应用nested和touch down PCR技术分离牦牛CAPN1大亚基基因EST

Isolation of EST for CAPN1 of Bos grunniens with nested and touch-down PCR

钙激活中性蛋白酶1(CAPN1)基因是影响肉嫩度的侯选基因。根据GeneBank中已公布的CAPN1基因cDNA序列设计同源PCR引物,通过RT,nested和touch-downPCR技术从牦牛肌肉组织总RNA中分离目的序列,与pGEM-T-easy载体连接后转化DH5α感受态菌株,通过PCR扩增、限制性酶切和测序分析鉴定阳性克隆。分离到的基因片段为牦牛CAPN1大亚基的部分编码序列,在GeneBank中的注册号为AY197555,与奶牛、人、食蟹猴、小鼠、绵羊以及大鼠相应序列的同源性分别为98%,89%,89%,83%,96%和81%,在不同物种之间具有较强的保守性。CAPN1基因EST的分离为进一步研究牦牛CAPN1基因奠定了基础。此研究表明,在总RNA质量不理想的情况下,同时采用这2种技术可以较大限度地获取目的序列。

CAPN1 gene was investigated as candidate gene affecting meat tenderness.With the primers designed according to cDNAs encoding CAPN1 published in GeneBank,RT,nested and touch-down PCR were used to isolate EST of total RNAs from muscle of Bos grunniens.The PCR product was ligated to pGEM-T-easy vector,and then E.coli DH5α strain was transformed.Positive clones were identified by restriction endonuclease,PCR and sequencing.The partial sequence encoding CAPN1 of Bos grunniens was identified,the corresponding ESTs of Bos taurus,Homo sapiens,Macaca fascicularis,Mus musculus,Ovis aries and Rattus norvegicus shared 98%,89%,89%,83%,96% and 81% homology with that of Bos grunniens respectively.It indicated that the EST was comparatively conservative between different species.Isolation of the EST has made substantial basis for further research on CAPN1 gene of Bos grunniens.The result of this research suggests that the combining use of nested and touch-down PCR is an effective method in EST isolation even though total RNAs are partially degraded.