Bioinformatics Analysis,Synthesis Of VvXDH1,VvOPR1 In Vitis Vinifera And Functional Identification

Grapevine（Vitis vinifera）is of high cultural value for the occupation in the fruit production.However,the abiotic stresses,such as salinity,high temperature,drought,heavy metals and chemical toxicity are serious treats to the quality and yield of grapevine.The grafting and cutting propagation were usually used in grape production,which not only shorten the time required for breeding but also maintain the stability of grape varieties.Therefore,it is of great practical significance for the cultivation of grape and other fruit trees to explore the function of grape resistance genes and to cultivate the grape rootstock with strong stress resistance through genetic engineering technology.Xanthine dehydrogenase（XDH）acts as one key enzyme in the purine catabolism and catalyzes hypoxanthine,xanthine to urate,XDH is associated with various metabolism.12-OXO-phytodienoic acid reductase（OPR）is a key enzyme in the biosynthesis of jasmonic acid（JA）,and the evolution and function of the OPR gene family in the whole plant genome is still unclear.We developed bioinformatic analysis of VvXDH1,VvOPR1 and predicted the functions of this two genes,respectively.Then we synthesized VvXDHIS,VvOPR1S via PTDS method based on the amino acid sequences released on NCBI.And we studied the function of the XDH1 gene（VvXDH1）and OPR1 gene from V.vinifera（VvOPR1）by developing the transgenic Arabidopsis,rice plants.We hope that both VvXDH1 and VvOPRl gene will be used in the genetic engineering breeding in grapevine and other plants and will offer theoretical basis for the regulation of gene expression.The main results are as follows:1.Sequence was analyzed and found the full-length cDNA of VvXDH1 has a 4077bp open reading frame（ORF）and encode a protein consisting in 1358 amino acids.At the amino sequence level,VvXDH1 has 76.26%sequence similarity to AtXDH1,and the phylogenetic tree of VvXDH1 gene matches well this.VvXDH1 is a homodimer and each subunit contains functional domains:four Moco,two 2Fe/2S,one NAD binding and one substrate specificity center.VvOPRl has 76.78%,70.30%sequence similarity to LeOPR1,AtOPR1,respectively.The phylogenetic tree and three dimensional structure of VvOPR1 were analyzed,it was suggested that VvOPR1 gene might not be involved in JA biosynthesis pathway and play a role in the defense signaling pathway.In this experiment,with the grape "Minicure Finger" as materials,we identified the expression level of VvXDH1 and VvOPR1 in different organs via qRT-PCR.The results showed that VvXDH1,VvOPR1 expressed in various organs of grape,and the expression level of VvXDH1 was found in leaves,followed by the stem;the expression level of VvOPR1 was higher in the roots and stems,the leaves showed lower VvOPR1 expression level.2.According to the amino acid sequence（XP₀02285473）,we successively synthesized XDH1 gene from V.vinifera using PTDS method.We constructed the plant expression vector YN36 and the constructs were introduced into Arabidopsis thaliana Columbia by floral dip method.We analyzed the function of VvXDHIS gene in A.thaliana.The transgenic Arabidopsis showed significantly higher germination rate,root length and fresh weight;The XDH1 activity of transgenic plants is higher than that of wild-type.The elevated tolerance of transgenic plants to salt stress was confirmed by the increase of water content,chlorophyll,proline,ABA,allantoin,the activities of antioxidant enzymes and the decline of MDA.In addition,the expression of AtNCED3,AtZEP,AtP5CS,AtSOD,AtPOD,AtCAT in transgenic plants was more significantly upregulated than that in wild-type,respectively.All the conclusions confirmed the overexpression of VvXDHIS gene in A.thaliana has elevated the tolerance to salt.3.According to the VvOPRl amino acid sequence,we successively synthesized OPR1 gene from V.vinifera（VvOPRIS）using PTDS method.VvOPRIS over-expressed in A.thaliana and rice via Agrobaceterium tumdfaciens mediated transformation.Solution culture experiments were carried out to study the effect of copper（Cu）,zinc（Zn）on germination,growth,physiological and biochemical characteristics,Cu,Zn accumulation of transgenic VvOPRIS A.thaliana and rice.And the expression level of antioxidative enzymes in transgenic rice was detected via qRT-PCR to further explore VvOPR1 function.This paper set Cu disposal,Zn disposal and the composite disposal of Cu,Zn.The results showed that the germination rates,root length and fresh weight of transgenic A.thaliana were obviously higher than those of wild-type Arabidopsis,this phenomenon was also detected in transgenic rice.Upon Cu,Zn treatment,the decline in chlorophyll content,the Chl a/Chl b ratio and the ratio of Fv/Fm in transgenic rice were to a lesser degree than those of wild-type rice,the MDA content,conductivity increased in transgenic rice and showed a lesser degree than those of wild-type rice.The activity of SOD,POD increased by a higher degree and the activity of CAT declined by a lower degree in the transgenic rice,and the expression level of the three antioxidative enzyme genes in transgenic rice were significantly higher than those in wild-type rice,in addition,the transgenic rice showed a higher transport capacity of Cu,Zn.All these results implied that transgenic rice may have higher tolerance to Cu,Zn stress by maintaining higher photosynthetic mechanism,transport capacity of Cu,Zn and improving protective antioxidant activity and expression.Taken together,VvXDH1,VvOPR1 of grapevine showed enhance saltnity,heavy metal tolerance.The results of this research not only provide insight into the functions of grapevine geens,but also add new information on obtaining some new grape stock species with excellent stress tolerance via genetic engineering and asexual propagation.And VvOPR1 enhanced the tolerance of salinity and heavy metal,which provides a new insight for the phytoremediation for saline alkali soil,heavy metal contaminated soil.