西藏大花红景天RcCATs与RcSODs基因克隆及功能分析

Cloning and fuction analysis of RcCATs and RcSODs genes in Tibet Rhodiola crenulata

关键词:  大花红景天, 过氧化氢酶, 超氧化物歧化酶, 生物信息学, 表达模式, 互作蛋白

Key words:   Rhodiola crenulata, catalase, superoxide dismutase, bioinformatics, expression pattern, interaction proteins

摘要:

为探究过氧化氢酶(CAT)和超氧化物歧化酶(SOD)基因家族成员在西藏大花红景天高原恶劣生境适应性中的作用,利用生物信息学和qRT-PCR技术对其RcCATs和RcSODs基因家族成员的序列和表达模式进行了分析,并构建RcCAT和RcSOD1的pYES2.0表达载体与pGBKT7诱饵载体,分别进行了酵母胁迫分析和拟南芥酵母文库中互作蛋白的筛选。结果显示:(1)大花红景天中共有2条RcCAT基因,3条RcSOD基因和1条Cu/Zn SOD铜伴侣基因RcCCS。生物信息学分析显示上述基因的氨基酸序列与其同源基因具有较高的相似性(66.37%～94.51%),且均没有跨膜结构域,亚细胞位置预测RcCATs位于过氧化物酶体,RcSODs和RcCCS位于细胞质或线粒体。(2)qRT-PCR结果显示6个基因在根、茎、叶三个器官中均有表达且主要在叶片中表达,低温胁迫和植物激素ABA对基因表达量的影响显著。(3)酵母胁迫结果显示异源表达RcCAT和RcSOD1基因的阳性酵母菌株在冷、热、盐、碱、重金属和H₂O₂胁迫下的细胞活力均比pYES2.0空载菌株高。(4)通过酵母双杂交共筛选到4个与RcCAT互作明显的基因AtbHLH121(AT3G19860)、AtCPCK2(AT2G23070)、AtGRP4(AT5G50750)和AtRAPTOR1B(AT3G08850),3个与RcSOD1互作明显的基因AtEMB(AT5G11890)、AtMBP2(AT1G52030)和AtRH8(AT4G00660)。综上结果表明,大花红景天能够通过RcCATs和RcSODs基因广泛参与调控其生长发育、胁迫响应等代谢途径来适应高原恶劣的环境。

Abstract:

In order to explore the role of catalase(CAT)and superoxide dismutase(SOD)in plateau harsh environment adaptation of Tibet Rhodiola crenulata, RcCATs and RcSODs gene family members were analyzed by bioinformatics and qRT-PCR. Spot assay was conducted to study the responses of yeast cells expressing the RcCAT and RcSOD1 genes under abiotic stress. Yeast two-hybrid was conducted to screen interacting proteins from Arabidopsis yeast library respectively by constructing bait vectors of RcCAT and RcSOD1. The results were as follows:(1)There were two CAT genes(RcCAT), three SOD genes(RcSOD), and one Cu/Zn SOD copper chaperone gene(RcCCS). Bioinformatics analysis showed that the above six genes held high sequence identity(66.37%-94.51%)with other homologous species. All genes had no transmembrane domain and held multiple phosphorylation amino acides. Subcellular localization predicted that RcCATs were located in peroxisoma, RcSODs and RcCCS were located in cytoplasm or mitochondria.(2)qRT-PCR analysis showed that RcCATs and RcSODs were constitutively expressed in three organs like root, stem and leaf and held the high expression levels in leaf, and all genes expression levels could also be regulated by low temperature and plant hormones(ABA). RcCAT was significantly up-regulated under cold treatment condition with the highest expression in leaf more than two times higher than root. RcCAT, RcSOD2, RcSOD3 and RcCCS expression patterns were similar under ABA treatment condition.(3)In addition, spot assay showed that the recombinant RcCAT and RcSOD1 yeast cells showed a higher cell viability than the pYES2.0 yeast cells in under cold, hot, NaCl, Na₂CO₃, Co²⁺ and H₂O₂.(4)The pGBKT7-RcCAT and pGBKT7-RcSOD1 bait plasmid without toxicity and auto-activation were constructed to perform yeast two-hybrid screening, then four significant interactional genes with RcCAT were screened, which were AtbHLH121 (AT3G19860), AtCPCK2 (AT2G23070), AtGRP4(AT5G50750)and AtRAPTOR1B (AT3G08850). Total three significant interactional genes with RcSOD1 were screened, which were AtEMB (AT5G11890), AtMBP2 (AT1G52030)and AtRH8 (AT4G00660). These results illustrate that RcCATs and RcSODs play an important role in regulating growth and promoting resistance to environmental stresses in Tibet Rhodiola crenulata, and laid the foundation for in-depth study of the adaptive mechanism of R. crenulata with plateau environment.