Drought Tolerance Function Analysis Of VyUSPA3 And VyCAS Gene From Chinese Wild Vitis Yeshanensis

Grapevine（Vitis vinifera L.）is one of the important fruit tree crops and is widely cultivated in the world,and has good fruit quality,but poor resistance to abiotic stresses.Drought is one of the major environmental stress factors affecting grape growth and development.Chinese main grape producing areas are located in the arid and semi-arid regions of northwest China.Low annual rainfall and drought severely restrict the development of the grape industry in these regions.China is one of the origins of grapes,with abundant wild grape resources.Among them,V.yeshanensis accession’Yanshan-1’has strong drought resistance and is an important drought-resistant gene resource.In a previous study,the transcriptome sequencing analysis of’Yanshan-1’and the drought-sensitive North American V.riparia acc.’He’an（♀）’under drought stress was performed,and a large number of differentially expressed genes were obtained.We selected two of them as the research objects,which were universal stress protein A（USPA3）and calcium sensing receptor（CAS）,respectively.On this basis,we cloned the two genes from’Yanshan-1’,and analyzed their biological functions in grape,as well as preliminarily explore the molecular mechanism for regulating drought resistance of grape.This study will lay a foundation for understanding the molecular mechanism of drought resistance of’Yanshan-1’,and breeding drought-resistant grape varieties by molecular breeding.The main results obtained are as follows:1.A total of 21 USPA genes were identified in the genome of Vitis vinifera L,and divided into five branches based on phylogenetic tree analysis.Bioinformatics analysis of Vv USPA gene family showed that the Vv USPA gene family was structurally conservative.A total of 16 pairs of homologous USPA genes between Arobidopsis and grape were identified by synteny analysis.There are three pairs of homologous Vv USPA genes in grape genome,which may be caused by gene duplication events during grape evolution.The prediction results of promoter cis-acting elements and upstream micro RNA suggest that Vv USPA may be involved in plant responses to abiotic stress and plant hormones.Tissue expression analysis showed that most USPA genes are more expressed in roots,flowers,and fruits than in tendrils,stems and leaves.When subjected to drought and exogenous abscisic acid（ABA）and methyl jasmonate（Me JA）treatments,most USPA genes were up-regulated in different degrees,while the expression levels of all USPA genes changed after ethephon（ETH）treatment,indicating that Vv USPA family genes may be involved in plant responses to abiotic stress and these plant hormones.Five Vv USPA genes（Vv USPA2,Vv USPA3,Vv USPA11,Vv USPA13 and Vv USPA16）selected were heterologous expressed in Escherichia coli.It was found that heterologous expression of Vv USPA3 in E.coli improved the growth rate of E.coli under osmotic（PEG or mannitol treatment）stress.This indicated that Vv USPA3 could improve the tolerance of E.coli to osmotic stress.2.Vy USPA3 gene,which is located on chromosome 1,was cloned from’Yanshan-1’with a length of 1,931 bp.The coding sequence（CDS）of Vy USPA3 is 495 bp in length and encodes 164 amino acids.Bioinformatics analysis showed that there is a USP domain and an ATP-binding site sequence G-（2X）-G-（9X）-G（S/T）at the carboxyl terminal of Vy USPA3,belonging to an ATP-binding protein.Secondary structure analysis revealed that Vy USPA3has fiveβ-folds separated by fourα-helices.The promoter sequences of Vy USPA3 and Vv USPA3（accession no.XM＿002283354）were 10 base pairs different,and the similarity of amino acid sequences was 100%.Subcellular localization showed that Vy USPA3 is localized to the nucleus and cytoplasm.Potted seedlings of V.vinifera cultivar’Thompson Seedless’were treated with low temperature,high temperature,Na Cl,oxidative stress（H₂O₂）,and ABA,respectively.The results of real-time quantitative PCR（q RT-PCR）showed that Vv USPA3 responds to these treatments,indicating it is a stress-induced gene.Vy USPA3 was overexpressed and silenced in the meristematic callus of the’Thompson Seedless’grape and three overexpressed lines and two RNAi-silenced