Functional Characterization Of Chorispora Bungeana Calreticulins On Salt-Stress

Chorispora bungeana is a rare alpine plant species grown in headwater region in the middle piece of Tianshan Mountains in Wulumuqi, which could adapt to the ecotope with anoxia, blast, heavy radiation and acute temperature fluctuation caused by freeze thawing. Chorispora bungeana has no obvious morphologic characteristics responsible for stress adaption. The tolerance to environmental conditions mainly depended on cytolergy and gene regulation. Thus this plant species had been considered as a typical material for exploring molecular mechanism underlying plant abiotic stress response. In this research, we used Chorispora bungeana as the experimental material, and focused on the role of CbCRT1(CbCalreticulins,CbCRT1)in response to stress conditions. We tested the tissue expression specificity and subcellular localization. We also used the reverse genetic approaches such as overexperssion and knock-out of this gene in cell to study the concrete function of CbCRT in abiotic stress response. The results were as follow.Firstly, Expression analysis of CbCRT1in different tissues assessed by semi-quantitative (RT-PCR). Results showed that CbCRT1expressed in root, stem, leaf and fruit, with little lower in root. CbCRT1had no tissue specificity.Secondly, subcellular localization analysis of CbCRT1by linking GFP fluorescin in cell.We constructed pEarleyGate103-CbCRT1(P:35S-CbCRT1-GFP), and transiently expressed CbCRTl-GFP fusion in Nicotiana benthamiana epidermal cells. Results showed that subcellular localization of CbCRTl was on the cytoplasmsma membrane.Thirdly, cellular calcium ion response to NaCl, exogenous ABA treatment assessed by flow cytometer. Results showed that the concentration of cellular calcium ion rised0.5minutes after NaCl treatment,5minutes later, the concentration began to descend, until10mins after the treatment, when it leveled off as much as control. Trends of cellular calcium ion concentration under exogenous ABA treatment were similar to NaCl treatment,but changes were more obvious. This fluctuations revealed cellular calcium ion played second message in singal transduction during stress.Fourthly, anlysis of transcriptional expression and translational expression of CbCRT1under NaCl and exogenous ABA treatments during0-12h assessed separately by semi-quantitative (RT-PCR) and Western-blot. Results showed that the levels of transcriptional and translational expression of CbCRT1both increased under NaCl stress during0-12h. It revealed that CbCRT1played a positive role when plants encountered NaCl stress. The level of transcriptional expression decreased within3h, and then increased to control amount till12h under exogenous ABA treatments. It revealed that CbCRT1was regulated only in transcription level by exogenous ABA.Fifthly, analogical phenotype research was carried out on wild type and gene knock off mutant (crt1) in Arabidopsis thaliana. Phenotype during growth and development was detected. The results showed that no differences were detected between crt1and WT plants based on their vegetable growth. Meanwhile, under NaCl treatment, no differences were detected between crt1and WT plants on germination and the length of root.Lastly,15positive transgenic T2plants have been derived.we constructed plasmid(P:35S-CbCRT1-GFP).The expressional vector were transformed Arabidopsis thaliana by dipping flower.Above all, CbCRT1played active function during abiotic stress of plants. Research on CbCRT1provided a new genetic resources and tools for resisting stress of plants, which had theoretical significance and applied prospect.