Expression Analysis Of The Genes VpUIRP2 And VpUIRP3 Involved In The Resistance To Uncinula Necator From Chinese Wild Vitis Pseudoreticulata

Grape is one of the widely cultivated and economically important fruit crops across the world. Cultivated grapevine(V. vinifera L.) is of great quality but is susceptible to many pathogens. For instance, powdery mildew(PM) causes significant losses in yield and quality of grape berry. One better way to solve this problem is through screening for disease-resistant genes of grape to improve European grape resistance in definite direction. China, one of the origins of grapevine, conserved abundant wild grape germplasm resources of disease-resistance. In a previous study, Chinese wild grapevine genotype Vitis pseudoreticulata Baihe-35-1 was identified as resistant to multiple fungi, particularly to U.necator. This study screened for pathogen-related genes in a PM-inoculated cDNA library of Baihe-35-1. Among the ETSs identified, two ETSs were predicted to encode a putative RING finger protein, belonging to the E3 ligase family. Then homologous cloning was used to obtain coding sequences of VpUIRP2 and VpUIRP3. As well as genes' expression characteristics and resistance of transgenic European grapevine were further analyzed. The main results are listed as follows.1. VpUIRP2 and VpUIRP3 coding sequences(CDS) were cloned from Chinese wild V.pseudoreticulata accession Baihe-35-1. Located at chromosome 5, the VpUIRP2 CDS is 954 bp in length, encoding 317 amino acids, and its GenBank accession number is KU519335.Located at chromosome 8, the VpUIRP3 CDS is 1641 bp in length, encoding 546 amino acids,and its GenBank accession number is KU519336. Bioinformatics analysis showed VpUIRP2 and VpUIRP3 genes possess C3H2C3-type RING finger conserved domain.2. Inoculation with powdery mildew in Chinese wild V. pseudoreticulata accession Baihe35-1 displayed the expression of VpUIRP2 and VpUIRP3 present peaks respectively in 24 h and 6 h, suggesting the expression of VpUIRP2 and VpUIRP3 are induced by powdery mildew. Treatment with salicylic acid(SA) in Chinese wild V. pseudoreticulata accession Baihe 35-1 displayed the expression of VpUIRP2 and VpUIRP3 both present peaks in 3h,suggesting the expression of VpUIRP2 and VpUIRP3 are induced by SA. The CDSs of VpUIRP2 and VpUIRP3 were cloned into pBI221-GFP to construct the subcellular localization vectors. Results from subcellular localization assay showed VpUIRP2 genelocalized in cytoplasm whereas VpUIRP3 gene localized in the whole cell.3. Regeneration systems of V. vinifera cv. Thompson Seedless, Red Globe and Cabernet Sauvignon were optimized through organogenesis. Based on the comparative analysis of BAP concentration, an optimized method for meristematic callus germination was established.8.8?mol·L-1 BAP and 4.4 or 8.8?mol·L-1 BAP were supposed to be the most suitable for germination induction of Thompson Seedless at one month and two month, respectively. In addition, 4.4 or 8.8?mol·L-1BAP were supposed to be the most suitable for germination induction of Red Globe at one month and two month. The BAP and NAA concentration influence were further studied for increasing meristematic callus induction efficiency of Cabernet Sauvignon. It was of high efficiency for meristematic callus information of Cabernet Sauvignon after culturing on media IM1C(IM+2.668 mg·L-1 BAP), IM2(IM+4.000mg·L-1BAP) and IM3C(IM+7.336 mg·L-1 BAP).4. VpUIRP2 and VpUIRP3 genes were introduced into Red Globe and Thompson Seedless by genetic transformation via Agrobacterium. 13 transformed lines of Red Globe with VpUIRP2 gene and 2 transformed lines of Red Globe with VpUIRP3 gene were obtained.As well as 2 transformed lines of Thompson Seedless with VpUIRP2 gene and 2 transformed lines of Thompson Seedless with VpUIRP3 gene were obtained. Grape transformed lines were further tested by PCR and Southern blot. Results showed that 33.3% of the kanamycin-resisted plantlets with VpUIRP2 were transgenic lines. These data suggested that VpUIRP2 was introduced to Red globe and Thompson Seedless while VpUIRP3 transformation strains did not detect target bands.5. Transgenic and non-transformed lines were assayed for powdery mildew U. necator resistance. The hyphae growing on the leaves of transgenic plants at 168 hpi, were lower than in the controls. Furthermore, the number of conidiophores counted at 168 hpi by aniline blue staining and statistical analysis showed the significant difference between the VpUIRP2 overexpressing plant and control. To sum up, these data clearly demonstrate that overexpression of VpUIRP2 contributed to a reduction in the susceptibility of the transformed V. vinifera plants to U. necator.In conclusion, ubiquitin ligase genes VpUIRP2 and VpUIRP3 from Chinese wild V.pseudoreticulata Baihe 35-1 could be involved in the resistance to grape powdery mildew.Additionally,the genes VpUIRP2 and VpUIRP3 could be used as disease-resistant genes to study and improve the resistance of European grape variety.