Functional Analysis Of Kn-Type Dehydrins Wcor80 & Dhn14 In Wheat

Dehydrins belong to the LEA protein family with 9～200 kD molecular weight and generally come into being during the later period of embryonic development in higher plant. These proteins are expression inducible under the abiotic (low temperature, heavy metal, drought, salt) and biotic stresses (ABA, pathogens, fungi, insects), and therefore they are proteins synthesized by higher plants responding to external stresses. Previous researches have shown that dehydrins contain at least one conserved sequence, i.e., K fragment, and this fragment is a specific structure for the formation of a helix under stress. Dehydrins present significant hydrophilia and thermostability and they may play positive roles during the responses of plants to adverse stress. Studying the functions of wheat dehydrins and their expression regulatory mechanism under stress will speed up the wheat breed improvement and provide for practical guidance.Our study isolated the dehydrin genes DHN14 and WCOR80 from the cDNA of wheat cultivar Zhengyin No.1. The obtained DHN14 and WCOR80 genes possess ORFs of 338 bp and 282 bp respectively. We investigated the functions of these two dehydrins under stress and the role played by the K fragment when dehydrin functions through analyzing the expression pattern of these two genes under different stresses, the effects of dehydrins on growth conditions of E.coli under stress and the protective activities of LDH。 We will provide theoretical basis for the research of structures of dehydrins in higher plants and their functions under disadvantaged environment.We draw the following conclusions according to the research mentioned above.We cloned and isolated the full length of wheat dehydrin genes DHN14 and WCOR80 and both of the two encoded amino acid sequences contain two K fragment conserved sequences, clarifying the affiliation of DHN14 and WCOR80 to Kn dehydrin.1. We analyzed the genes we isolated using bioinformatics and we concluded that the DHN14 protein is an acidic protein with molecular weight of 24 kD and isoelectric point (pI) of 6.28 while the proportion of disordered structures reaches at 81%; and the WCOR80 protein is a neutral protein with molecular weight of 23 kD and isoelectric point (pI) of 7.14 while the proportion of disordered structures reaches at 83%2. Both of wheat dehydrins DHN14 and WCOR80 are expression inducible under the low temperature stress (4℃ for 0 h,12 h,24 h,48 h,72 h), and after the recovery of temperature, the express levels of the two genes present decreases to different degrees. These results indicate that dehydrins DHN14 and WCOR80 are low temperature induced proteins; under the external ABA treatment, DHN14 can be induced at 36 h and the expression level keeps increasing with time continuing, while the expression of WCOR80 does not present significant fluctuations. These results suggest that DHN14 is the ABA-dependent dehydrin while WCOR80 is not. Under the treatment of PEQ both of DHN14 and WCOR80 can be induced, indicating that they belong to the drought induced proteins.3. Heavy metal ions and H2O2 can suppress the growth of E.coli. When adding the bacteria carrying plasmids with DHN14 and WCOR80, the growth conditions under the heavy metal ions and H2O2 can be significantly improved. Our result indicates that DHN14 and WCOR80 proteins can increase the tolerance of E.coli to heavy metal ions and H2O2 and the ranking of protective activities is:DHN14 is better than WCOR80.4. We constructed the expression constructs of DHN14 and WCOR80, expressed and purified these proteins, validated the protective effects of them on the enzymatic activity of LDH. The positive and negative controls are BSA and sucrose with buffer, respectively. The results indicate that both of dehydrins DHN14 and WCOR80 possess protective effects on the enzymatic activity of LDH, and this conclusion provides theoretical basis for the function of dehydrins in higher plants.