西藏大花红景天RcCATs 与RcSODs 基因克隆及功能分析 postprint

Abstract: In order to explore the role of catalase (CAT) and superoxide dismutase (SOD) in plateau environment adaptation of Tibet Rhodiola crenulata, the RcCATs gene family members and RcSODs gene family members were analyzed by bioinformatics and real-time quantitative 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, three SOD genes, and Cu/Zn SOD copper chaperone gene (RcCCS), 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 located in peroxisoma, RcSODs and RcCCS 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 illustrated that RcCATs and RcSODs could 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 Rhodiola crenulata with plateau environment.