猪CAⅢ基因CDS区克隆、生物信息学及组织表达分析

Cloning,Bioinformatic Analysis and Tissues Expression Patterns of CDS of Carbonic anhydrase Ⅲ(CAⅢ) Gene in Pig

对猪碳酸酐酶Ⅲ(Carbonic anhydrase Ⅲ,CAⅢ)基因进行克隆及生物信息学分析,并分析该基因的时空表达特性。通过RT-PCR和克隆测序技术获得猪CAⅢ基因的CDS序列,综合利用多种生物信息学软件,对该基因编码的蛋白质生物学特性进行预测,采用qRT-PCR技术检测CAⅢ基因的组织表达谱和时序表达规律。结果表明,猪CAⅢ基因CDS区长783bp,编码260个氨基酸;CAⅢ蛋白为亲水性碱性蛋白,无信号肽,主要在细胞质中发挥作用;CAⅢ基因在心脏、肝脏、胰脏、肺、胃、背最长肌、皮下脂肪、脾脏、盲肠等9种组织中均有表达,但在背最长肌中表达量最高,极显著地高于其他组织。从初生到5月龄,大白猪背最长肌中CAⅢ基因mRNA表达量呈上升—下降—上升—下降的趋势,初生时最低,随后表达量逐渐升高,到3月龄时达到峰值,之后开始下降;马身猪中,从初生到5月龄,CAⅢ基因mRNA的表达量基本呈上升趋势,在5月龄时达到峰值,极显著地高于其他各个阶段。2个不同品种相比,3月龄和4月龄时,大白猪CAⅢmRNA表达量显著或极显著地高于马身猪;而在5月龄时,马身猪CAⅢmRNA表达量极显著地高于大白猪,其他阶段2个品种CAⅢmRNA表达量无显著差异。CAⅢ基因可能直接或间接参与猪骨骼肌的生长发育过程。

Carbonic anhydrase Ⅲ(CAⅢ) gene cloning and bioinformatics analysis were carried out,and the spatiotemporal expression characteristics of the CAⅢ gene were investigated in this study.The CDS sequence of CAⅢ gene of pig was obtained by RT-PCR and cloning and sequencing techniques,and the biological characteristics of CAⅢ protein were predicted by using various bioinformatics softwares.The mRNA expression patterns CAⅢ gene was conducted by qRT-PCR.The results showed that the CAⅢ CDS sequence of pig was 783 bp,which encoded a protein with 260 amino acids.CAⅢ protein was a hydrophilic basic protein with no signal peptide,which played a major role in the cytoplasm.CAⅢ gene was expressed in nine various tissues,such as in heart,liver,pancreas,lung,stomach,longissimus dorsi,subcutaneous fat,spleen,and cecum.The highest expression was in longissimus dorsi,which was greater than that in other tissues.The mRNA expression trend of CAⅢ gene in Large White pig was in the trend of ascending-descending-ascending-descending from birth to five-month-old.The expression was the lowest at birth,then increased with age increase,reached the peak at three-month-old,afterwards,the mRNA expression of CAⅢ gene was decreased.In Mashen pig,the mRNA expression of CAⅢ gene was increased with age increase from birth to five-month-old,and reached the peak at five-month-age,which was the greatest than those of other stages.Compared with the expression in these two pig breeds,the expression in Large White pig was greater than that in Mashen pig at three-month-old and at four-month-old.However,the expression in Mashen pig was greater than that in Large White pig at five-month-old.There was no significant difference between Large White pig and Mashen pig at other ages.The results in this study indicated that CAⅢ gene could participate directly or indirectly the process of growth and development of skeletal muscle in pig.