巴西橡胶树HbCytc1的克隆及表达分析

Cloning and Expression Analysis of HbCytc1 in Rubber Tree(Hevea brasiliensis Muell. Arg.)

橡胶树寒害和干旱逆境响应均与活性氧代谢和淬灭有关,研究表明细胞色素c1通过参与抗坏血酸的再生在抗旱、活性氧淬灭等逆境中发挥作用。为研究细胞色素c1基因在橡胶树寒害和干旱逆境响应中的作用机制,采用5'/3'末端快速扩增技术从巴西橡胶树‘热研7-33-97’叶片中克隆了一个细胞色素c1基因,通过生物信息学软件分析,可知其推导氨基酸含有特征性Cyt_c1家族结构域,将其命名为HbCytc1。HbCytc1全长1 351 bp,ORF933 bp。根据c DNA序列推导出该蛋白氨基酸,利用DNAman与其他植物中的同源Cytc1基因进行序列比对,发现橡胶树HbCytc1与蓖麻RcCytc1(XP_002509561.1)、毛果杨PtCytc1(XP_002313590.1)、克莱门柚CcCytc1(XP_006441506.1)和烟草NsCytc1(XP_009804309.1)氨基酸的相似性分别为91%、92%、89%和87%。系统进化分析显示,HbCytc1与RcCytc1、PtCytc1位于同一分支。表达分析结果显示,该基因在花、叶片和胶乳中表达,在树皮中不表达。在干旱、机械伤害和白粉菌侵染处理下,均显著上调该基因在橡胶树叶片中表达。结果表明HbCytc1受逆境反应诱导,参与橡胶树抗逆过程。为揭示细胞色素c1的功能和橡胶树抗逆响应的研究提供理论指导。

Cold damage and drought stress response are both related to active oxygen metabolism and quenching in rubber tree. Cytochrome c1 plays an important in drought tolerance and reactive oxygen quenching and other adverse effects by participating in the regeneration of ascorbic acid. In order to study the mechanism of cytochrome c1 gene in the response of cold damage and drought stress of rubber tree, rapid amplification technology of 5＇/3＇terminal was used to obtain the cytochrome c1 gene by cloning CATAS7-33-97 leaves of rubber tree. Through bioinformatic software analysis, characteristic Cyt_c1 family structure domain was observed in the deduced amino acid of the gene and was named as HbCytc1. It was 1 351 bp in length, containing a 933 bp ORF. According to the c DNA sequence, the amino acid of the protein was deduced, and then the sequence of the protein was compared with that of the homologousCytc1 gene in other plants by DNAman. The similarities of HbCytc1 in rubber tree between and RcCytc1 in Ricinus communis（XP_002509561.1）, PtCytc1 in Populus trichocarpa（XP_002313590.1）,CcCytc1 in Citrus clementina（XP_006441506.1）, and NsCytc1 in tobacco（XP_009804309.1） were 91%, 92%, 89%and 87%, respectively. Phylogenetic analysis showed that HbCytc1 was on the same branch with RcCytc1 and PtCytc1. Expression analysis result showed that the gene was expressed in flower, leaf and latex, while not in bark.Drought, mechanical wounding and powdery mildew infection treatments all upregulated its expression in rubber tree leaves at significantly level. These results suggested that HbCytc1 was induced by adversities, and played roles in stress response in rubber tree, which would provide theoretical guidance for the study of cytochrome c1 function and the stress resistance of rubber tree.