两个菜心CBL基因的克隆、生物信息学分析及其表达特性分析

Cloning,Bioinformation Analysis and Expression Characteristics of Two CBL Genes in Flowering Chinese Cabbage

本研究以‘油绿501’菜心(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee)为材料,采用同源克隆方法对其CBL1、CBL9基因进行克隆,运用DNAMAN和MEGA软件对其编码蛋白序列进行同源性分析和系统进化分析,并利用qRT-PCR技术分析二者在菜心组织中及不同氮素形态条件下的表达模式。结果表明:菜心CBL1、CBL9基因的cDNA全长均为642 bp,编码213个氨基酸,分别命名为BcCBL1和BcCBL9,具有CBL蛋白结合Ca2+所必需的EF-hand型结构域(EFh)。BcCBL1、BcCBL9编码的氨基酸序列与拟南芥(Arabidopsis thaliana)、大白菜(Brassica rapa)等物种的同源性均达95%以上;二者分别与大白菜的BrCBL1、BrCBL9遗传距离最近,并与拟南芥AtCBL1、AtCBL9共同聚类为一支。BcCBL1和BcCBL9在菜心各组织器官中均有表达,前者在菜心叶片中表达水平较高,后者在菜心荚果等组织中表达水平较高;两者表达受氮素形态及水平影响,前者在低浓度NO_(3)^(-)或NH_(4)^(+)处理后上调表达,后者表达随NH_(4)^(+)浓度增加而上调表达。该研究结果可为进一步研究菜心CBL1和CBL9在氮素吸收利用过程中的功能及其调控机制提供一定理论基础。

In this study,the CBL1 and CBL9 genes were cloned by homologous cloning method from flowering Chinese cabbage(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee)variety‘Youlü501’,whose homology and phylogenetic analysis of protein sequences were analyzed using DNAMAN and MEGA software.Additionally,the expression patterns of the two CBL genes in tissues of flowering Chinese cabbage and under different nitrogen forms were investigated by qRT-PCR method.The results showed that the full-length cDNA sequences of CBL1 and CBL9 genes were both 642 bp,which encoded 213 amino acids and named BcCBL1 and BcCBL9,respectively.Both the two CBL genes possessed EF-hand domain(EFh),which was necessary for CBL protein to bind Ca2+.Homology analysis showed that the homology of the two CBL genes of flowering Chinese cabbage with those of Arabidopsis thaliana and Brassica rapa were higher than 95%.Ac-cording to the results of phylogenetic analysis,it was found that BcCBL1 and BcCBL9 were the most closely related with BrCBL1 and BrCBL9 of B.rapa,and clustered into the same branch with AtCBL1 and AtCBL9 of A.thaliana,respectively.According to the results of qRT-PCR analysis,it showed that BcCBL1 and BcCBL9 were expressed in different tissues of flowering Chinese cabbage,BcCBL1 gene expressed higher in leaves and BcCBL9 gene expressed higher in pods.In addition,the expression levels of BcCBL1 and BcCBL9 were affect-ed by the forms and levels of nitrogen.BcCBL1 was up-regulated at the lower concentration of NO_(3)^(-) or NH_(4)^(+),while the expression of BcCBL9 was up-regulated with the concentration increasing of NH_(4)^(+).These results pro-vided theoretical bases for further studying the functions and regulation mechanisms of CBL1 and CBL9 of flow-ering Chinese cabbage in the process of nitrogen uptake and utilization.