大豆CML38-like基因的克隆及表达载体构建

类钙调蛋白(CaM-likeprotein,CML)在植物抵抗逆境中发挥了重要的作用,因此研究CML基因是植物抗逆分子育种的一个重要分子基础。本实验室在前期工作中筛选得到1个片段大小为264 bp的大豆CML基因,对该基因序列设计特异性引物,利用PCR技术对该基因进行扩增,并获得大小为264 bp的片段,利用无缝克隆技术连接到pMD18T载体上,获得克隆载体。通过对该基因的核苷酸序列分析,在NCBI上发现该基因序列与大豆Calcium-binding protein(CML38-like)基因序列相似度为98.53%。目前对CML38-like基因的功能还未见报道,本研究根据克隆测序得到的基因序列构建系统进化树并分析,随后对该基因的蛋白质二级结构和蛋白质三级结构进行预测,发现该基因可能对大豆抗旱有着一定的影响。最后通过克隆载体构建CML38-like基因的过表达载体以及RNA干扰表达载体。本研究为鉴定大豆CML38-like基因的功能和了解该基因对大豆的非生物胁迫反应的影响提供了理论依据。

Cloning and Expression Vector Construction of CML38-like Gene in Soybean

Calmodulin-like protein(CaM-likeprotein,CML)plays a crucial role in plant stress resistance,so study of CML gene is a significant molecular basis for plant stress resistance in molecular breeding.In the previous work,a soybean CML gene with fragment size of 264 bp was acquired by our laboratory.Specific primers were designed for this gene sequence.The gene was amplified by PCR,and a fragment of 264 bp was obtained.The fragment was connected to the pMD18T vector by seamless cloning technique,then the clone vector was obtained.By analyzing the nucleotides sequences,it was found that the similarity between this gene sequence and the soybean calcium-binding protein(CML38-like)gene sequence was 98.53%on NCBI.At present,the function of CML38-like gene has not been reported,in this study,the phylogenetic tree was constructed and analyzed according to the gene sequences obtained by cloning and sequencing.Based on those,we predicted the protein secondary structure and protein tertiary structure of the gene.It was found that this gene may have a certain effect on soybean drought resistance.Finally,the over-expression vector and the RNA interference expression vector of CML38-like gene were constructed by cloning vector.This study which provides a theoretical basis for identifying the function of soybean CML38-like gene and its effect on abiotic stress response of soybean.