Cloning And Functional Identification Of Dehydroascorbate Reductase Gene IbDHAR3 From Ipomoea Batatas(L.) Lam

Sweetpotato is the seventh largest food crop in the world,which has low price,high yield,and high biomass production,widely being grown in China.China is one of the largest sweetpotato producer in the world,a mass of soil was barren result from the complicated area and niche,which has a significant impact on sweetpotato yield.Hence,effective measures should be taken to solve this problem as soon as possible.Ascorbic acid（As A）is a pivotal antioxidant in plants with high contents,which as a major contributor to the removal of reactive oxygen species（ROS）.The plants could produce ROS result from drought,salt,high or low temperature,which cause severe damage to plants,subsequently,As A involved in both the enzymatic and non-enzymatic elimination of ROS,thereby enhancing plant stress resistance.Dehydroascorbate reductase（DHAR）plays a dramatical role in the regeneration process of As A,which also involved in the resistance of plants to biotic and abiotic stress,and increase the resistance of plants.Based on the sweetpotato transcriptome database,this study successfully cloned the full length of IbDHAR3 gene from Xushu 18 leaves by RT-PCR,the sequence and expression patterns were analyzed respectively.The expression vector was constructed for subcellular localizationin Arabidopsis thaliana.In addition,the T3 generation of transgenic Arabidopsis was applied for stress resistance.To further investigated the function of IbDHAR3,the transgenic sweetpotato was constructed with introducing this gene into it,and its resistant ability and mechanisms was addressed.The results are as follows:（1）A 813 bp IbDHAR3 gene was successfully cloned,encoding 270 amino acids,among which included 52-amino acid chloroplast transit peptide,as well as two glutathione-S-transferase（GST）N-terminus and GST-C-DHAR domains without signal peptide.The predominant structure is α-helix,contains β-sheet,and its tertiary structure was predicted to be a monomeric protein.IbDHAR3 is most likely localized in the chloroplast and is a hydrophilic protein using subcellular localization;（2）The relative expression pattern of IbDHAR3 gene was analyzed by qRT-PCR under distinct tissues in different abiotic stress conditions.The results showed that it was expressed to varying degrees in each tissue,but with highest expression levels in leaves,meanwhile,the IbDHAR3 gene can be induced to up-regulated via ABA,drought,salinity,and high temperature;The Gateway vector technology was used to successfully construct the expression vector IbDHAR3-p GWB5,which was transformed into Agrobacterium and transiently expressed in tobacco leaves.The confocal results revealed that the IbDHAR3 protein subcellular localized in the chloroplast;（3）Transformation of Arabidopsis thaliana with dip flower method,6 IbDHAR3 transgenic T3 generation Arabidopsis lines were screened by suitable antibiotics,the IbDHAR3 transgenic Arabidopsis lines（OE5,OE13）were selected for stress resistance studies.Compared to wild type（WT）,the seeds germination rate,root length,and fresh weight of Arabidopsis transgenic lines were higher under the high salt and osmotic stress.;under drought and high salt stress conditions,however,the NBT and DAB staining tests indicated that the color of the transgenic line leaves were lighter than WT,and the content of O2·-and H2O2 were lower than WT in the condition of drought and high salt.Under the drought stress,the relative expression level of At GR gene was elevated in transgenic lines,and the levels of MDA and As A were higher in WT.The activity of DHAR,ascorbate peroxidase（APX）and superoxide dismutase（SOD）in Arabidopsis overexpression lines was prominently higher than that in WT.Together,these results indicate that the overexpression of IbDHAR3 in Arabidopsis enhances the resistance of Arabidopsis,this laid a theoretical foundation for further exploring the identification of stress resistance of IbDHAR3 transgenic sweetpotato;（4）10 IbDHAR3 transgenic sweetpotato lines were obtained by using Agrobacterium-mediated genetic transformation method;and two transgenic lines（SD6,SD10）with higher relative expression levels were selected for further study.The results of the leaf disc stress test illustrated that: under high salt and osmotic stress conditions,the chlorophyll content in the leaf discs of the transgenic lines was higher than that of the wild type in different degrees;the increments of relative plasma membrane permeability of the transgenic lines was lower than that of the wild type under oxidative stress,and also the leaf discs were darker in transgenic lines than WT via DAB and NBT staining,this result suggested that wild type leaf discs were more oxidatively damaged than transgenic lines.The pot test results signified that: The Fv/Fm of transgenic sweetpotato lines is higher than WT under high salt and drought stress conditions,while the H2O2 and MDA content of WT are higher;The contents of APX,SOD,peroxidase（POD）and catalase（CAT）were higher than those of the wild type,in addition,the DHAR contents of the transgenic lines were higher than WT,and the ratio of As A/DHA were higher in transgenic lines.Taken together,these results proved that the overexpression of IbDHAR3 gene in sweetpotatoes enhances the ability to scavenge reactive oxygen species（ROS）,for instance,H2O2,result from promoting the circulation of As A-GSH and the mechanism of relative antioxidant enzyme systems,and then strengthen the resistance of sweetpotatoes.In summary,the molecular mechanism of IbDHAR3 gene in sweet potato stress response is clarified,which lays the foundation for in-depth analysis of the function of this gene and its application in molecular genetic improvement of sweetpotato.