Cloning Of Dehydrin And The Western-blot Analysis Of Expression Patterns Under Abiotic Stress In Tea Plant [C.sinensis(L.)O.Kuntze]

Tea plant is a perennial woody plant,its growth and development is influenced by many abiotic stress.In order to breed high quality and yield tea cultivares with high resistance to stress,we usually use optimum cultivation measures for a better culture environments.However,the hereditary character of cultivar is the key factor.Studying the mechanism of tea plant stress resistance and identifying stress-related genes and proteins are of great significance to the molecular breeding of tea.We researched the expression pattern of the LEA gene and protein family of tea leaves under different abiotic stress by using RACE and Western-blot technology.Results are as follows:1.In this study,the cDNA full-length of CsDHN3 in Camellia sinensis,was 869 bp,with 627 bp ORF encording 208 amino acid,which formed a 21.7 kD polypeptide with a predicted isoelectric point(pI)of 6.95.The deduced CsDHN3 protein was hydrophilic protein,no signal peptide,secretory protein.Phylogenetic analysis also showed that it had the closest relationship with oil camellia.To study expression levels of CsDHN3 on different tissues(buds,mature leaves,flowerets,root,mature seeds)using qRT-PCR technology.The results indicated that CsDHN3 exhibited tissue-specificity and mainly distributed in mature seeds.Under the different dehydration process,the expression level of CsDHN3 in mature seeds increased rapidiy to 2.13 times of control,Water content of 20.04% for 30 hours,then the expression level were decreasing all time after 30 hours,With the prolongation of dehydration stress.2.Comparison of three methods(TCA/Acetone precipitation ? Kit method ?phenol-methanol/ammonium acetate precipitation)of total protein extracted from tea leaves,within the phenol-methanol/ammonium acetate precipitation method was suitable for total protein extraction from tea leaves,and met the requirements for Western-blot analysis,together with short time,high concentration,clear electrophoretic bands,and no contaminated background in SDS-PAGE.3.Under the abiotic stress(low temperature,drought,ABA,BR)treatment,Ten kinds of dehydrin proteins in tea plant with the molecular weights ranging from 14-95 kD,were found in different stress.The expressions leverls were relatively stable in the 3 kinds of dehydrin proteins(molecular weights 95,65 and 48 kD).The 58 kD dehydrin occurred only in drought treatment,and showed a continuous expression in drought stress,which achieved the highest expression in 25 day,Afterrewatering returned to the control level.The highest expression of 37 kD dehydrin was found after ABA treatment 12 h and then decreased.The highest expression of 34 kD dehydrin was only found after ABA treatment 12 h and then decreased.,which both cold stress and drought stress rose.The 28 kD dehydrin showed a continuous expression in cold stress and then decreased in BR treatment.The 26 kD dehydrin occurred only in drought stress.The highest expression of 21 kD dehydrin was only found after ABA treatment 24 h and then decreased in cold,drought and BR treatment.