猪背最长肌肌红蛋白基因片段的克隆及甘氨酸铁对其表达的影响

Cloning of myoglobin(Mb) gene of M. longissimus dorsi and effects of iron glycine on its expression in weanling pigs

从杜长大断奶仔猪背最长肌中克隆肌红蛋白(Mb)基因的部分序列并探讨甘氨酸铁对其表达的影响.从仔猪背最长肌提取总RNA,用RT-PCR扩增Mb基因,获得l条约335 bp的片段,以pGEM-T Easy为载体,将该基因片段克隆到大肠杆菌(Escherichia coli)DH5α中.从筛选的阳性克隆中分离出Mb基因,测定其序列.分析表明,该片段为MbcDNA的部分序列,编码111个氨基酸组成的多肽.研究得到的基因片段与报道的猪背最长肌MbcDNA部分序列相似性达到99%.以Mb基因片段克隆为基础,构建优化的半定量RT-PCR法,以18S rRNA为内标,研究不同甘氨酸铁添加量对Mb基因表达的差异.结果表明,与对照组相比,饲养35 d后,60、90和120 mg?kg-1甘氨酸铁组(以铁计)仔猪背最长肌Mb的相对表达量增加了97.35%、108.87%和86.17%(P<0.05).据此认为,甘氨酸铁在一定程度上可以调控肌红蛋白基因表达,改善肉色.

The experiment was conducted to investigate cloning of myoglobin （Mb） gene of M. longissimus dorsi and effects of iron glycine on its expression in weanling pigs. Genome RNA was extracted from M. longissimus dorsi of swine and amplified using RT-PCR. A DNA fragment about 335 bp in length was obtained and the PCR product was cloned into pGEM-T Easy vector. The Mb gene was isolated and sequenced from the screened positive clones. Result of sequence analysis shows that this fragment was the partial sequence of Mb cDNA and coded 111 amino acid residues. The gene homology of the obtained fragment was up to 99 % compared with that of reported Mb gene sequence in the adipose of porcine. Based on the Mb gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Twenty-four piglets （Duroc × Landrace × Yorkshire） were used to determine Mb mRNA expression after 35 days treated with iron glycine （0, 30, 60, 90, 120 mg.kg-1）. Results show that 60, 90 or 120 mg. kg-1 iron glycine could increase the Mb mRNA expression obviously, which were improved by 97.35%, 108, 87% and 86.17%（P〈0.05）, respectively. It was indicated that iron glyeine could affect meat color by regulating Mb mRNA expression.