Establishment And Optization Of VIGS System With PDS Gene In Dianthus Chinensis L.

VIGS (Virus induced gene silencing) is a powerful tool for analyzing gene function rapidly and extensively in plants. Dianthus chinensis L. possesses high stress-tolerance. Therefore, it can be utilized for plant genetic improvement by studying its mechanism and obtaining related genes. Such will be benefit to increase plant species and gain the aim of conservation-minded garden. In order to identify, analyze and utilize its tolerant genes in the future the VIGS system of D. chinensis will be built and optimized in our study. Firstly we used a Tobacco Rattle Virus (TRV) vector containing different sizes of phytoene desaturase (PDS) gene fragments to infect D. chinensis seedlings, and then evaluated and optimized the VIGS systems based on the phenotypes. Finally the expression level of PDS gene in the positive plants was analysed by Real-time PCR. The main results were as follows:1.The 1651bp fragment of DcPDS was obtained through RT-PCR (Genebank accession:KU318404)。2. The VIGS system was built in D. chinensis and the expected phenotype were obtained by silencing of the PDS gene. By evaluating the silence efficiency of TRV vectors containing different sizes of PDS gene fragments (pTRV2/DcPDS260, pTRV2/DcPDS509 and pTRV2/DcPDS762), the seeding age, acetosyringone(AS) concertration and pre-culture temperature, we found the photo-bleaching phenotype appeared on half-leaf or on entire-leaf when seedings with 2-3 pairs of true leaves were firstly pre-cultured at 10-15℃ for 7 days and then cultured under normal conditions after they were infected by TRV2 containing DcPDS509 in 40μM AS.3. The photo-bleaching phenotype was resulted from PDS gene silencing in D. chinesis by Real-time PCR The RNAs isolated from leaves of the silenced, the control and the mock were used to synthesize cDNA. Real-time PCR was carried out with PDS gene-specific primers and relative mRNA levels were calculated using the 2-△△CT method against the internal reference gene ACTIN. The results showed that compared with the control the mRNA quantity of PDS gene significantly declined in the silenced and the difference between the control and the mock was not significant.