| Chrysanthemum(Chrysanthemum grandiflorum (Ramat.) Kitam.), as one of the four most popular cut flower over the world and one of the ten most famous flowers in China, provides very high ornamental and economic value, which possess the important status on flower industry. However, low temperature in winter in the North and Region of Yangtze in China seriously affects growth and development of Chrysanthemum and leads to lower ornamental characteristics, which has become a limitive factor on Chrysanthemum industry development. The research on cold-tolerance and new cultivar breeding is a fundamental approach to solve the problem. Chrysanthemum related genera carrying stress-resistent ability that is in absence in cultivar can play important worthiness for broadening germplasm and gene pool of Chrysanthemum. Therefor, cold-tolerance selection of Chrysanthemum related species is performed to achieve resistant germs based on the further analysis about their physiological and molecular mechanisms for cold-tolerance, which is benefit for pushing forward the germplasm enhancement and new cultivar breeding of Chrysanthemum. The contents and results on this research are as follows:1. Evaluation for cold-tolerance of45Chrysanthemum related genera was analyzed based on the method of Semi-lethal temperature (LT50) of rhizomes leaf. Cold tolerance was classified, strong cold tolerance (LT50<-20℃):C.zawadskii, C.indicum (shenyang), C. dichrum, O. taihangensis and C.indicum (beijing); moderate cold tolerance (-20℃<LT50<-10℃):A. vulgaris, C.indicum (chengdu), C.nankingense; weak cold tolerance (LT50>-10℃):C.makinoi, C.indicum (shennongjia), A.spathulifolius. Further confirmation via rhizomes recovery growth exhibited negative correlation between survival rate after freezing-treatment and LT50, which indicated that LT50could be a reliable index for cold-tolerance evaluation of Chrysanthemum related genera.2. The difference of cold-tolerance between strong cold-tolerant C. dichrum and weak cold-tolerant C.makinoi under cold acclimation was analyzed. The results showed LT50presented different degree decrease under cold acclimation between the two Chrysanthemum species, C. dichrum varing from-7.3℃to-23.5℃and C.makinoi from-2.1℃to-7.1℃. Activities of antioxidant enzymes (SOD, CAT and APX) and proline content were measured, and antioxidant isoenzymes gene (Cu/Zn SOD、Fe SOD、Mn SOD、 CAT and APX), proline related-metabolism gene (P5CS, OAT and PDH) and cold-regulated gene (two DREB family genes, two COR413family gene and two CSD family gene) obtained from Chrysanthemum EST library were used for quantitive expression analysis. The results showed that cold acclimation promoted the activities increase of SOD, CAT and APX and proline accumulation, and C. dichrum presented higher varing degree than C.makinoi. Gene expression assay indicated that SOD activity promotion resulted from Mn SOD expression increase, CAT and APX expression exhibited consistent changes with their enzymes activities, P5CS and PDH expression showed similar alteration with proline content. Higher extent was detected on Mn SOD、CAT、APX and P5CS expression in C. dichrum comparing to in C.makinoi. Cold-regulated gene analysis indicated DREBs, COR413s and CSDs expression up-regulated under cold acclimation, and C. dichrum presented higher increase degree than C.makinoi. Further analysis revealed cold-tolerance improvement during the early days resulted from the functions of antioxidant enzymes, proline content and cold-regulated gene expression, and improvement during the late days origined from Mn SOD, COR413s and CSDs expression.3. DREB family genes play important roles in regulating cold-tolerance. To investigate the upstream regulating passway of DREB gene, a1474bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB-site, GT-1and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-β-glucuronidase (GUS) gene fusion, serially5’-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream430and351bp. This80bp fragment enhanced GUS expression under salinity stress when fused to-90/+8CaMV35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature responsive element was located between positions-430and-390, and a salinity inducible one between-385and-351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.4. ICE family genes are very important for regulating cold-tolerance passway. An ICE family homologue gene CdICE1was separated from Chrysanthemum dichrum, encoded471amino acids, which showed high similarity with ICE homologues from other species. Quantitive analysis revealed that CdICE1expression was induced under low temperature, salt and ABA treatment. CdICE1promoter within1682bp region upstream translation initiation site was identified, including some stress-responsve elements by sequence analysis. The assay that35S::CdICE1-GFP expression vector was bombarded into onion epidermal cells showed CdICE1was located in nucleus. CdICE1were expressed in Pichia pastoris system and purified, and then the specific binding of CdICE1to MYC element within the CdDREBa promoter was found through EMSA assay. Yeast one-hybrid assay presented that CdICE1had transcriptional activation activity. These data showed ICE1-DREB passway would exist in Chrysanthemum dichrum.5. For identifying the cold-regulated functions of CdICE1, CdICE1was transformed to Arabidopsis and freezing-tolerance was detected by phenotype analysis, then molecular mechanism of freezing-tolerance was further explored. The results indicated that Arabidopsis freezing-tolerance was enhanced after different temperature acclimation, and heterologous expression CdICE1improved the freezing-tolerance under cold acclimation. Under4℃acclimation, CdICE1promoted expression of CBFs and their downstream freezing-tolerance gene including COR15a, COR6.6and RD29a, which revealed similar function with Arabidopsis AtICE1. However, under16℃acclimation, CdICE1only increased mildly three freezing-tolerant genes, while no changes on CBFs expression.16℃acclimation downregulated miR398and upregulated CSD1and CSD2expression, miR398negatively modulated freezing-tolerance and CSDs positively regulated freezing-tolerance, which indicated miR398-CSDs pathway played a role in16℃acclimation inducing freezing-tolerance. Overexpression CdICE1plants revealed significant reduction of miR398under16℃acclimation, comparing to WT plants. CdICE1mediated miR398-CSDs passway and then led to freezing-tolerance. Therefor, CdICE1affected freezing-tolerance via two different passway under two different cold acclimation conditions.6. ICE1modification was found in previous research. To improve the research on CdICE1regulating mechanism, CdICE1-interacting proteins were screened by yeast two-hybrid system. A cDNA yeast mixture library was successfully constructed according to Clontech kit. A total of64candidate positive clones were sequenced and analyzed through homology analysis using the BLAST in NCBI, which showed that those candidate proteins were related to photosynthesis, oxidative stress and post-translational modification. Function prediction of the candidate proteins suggested that CdICE1was possibly involved in several stress signal transduction pathways and played an important role in regulation stress resistance. |
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