酿酒酵母抗镉基因CAD1的克隆和分析

目的:克隆CAD1基因,对其结构和功能进行预测,为后期研究利用该基因用于重金属镉的解毒作用及其他有毒物质的抗性研究打下基础。方法:使用PCR技术对酿酒酵母BZL06的CAD1基因序列进行全长克隆,再对其进行染色体定位和亲缘关系分析,使用在线分析工具ExPASy、ProtParam等对其编码蛋白质的一级结构、二级结构、三级结构、结构域和蛋白互作进行预测分析。结果:该CAD1基因位于Ⅳ染色体1318046~1319275;全长1230 bp,可编码409个氨基酸,亲缘关系分析表明该基因与酿酒酵母CEN.PK113-7D菌株CAD1基因(GenBank序列号为EIW11633.1)最为接近,同源性达到99%;编码的蛋白质为在细胞核中进行代谢调控的不稳定的亲水蛋白;存在明显的卷曲螺旋区域,不存在跨膜结构,二级结构预测中发现该蛋白主要存在α-螺旋和随机卷曲,β-转角与延伸链分散分布;预测存在bZIP和PAP1结构域;蛋白互作分析中相关系数0.7以上的蛋白有9个,其中相关系数为0.937的SKN7蛋白是CAD1蛋白。结论:分析结果得出克隆CAD1基因正确,其编码的蛋白结构不稳定,在细胞核中代谢,含有与重金属镉与其他有毒物的过表达中存在抗性的表达紧密相关的结构域bZIP和PAP1,在表达抗性时并与SKN7蛋白功能联系密切。

Cloning and Analysis of Cadmium Resistant Gene CAD1 of Saccharomyces cerevisiae

Objective:The CAD1 gene was cloned and its structure and function were predicted.It laid a foundation for later research on the detoxification of heavy metal cadmium and the resistance of other toxic substances.Methods:The full-length clone of the CAD1 gene sequence of Saccharomyces cerevisiae BZL06 was performed by PCR,and then the chromosomal location and phylogenetic relationship were analyzed.The primary and secondary structures,tertiary structure,domain and protein interaction of the protein were predicted by online analytical tools such as ExPASy and ProtParam.Results:The CAD1 gene was located on chromosomeⅣ1318046~1319275;The full length was 1230 bp,encoding 409 amino acids.The phylogenetic analysis indicated that the CAD1 gene of the S.cerevisiae CEN.PK113-7 D(EIW11633.1)was the closest one and the homology between them was 99%.The encoded protein was an unstable hydrophilic protein which was metabolically regulated in the nucleus;there was a distinct coiled-coil region,and there was no transmembrane structure.In the prediction of the structure,it was found that the protein mainly containedα-helix and random coil,β-turn and extended chain dispersion distribution;bZIP and PAP1 domains were predicted to be existed;the result of protein interaction analysis showed that there were 9 proteins whose value was more than or equal to 0.7,and the interaction index of SKN7 protein was the highest one(0.937).Conclusion:The results showed that the cloned CAD1 gene was correct,and its encoded protein was structurally unstable and metabolized in the nucleus.It contained bZIP and PAP1 domains,and closely related to the expression of resistance in the overexpression of heavy metal cadmium and other toxicants.It was closely related to the function of SKN7 protein when expressing resistance.