,以β-actin为内标,构建优化的半定量RT-PCR方法,研究高中低(A:13.38MJ·kg-1、B:12.13MJ·kg-1、C:10.87MJ·kg-1)3种不同能量对朗德鹅21日龄和70日龄2个时期,肌肉组织MSTN基因表达的差异;同时用放免法测定21、70日龄的血清GH和IGF-I浓度。【结果】克隆朗德鹅MSTN基因cDNA的部分序列,其片段大小为1128bp,编码375个氨基酸组成的多肽,与已发表的鸡、鸭、鹅的MSTN核苷酸相似性分别为94%、94%、99%;氨基酸的相似性分别为98%、97%、98%;21日龄时,朗德鹅公鹅、母鹅MSTN表达量差异不显著;70日龄时朗德鹅公鹅MSTN的表达量情况为能量A>C>B,朗德鹅母鹅MSTN的表达量情况为C>B>A;对于朗德鹅21～70日龄阶段的生长,MSTN基因的表达量和血清IGF-I的变化基本一致;与血清GH含量之间并不存在很大关联,GH和能量的高低呈正相关。【结论】日粮能量对21日龄后朗德鹅MSTN基因的表达有影响,且MSTN基因表达量和血清IGF-I具有相关性,和血清GH无较大相关性。

[Objective] The myostatin gene of Landes goose was amplified by reverse transcription-polymerase chain reaction （RT-PCR）, and the relationship between MSTN gene expression and different energy diets, or IGF-I, GH levels in serum was researched in order to know the function of MSTN gene and provide information for molecular nutriology and breeding of water fowl. [ Method ]Genome RNA was extracted from muscle of Landes goose, MSTN gene mRNA was amplified by RT-PCR, the PCR product was cloned into pGEM-T vector and transformed into Escherichia coli. The MSTN gene was isolated and sequenced from the positive clones screened. Based on the MSTN gene clone, β-acfin as inner control, an optimal semi-quantitative RT-PCR method was constructed to study the difference of MSTN gene expression at 21st and 70th day of Landes goose fed with different energy diets, and IGF-I and GH in serum were determined. [Result] The unique DNA fragment about 1 128 bp in length was obtained. Sequence analysis revealed that it has a length of 1128 nucleotides which encodes a peptide of 375 amino acids. Compared with homologous sequence of chicken, duck and goose, it displayed a fairly high degree conservation. The＇homology of the nucleotide sequence were 94%, 94%, and 99%, respectively; the homology of the amino acid sequence were 98%, 97%, and 98%, respectively; at 21st day and 70th day, the results showed that the expression of MSTN in female Landes geese was a little higher than male at 21st day; the expression of MS TN in male was A〉 C 〉 B at 70th day, female was C 〉B 〉 A at 70th day; In the period of 21-70 days, the expression of MSTN was changed with IGF-I in serum; it was not associated with GH in serum, GH was significantly correlated with energy. [Conclusion] Energy influences MSTN gene expression of Landes goose after 21 days; and MSTN gene expression is associated with IGF-I in serum, but it is not associated with GH in serum.