Research On Disease Resistance Of Transcription Factor JAZ And TLP Gene In Chinese Wild Grape Vitis Quinquangularis

Grape(Vitis vinifrea L.)as one of the representative world's fruit is highly vulnerable to external climate and insect pests.European grapes considered special are highly susceptible to powdery mildew,Biotrytis cinerea,downy mildew and anthracnose,and Chinese wild grape show the high resistance or immunity to various pathogens.Based on the previous research of our team,on the one hand,transcription factors VqJAZ4 and VqJAZ9 from Vitis quinquangularis ‘Shang-24' are analyzed for the anti-disease function,and the mechanism of regulating the anti-disease of VqJAZ4 and VqJAZ9 are studied using the transgenic Arabidopsis lines inoculated by powdery mildew and B.cinerea.The promoter sequence of VqJAZ4 is cloned and analyzed.Then screening of genes interacted with VqJAZ4 is conducted using Yeast Two Hybrid technology.On the other hand,gene identification and bioinformatics analysis of grape TLPs gene family are conducted to predict and locate the response of TLPs to different pathogens.VqTLP15 and VqTLP29 from ‘Shang-24' are homology-based cloned,and VqTLP15 and VqTLP29 are analyzed for the response to powdery mildew,B.cinerea and DC3000 using transgenic Arabidopsis lines.The main results are as follows:1.The full length of VqJAZ4 is homology-based cloned from disease-resistant Vitis quinquangularis ‘Shang-24' using RT-PCR.The open reading frame(ORF)of VqJAZ4 is 861 bp,which encodes 286 amino acids.On the base of construction of VqJAZ4 over-expression vector,transgenic Arabidopsis lines increase resistance to powdery mildew and decreased resistance to B.cinerea.Expression analysis of related genes comes to the conclusion,VqJAZ4 transgenic lines are co-regulated by SA and JA signaling pathways to participate in the resistance to powdery mildew,and transgenic lines show the decreased resistance to B.cinerea because of significantly inhibition of the MYC2-diameter in JA signaling pathway by VqJAZ4.Encoding region of VqJAZ4 does not have self-activating activity.After mating with the Yeast Two-Hybrid Library,GDSL lipase is found out to interact with VqJAZ4.Finally,38 positive transgenic grape lines have been successfully obtained via transmutation of VqJAZ4 into disease-susceptible Vitis vinifera ‘Thomson Seedless' somatic embryos.Besides,1,465 bp promoter sequence of VqJAZ4 homology-based cloned from ‘Shang-24' contains several cis-acting elements involved in the response to stress,plant hormone and defense reaction,which suggests that VqJAZ4 promoter may play an important role in response to grape biotic and abiotic responses.2.The 807 bp ORF of VqJAZ9 is homology-based cloned from disease-resistant Vitis quinquangularis ‘Shang-24',which encodes 268 amino acids.On the base of construction of VqJAZ9 over-expression vector,transgenic Arabidopsis lines improve resistance to powdery mildew via regulating the related genes expression of SA and JA signaling pathway.VqJAZ9 transgenic Arabidopsis lines decreased resistance to B.cinerea via significantly inhibition of the MYC2-diameter in JA signaling pathway,which is similar to VqJAZ4.The coding region of VqJAZ9 does not have self-activating activity.3.A total of 33 members of TLPs are identified from the grape genome database,and they are named VvTLP1 to VvTLP33 based on their distribution and relative linear order on the chromosomes.29 out of 33 TLPs contain the complete and typical thaumatin-domain.The grape VvTLP family is divided into 4 subfamilies: Type I,II,III and IV,based on the analysis of a phylogenetic tree,which occupies the proportion of 27.27%,54.55%,3.03% and 15.15%,respectively.Analysis of the structure of gene and protein shows the diversity evolutionary relationship between VvTLP.Tandem duplication and synteny analysis show that a total of 18 genes(VvTLP1,VvTLP2,VvTLP3,VvTLP6,VvTLP7,VvTLP8,VvTLP9,VvTLP10,VvTLP11,VvTLP12,VvTLP13,VvTLP15,VvTLP16,VvTLP25,VvTLP26,VvTLP27,VvTLP31 and VvTLP32)clustered into 6 tandem duplication event regions on grape chromosome 1,2,3,4,15 and 18,and a synteny analysis of the A.thaliana and grape TLP genes further revealed the 20 syntenic relations that contain 15 AtTLP and 12 VvTLP.A systematic expression analysis using PCR of all the 33 VvTLP in grape is conducted following inoculation with anthracnose,powdery mildew and B.cinerea.Three pathogens are used to infect the anthracnose-resistant grape ‘Shang-24' and anthracnose-susceptible grape ‘Red Globe',powdery mildew-resistant grape ‘Shang-24' and powdery mildew-susceptible grape ‘Hunan-1',and B.cinerea-resistant ‘Shuangyou' and B.cinerea-susceptible grape ‘Red Globe',respectively.The expression levels of 23 genes increase following the anthracnose inoculation,the expression levels of 14 genes increase following the powdery mildew inoculation,and the expression levels of 19 genes increase following the B.cinerea inoculation.Among these genes,the expression levels of 6 genes(VvTLP3,VvTLP6,VvTLP7,VvTLP8,VvTLP12 and VvTLP29)are simultaneously up-regulated following the three pathogens inoculations.4.The full lengths of VqTLP15 and VqTLP29 are homology-based cloned from disease-resistant Vitis quinquangularis ‘Shang-24' using RT-PCR.The ORFs are 891 bp and 735 bp,which encode 296 and 244 amino acids,respectively.High sequence homology is analyzed between VqTLP15 with GSVIVT01018769001 known as homologous gene from grape ‘Pinot noir' and VqTLP29 with GSVIVT01008423001.Analysis of protein sequence of VqTLP15 and VqTLP29 shows the cleavage site of signal peptide.Both VqTLP15 and VqTLP29 are located in the cytoplasmic matrix of grape mesophyll protoplasts.On the base of construction of over-expression vector,VqTLP15 transgenic Arabidopsis lines improved resistance to powdery mildew and DC3000,and decreased resistance to B.cinerea.Result of the expression analysis of related genes shows that over-expression of VqTLP15 have an effect on SA and JA/Eth signaling pathways so that transgenic lines make changes for immune response to powdery mildew,DC3000 and B.cinerea.The phenotypes of VqTLP29 transgenic Arabidopsis lines are similar to that of VqTLP15.VqTLP29 transgenic lines also improved resistance to powdery mildew and DC3000,and decreased resistance to B.cinerea.Result of the expression analysis of related genes shows that over-expression of VqTLP29 as a regulatory factor could make changes for multifarious immune response to powdery mildew,DC3000 and B.cinerea via both SA and JA/Eth signaling pathways.