青稞类钙调素蛋白基因CML19的克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of CaM-like Protein Gene CML19 from Tibetan Hulless Barley(Hordeum vulgare L. var. nudum HK.f.)

根据鉴定得到青稞类钙调素蛋白基因CML19的序列设计引物,以青稞叶片的cDNA为模板,扩增得到目的片段,对目的基因序列进行鉴定和分析,进一步构建该基因的原核表达载体pET28a-CML19,转入大肠杆菌BL21(DE3),利用IPTG诱导外源蛋白质表达并检测。结果表明,扩增得到CML19的CDS序列全长为447bp,编码的蛋白质含有148个氨基酸,分子质量为16.46ku,理论等电点(pI)为4.40,总平均亲水性(GRAVY)为-0.176,不稳定系数为49.59,存在典型的EF手结构域;系统进化分析表明,青稞CML19与山羊草具有较近的亲缘关系;原核表达蛋白结果显示,重组蛋白主要以包涵体形式存在于沉淀中。初步阐明青稞CML19基因的序列特征,为进一步制备抗体、探讨CML19基因的功能奠定基础。

The primers were designed according to the sequence of the identified Tibetan Hulless bar- ley CaM-like protein gene CML19 , and target fragment was amplified from the cDNA of the barley leaf. After that, target sequence was sequenced and analysed. Prokaryotic expression vector pET28a- CML19 was constructed and transformed into E. coli BL21（DE3）. At the same time, positive bacteria was induced by IPTG to express and detect foreign proteins. The results showed that, the full CDS of CML19 was amplified and the size was 447 bp. According to the results of bioinformatics analysis,CML19 encodes a protein containing 148 amino acids with a relative molecular mass of 16.46 ku, a theoretical isoeleetric point （pI） of 4.40, a total average hydrophilicity （GRAVY） of -0. 176, an un- stable coefficient of 49.59, aEF-hand domain. Phylogenetic analysis showed that the CML19 had a close genetic relationship with Aegilops tauschii. The results of protein expression showed that the recombinant protein was mainly precipitated in the form of inclusion bodies. In this study, we first elucidated the sequence characteristics of CML19 gene in Tibetan Hulless Barley, laying a foundation for further preparation of antibody and exploring the function of CML19 gene.