Cloning And Functional Analysis Of A WRKY3 Transcription Factor In Vitis Quinquangularis

Grapevine is an important global economic fruit crop which has been widely cultivated in China and abroad. Vitis vinifera cultivars that have excellent quality tend to be susceptible to a variety of pathogenic bacteria. Conventional cross-breeding can cultivate high disease-resistant grape varieties, but takes long breeding cycle, with low efficiency and complicated character of hybrid offspring separation. Genetic engineering methods to improve grape disease-resistance has gradually been widespread concerned. WRKY is a novel transcription factor that was found recent years. It was closely related to kinds of biotic and abiotic stresses. According to the previous research results of our group, we cloned a disease-resistant gene VqWRKY3 from Chinese wild Vitis quinquangularis cv. ‘Shang–24',constructed an over-expression vector and transfered into Arabidopsis thaliana and the disease-resistant ability of the transgenic Arabidopsis was studied. The main results are described as following:1. Results of real-time PCR showed that expression of VqWRKY3 was induced by powdery mildew in Vitis quinquangularis cv. ‘Shang–24' leaves. The expression level of VqWRKY3 was highly induced by powdery mildew at 24 h treatment, and then start to decrease after 24 h. Furthermore, real-time PCR were performed to investigate the expression profiles VqWRKY3 in different organs of Vitis quinquangularis cv. ‘Shang–24'. VqWRKY3 showed distinct expression patterns in root, stem, leave, flower, tendril and fruit of Vitis quinquangularis cv. ‘Shang–24' seedlings, with the highest expression in root and tendril, and the lowest in stem, leaf and fruit.2. In the present study, a 570 bp fragment containing a complete open reading frame(ORF) of VqWRKY3 was cloned from Vitis quinquangularis cv. ‘Shang–24'. Sequence analysis indicated that the VqWRKY3 encodes a protein of 188 amino acids. The coding sequence of the VqWRKY3 in Vitis quinquangularis cv. ‘Shang–24' shared 98.6% identity with that identified from the V. Vinifera cv. PN40024 and the protein sequence shared 97.3%identity.3. Inoculate the transgenic A. thaliana plants that over-expressing VqWRKY3 with powdery mildew, Pst DC3000 and Botrytis cinerea. Results show that over-expression ofVqWRKY3 can improve the resistance to powdery mildew and Pst DC3000 but reduce the resistance to Botrytis cinerea of transgenic Arabidopsis thaliana. The expression profile of several defense response genes were tested in transgenic plants post pathogens inoculation using RT-PCR. Results showed that the VqWRKY3 may act as a positive regulator in SA signal pathway, and a negative regulator in JA signal pathway. The transgenic and wild type Arabidopsis were injected with flg22, LPS and Pst DC3000 to observe the accumulation of callose. Result shows that the accumulation of callose in transgenic leaves was more than that in wild type after injection with Pst DC3000, flg22 and LPS.