蓖麻钙依赖蛋白激酶29基因克隆与表达分析

Cloning and Expression Analysis of Castor Calcium Dependent Protein Kinase 29 Gene

为探讨蓖麻钙依赖蛋白激酶29基因(RcCDPK29)在蓖麻耐盐中的作用,以蓖麻叶片为材料,设计特异性引物,克隆蓖麻钙依赖蛋白激酶29基因(RcCDPK29),并对所得序列进行生物信息学分析。结果表明,蓖麻钙依赖蛋白激酶29基因(RcCDPK29)序列全长1590 bp;编码528个氨基酸;蛋白分子量为59.74 ku;等电点(pI)值6.21;是典型的非跨膜蛋白;亲水性数值为负值,属于亲水性蛋白;RcCDPK29蛋白α-螺旋占比最高有228个;RcCDPK29与拟南芥CDPK(SMTL ID:3q5i.1)相似度为40.41%,具有较高可信度(>30%)。将木薯、麻枫树、巴西橡胶树、柑橘、石榴、毛果杨与蓖麻RcCDPK29氨基酸序列进行同源性比对。其中,与麻枫树的同源性最高,为82.29%。RcCDPK29蛋白包含1个Ser/Thr蛋白激酶催化结构域和4个与Ca^(2+)结合的EF-hand型结构域。通过qRT-PCR技术,分析RcCDPK29在不同水平盐胁迫下蓖麻不同组织中的表达,结果表明,RcCDPK29基因主要在茎中表达,盐处理12 h表达量最高。随着盐处理时间的延长,RcCDPK29基因根的表达量逐渐下降,分别在2,8,24 h达到最低,与0 h差异显著;叶的表达量在2 h的表达量最低且与0 h相比差异显著。根据RcCDPK29全长设计带有SmaⅠ和XbaⅠ酶切位点的引物扩增出序列全长,用SmaⅠ和XbaⅠ进行双酶切后与表达载体pCG-3300连接。成功构建了CRcCDPK29的表达载体。因此,RcCDPK29在蓖麻受到盐胁迫时起重要作用。

To investigate the role of RcCDPK29 in salt tolerance of castor,we designed specific primers to clone the calcium-dependent protein kinase 29 gene(RcCDPK29)from castor leaves and performed bioinformatics analysis of the resulting sequence.The results showed that the sequence of RcCDPK29 was 1590 bp,encoding 528 amino acids,with a molecular weight of 59.74 ku and an isoelectric point(pI)value of 6.21.It is a typical non-transmembrane protein with a negative hydrophilic value and a hydrophilic protein with a maximum of 228α-helices.The similarity of RcCDPK29 to Arabidopsis CDPK(SMTL ID:3q5i.1)was 40.41% with high confidence(>30%).The amino acid sequences of Manihot esculenta,Jatropha curcas,Hevea Brasiliensis,Citrus sinensis,Punica granatum,Populus trichocarpa and Ricinus communis RcCDPK29 were compared for homology.Among them,the highest homology with Jatropha was 82.29%.The RcCDPK29 protein contained a Ser/Thr protein kinase catalytic domain and four Ca^(2+)-binding EF-hand-type structural domains.The expression of RcCDPK29 in different tissues of castor under different levels of salt stress was analyzed by qRT-PCR.The results showed that the RcCDPK29 gene was mainly expressed in the stem,with the highest expression at 12 h of salt treatment.With the extension of salt treatment time,the expression of RcCDPK29 gene in roots gradually decreased and reached the lowest at 2,8,24 h,respectively,with significant differences from 0 h;the expression of leaves was the lowest at 2 h and significantly different compared with 0 h.The full length of RcCDPK29 was amplified by designing primers with SmaⅠ and XbaⅠ digestion sites,and then ligated with the expression vector pCG-3300 after double digestion with SmaⅠ and XbaⅠ.The expression vector of RcCDPK29 was successfully constructed.Thus,RcCDPK29 plays an important role when castor is subjected to salt stress.