Characterization Of DsCOR Protein From Descurania Sophia(L.) Webb And Functionin Research In Arabidopsis Mutant

Temperature is one of important environmental factors for plantsbecause temperature limits the geographical distribution and growing of mostplant species. Low temperature may hurt plants variously even make themdead. Every year low temperature stress caused adverse effects on the growthof plants and the productivity of crops and made large loss.The enhancementof freezing tolerance for crops has significant meanings for the improvementof crops output.In 1970, Weiser found that cellular gene expression changed during coldacclination.Research on plant cold tolerance developed to molecular level.Atpresent, people has carried out widespread research on cold inducegenes.COR gene (cold-regulated gene) is a kind of gene could enhancefreezing tolerance for plants.Its expression need to be induced by the stressesjust as low temperature, drought or salt loading.The COR gene which isstudied most clearly is COR15a cloned from Arabidopsis thaliana.Just like Arabidopsis thaliana, Descurania sophia(L.) Webb iscruciferae.But Descurania sophia(L.) Webb has much more tolerance to lowtemperature than Arabidopsis thaliana.This kind of plant can blossom at 0℃, and the seeds can grow at -40℃.But at present there is very littleresearch about it. We use Descurania sophia(L.) Webb as our material to research its cold-resist mechanism at the gene level in order to develop thisgood cold-resist material. It is useful to improve freezing tolerance for otherplants though genetic engineering.Our lab has made some research on physiological nature of Descuraniasophia(L.) Webb and successfully cloned the gene DsCOR (cold-regulatedgene) from Descurania sophia(L.) Webb.Bioinformation analysis suggestedthe DsCOR gene was high homologous to Arabidopsis thaliana COR15a.Onthis base, we made further research about the character of DsCOR protein,and analyzed the founction of DsCOR gene in Arabidopsis mutant .Themain results are as followed:1. DsCOR mRNA was induced only transiently by low temperature stress.In order to analyze the effects of low temperature on the gene expression ofDsCOR, semi-quantitative RT-PCR was performed with an internal standardof 18S rRNA from Descurania sophia(L.) Webb. (1) The results showedDsCOR mRNA levels correlated with the low temperature. The transcript ofDsCOR which could not be detected at normal temperature, were induced bya 10min low temperature treatment of 4℃. When plant was treated with lowtemperaturement 1h, the transcript amounts of DsCOR was highest. Aslong as low temperature treatment, the transcript amounts of DsCOR kepthighest. (2) The result showed that DsCOR mRNA was induced onlytransiently by low temperature. The mRNA amounts began to reduce aftertransferring the plants from low temperature to normal temperatureconditions. The results of such a recovery experiment demonstrated thatDsCOR mRNA was short-lived. Under the treatment of low temperature,the mRNA accumulated rapidly in 10min. When temperature rised to thenormal condition, the mRNA degraded quickly within 30min.Then as longas normal temperature, DsCOR mRNA expression was gradually recoveredto the normal level. (3) At the critical temperature of 8℃the expression ofDsCOR mRNA could be detected. Ⅱ. Using E.coli expression and protein purification system, DsCOR genewas expressed and purified. In order to study the mechanism of DsCORenhancing plants cold tolerance, E.coli expression and protein purificationsystem was used in the paper. The ORF of DsCOR was amplified by PCR,and cloned into expression vector pET32a to construct recombinantexpression plasmid pET32a-DsCOR. The recombinant plasmid wasstransformed into E.coli BL21 and induced to express recombinant proteinwith IPTG. According to hydrophilic character, The fusion protein wasfurther purified through (1) boiling and affinity chromatography(2)electroelution in dialysis bags; concentrated by PEG; analyzed bySDS-PAGE. Restriction enzyme digestion and DNA sequencing analyssuggested that the recombinant expression plasmid contained correct codingregion of DsCOR. SDS-PAGE demonstrated that the recombinant proteinwas expressed w ith the expected molecular weight at 31kD.The E.coli wasinducd by 1mmol/L IPTG at 37℃for 3 hours, the most recombinant proteincan be obtained.After purified and concentrated, the concentration ofDsCOR fusion protein was 1.11mg/mL.The basic works would help us to dofarther research in DsCOR protein.Ⅲ. Research about protein resistance to heat, acid, alkali and UVdemonstrated DsCOR fusion protein has strong stability. After boiling 4h,DsCOR protein remained good dissolved state. The acid and alkali resistanceof DsCOR protein was strong and the pH range was 3.0-10.5.The longesttime DsCOR protein could tolerate UV is 100μmol m^-2s^-1 for 12h. IfDsCOR protein was exposed to UV longer than 12h, the protein wouldbegin its degradation. The strong stability of antifreeze protein DsCOR wasvery important in enhancing freezing tolerance for plants.Ⅳ. A rabbit was immunized with the purified DsCOR fusion protein toproduce polyclonal antibody, and the production of antibody was confirmedby EL ISA .Rabbit polyclonal antibody was obtained, the titer of which was 1:12800. Western bloting results indicated that DsCOR protein is induced bylow temperature and the expression of DsCOR gene on protein level isregulated by low temperature. This result is agreement with chapter one.Ⅴ. By the floral-dip method, DsCOR gene was transformed intoArabidopsis COR15a mutation. In order to assure the DsCOR gene function,we used an Arabidopsis COR15a gene mutant (RIKEN BioResource CenterNo.PST15781) as transgenic plant material. Firstly, the vectorpCAMBIA2301G-DsCOR was constructed. Then thepCAMBIA2301G-DsCOR vector was introduced into Agrobacteriumtumefaciens EHA105 using the freeze-thaw method. Thirdly, theArabidopsis COR15a mutation was transformed on a large scale by thefloral-dip method. Finally, we got a lot of transformed seed.Ⅵ. Constitutive expression of DsCOR genes in Arabidopsis mutant restoredthe low temperature tolerance of the plants to the wild-type level and thefindings indicated that DsCOR fulfill a function to enhance freezing toleranceof plants. We got 68 Kmamycin resistant plants. The transformed plants weretransplanted into vermiculite: sand (1:1) medium. Transgenic plants withDsCOR gene were obtained and their target gene was detected through PCRanalysis in this study. For function analysis, the Arabidopsis mutant wasexposed to low temperature stress, resulting in leaf bleaching, wilting andextensive freezing damage. Constitutive expression of DsCOR genes inmutant after low temperature stress resulted in DsCOR accumulation andrestored the freezing tolerance of the plants to the wild-type level. Ourfindings indicated that DsCOR could play a part in enhancing the tolerance tolow temperature stress of the plants.Our research about DsCOR gene and DsCOR protein has significantmeanings for making clear the reason why Descurainia sohia has strongfreezing tolerance and the molecular mechanism of cold acclimation. DsCORantifreeze geen and DsCOR antifreeze protein will have good prospect for improving the cold resistance of plants through genetic engineering method.