Prokaryotic Expression Of Cold Adapted Gene Cpcor413pm1 From Chimonanthus Praecox And Its Overexpression In Arabidopsis Thaliana

Low temperature damage has caused great losses to the world’s agricultural production.In the process of plant resistance to low temperature,there will be a variety of protective proteins expressed in the bodies.COR（cold-regulation）genes belong to plant cold regulation genes,which play a stabilizing role in cell lipid membrane,so that plants can resist certain cold.In addition,previous studies have shown that Cpcor413pm1 protein is related to abiotic stress resistance of plants.In order to clarify the specific function of Cpcor413pm1 gene in the cold resistance of Chimonanthus praecox,the recombinant protein Cpcor413pm1 of Chimonanthus praecox was obtained by prokaryotic expression system,and the stress resistance of E.coli overexpressing Cpcor413pm1 gene was analyzed;meanwhile,the Cpcor413pm1was verified by in vitro enzyme activity test.The protective effect of protein on superoxide dismutase（SOD）under low and high temperature stress.In addition,the plant overexpression vector was constructed and transformed into Arabidopsis thaliana for abiotic stress function analysis.Finally,we hope that through the study of Cpcor413pm1 gene function,we can deepen the understanding of cor413 gene family function,and provide theoretical clues for the study of cold resistance of plants.The results are as follows:（1）The full-length c DNA of cold adapted protein gene of Chimonanthus praecox with complete ORF（Open Reading Frame）was amplified by PCR and named Cpcor413pm1（GenBank access number:dq359747）.The c DNA contains an open reading frame of606 BP,encoding 201 amino acids.The predicted molecular weight of the protein is22.69 kDa.It contains 101 hydrophobic amino acids,accounting for 50.25%of the total amino acids,41 polar amino acids,19 basic amino acids,and 12 acidic amino acids.The theoretical molecular weight is 22.69kDa,the predicted is oelectric point is 9.55,the half-life is 30h,and the instability coefficient is 23.59.（2）The Cpcor413pm1 gene of Chimonanthus praecox was success fully constructed into the prokaryotic expression vector pET32a（+）and transformed into the host E.coli BL21（DE3）chemically cell for expression.The fusion protein was expressed under the optimal conditions of temperature 37℃,IPTG induction concentration 1.0 mM and induction time 6 h.SDS-PAGE showed that the fusion protein was soluble,and its molecular weight was 41 kDa,which was consistent with the expected value.（3）Cpcor413pm1 protein was purified by Ni²⁺affinity chromatography.SDS-PAGE showed that the specific band was about 41 kDa.（4）The protective effect of Cpcor413pm1 protein on superoxide dismutase（SOD）in Chimonanthus praecox under low and high temperature stress was determined in vitro.It was confirmed that SOD activity could be maintained under low and high temperature stress under the protection of Cpcor413pm1 protein.（5）The results showed that Cpcor413pm1 protein could respond to low temperature,high temperature,high salt and drought stress.Over expression of Cpcor413pm1 protein could improve the tolerance of E.coli to abiotic stress such as low temperature,high temperature,high salt and drought.（6）The plant over expression vector pcambia 2301g-Cpcor413pm1 was constructed and transformed into Arabidopsis thaliana by Agrobacterium mediated inflorescence infection.Three lines with high,medium and low expression levels were selected by QRT PCR for phenotype observation and abiotic stress experiment.The results showed that the over expression of Cpcor413pm1 gene did not significantly affect the growth and development of Arabidopsis plants.The results showed that Cpcor413pm1 transgenic Arabidopsis had stronger cold resistance,drought resistance and high salt resistance than wild-type Arabidopsis.In conclusion,Cpcor413pm1 gene can play a role in abiotic stress,such as cold resistance,drought resistance and high salt resistance.