lines were obtained.After 21 days of drought stress,all the leaves of wild-type（WT）lines withered,while only some leaves of the overexpressed lines wilted slightly,which was in sharp contrast to the severe leaf wilting and curling of RNAi-Vy USPA3 lines.Meantime,overexpression of Vy USPA3 could promote root growth and reduce stomatal apeture and leaf water loss,increase proline content and antioxidant enzyme activity（POD,SOD and CAT）,as well as reduce the accumulation of malondialdehyde,H₂O₂and O₂^-of transgenic plants under drought stress.In addition,some drought-related genes（RD22,RD29B,ERD14,DREB2A,NCED1 and KIN2）were up-regulated under drought stress.RNAi-silenced lines showed the opposite.It indicates that Vy USPA3 confers drought tolerance of transgenic grapes.3.Three interacting proteins of Vy USPA3 were screened by yeast two-hybrid,which were ERF105,PUB24 and NF-YB3.The interaction between them was further confirmed by bimolecular fluorescence complementation assays.Moreover,the interaction between Vy USPA3 and ERF105 or PUB24 primarily occurred in the nucleus,while the interaction with NF-YB3 happened mainly in the cytoplasm.Results of q RT-PCR showed that the expression levels of ERF105 in transgenic grapes were down-regulated,the expression levels of NF-YB3 were up-regulated,and the expression levels of PUB24 down-regulated under drought stress.It was speculated that Vy USPA3 could regulate drought tolerance of grapes by synergistic with NF-YB3 and antagonistic with ERF105.4.Vy CAS gene（accession no.OK628347）was isolated from’Yanshan-1’with a full length of 3,040 bp.The CDS length of Vy CAS is 1,200 bp,which encodes a protein of 399amino acids.Bioinformatics analysis showed that Vy CAS was located on chromosome 17,and Vy CAS protein had a RHOD domain（PF00581）at the carboxyl terminal and a transmembrane domain.The sequence similarity between Vy CAS and Vv CAS was 99%,with only two base pairs differences,while the amino sequences were identical.A phylogenetic tree analysis showed that Vy CAS is highly similar to the CAS of Vitis vinifera,Myrica rubra,Malus domestica and Ziziphus jujuba.Vy CAS is localized to chloroplasts and punctured structures based on subcellular localization.Potted seedlings of’Thompson Seedless’grape were treated with Ca²⁺,low temperature,Na Cl,ABA,and SA,respectively.Results of q RT-PCR showed that Vv CAS could respond to these treatments,indicating it is a stress-induced gene.Vy CAS was overexpressed in the meristematic callus of’Thompson Seedless’grape,and three transgenic lines were obtained.After 14 days of drought stress treatment,the leaves of WT showed completely wilting and yellow,while a few leaves of the transgenic lines had withered,indicating that overexpression of Vy CAS can improve drought tolerance of transgenic grapes.Compared with the WT,transgenic plants had smaller stomatal aperture and stomatal conductance,lower transpiration rate,higher leaf relative water content,lower malondialdehyde content and electrolyte leakage,higher CAT,POD and SOD activities,and less H₂O₂and O₂^-accumulation under drought stress.Meanwhile,some drought-related genes（RD22,DREB2A,NAC72,SRK2A,CBL1 and CYP707A2）presented higher expression levels in transgenic grape plants.In addition,Arabidopsis thaliana with a At CAS mutation was more sensitive to drought stress than WT,it was further demonstrated that CAS gene was positively regulating drought tolerance in plants.5.The Vy CAS promoter was cloned from’Yanshan-1’grape.After analysis of cis-acting elements,it was found that the Vy CAS promoter contained an ABA response element（ABRE）and an SA response element（TCA-element）.Then,the Vy CAS promoter was fused with a vector containing a GUS reporter gene.The Vy CAS promoter was induced by ABA and by SA after transient transformation in tobacco leaves.By predictive analysis using the JASPAR website（http://jaspar.genereg.net/）,it was found that the Vy CAS promoter sequence contains three MYB59 binding sites（MA1042.1）.It was showed that Vy MYB59 could bind directly to the promoter of Vy CAS by yeast one-hybrid test,and further experiments by a luciferase and a GUS activity assay indicated that Vy MYB59could negatively regulate Vy CAS promoter activity.