Comperative Study On The Cold Resistance Of 'Soft-Seed Punica Granatum L. From Tunisia' And Cloning Of PgCBF2 Gene In Pomegranate

‘soft-seed Punica granatum L.from Tunisia' with poorly cold resistance was introduced into China sixty years ago.To expand the suitable region of ‘soft-seed Punica granatum L.from Tunisia',the varieties of annual branches cutting seedlings as test materials in this study.Several physiological indexes related cold resistance,including the relative conductivity,MDA,soluble sugar,soluble protein,O2-,H2O2,Pro,CAT,SOD,POD and restore growth,was compared in different low temperature(-6?,-9?,-12?)under different duration of treatment.Furthermore,PgCBF2 gene was cloned in Pomegranate,and then analyzed by using bioinformatics.The overexpression vector of PgCBF2 gene was constructed and transformed into Arabidopsis to identified gene function.It laid the foundation for pomegranate cold hardiness research and creating new germplasm of Pomegranate with highly cold resistance by using genetic engineering.The main results are as follows:1.Study on cold hardiness research of Pomegranate under different low temperature.(1)As the extension of stress time,the temperature conductivity,the contents of MDA,soluble protein and soluble sugar were risen,while the contents of CAT,POD,SOD,Pro and H2O2 were showed a trend of rise.(2)According to various physiological indicators reflect the situation of comprehensive,the ability of enduring low temperature time of ‘soft-seed Punica granatum L.from Tunisia' was decreased as temperature decreasing.‘soft-seed Punica granatum L.from Tunisia' was could tolerated 54 h,24 h and 12 h under-6?,-9? and-12?,respectively.(3)According to the shoot after frozen recovery rate values,‘soft-seed Punica granatum L.from Tunisia'could tolerated 54 h under-6?,most patient 24 h under-9?,while it couldn't survived under-12? over 12 h.(4)According to the results,it is recommended that ‘soft-seed Punica granatum L.from Tunisia' is not suitable for cultivation in the area under-12? or using protective cultivation measures in order to preventing ‘soft-seed Punica granatum L.from Tunisia' freeze injury occurs,or affect the economic benefits.2.Gene cloning and functional analysis of PgCBF2.(1)PgCBF2 gene was cloned from Pomegranate by blasting the reference sequence information of Arabidopsis thaliana At CBF2 from NCBI.The open reading frame of PgCBF2 gene is 699 bp and encoding 232 amino acids,excluding introns.The molecular mass of the protein is 25473.35 k Da,the isoe-lectric point is 5.09,is hydrophilic protein.The AP2 characteristic structure domain was found in the sequence conservative regional analysis.The results of evolutionary tree analysis showed that all homologous proteins of PgCBF2 belong to the CBF family.Subcellular localization prediction results indicated that the gene is expressed in the nucleus.(2)The PgCBF2 gene was expressed through inducing low temperature by RT-PCR analysis after the phloem of the branches was treated with low temperature.(3)The overexpression vector of 35S:PgCBF2 were constructed and transferred into wild-type Arabidopsis thaliana.3 overexpression PgCBF2 Arabidopsis plants were successfully obtained and subsequent research was in progress.