Establishment Of Virus-Induced Gene Silence In Cotton And Its Application In Cotton Resistance To Verticillium Wilt

Verticillium wilt, one of the most devastating plant diseases worldwide, is caused by the fungi Verticillium spp.In recent few years it spread rapidly in South and North cotton-growing areas in China,the damage is also increasing year by year and has become the main obstacle in the production of cotton.The molecular mechanism underlying cotton resistance to verticillium infection is still not very clear. So finding Verticillium wilt resistance genes can accelerate the process of cotton breeding for disease resistance.Using the method of virus-induced gene silence to silence the target gene and observing its effect on cotton verticilium wilt resistance has a important significance in the study of the molecular mechanisms of cotton resistance to verticillium wilt. We have successfully established the VIGS system in cotton and its application in cotton resistance to Verticillium wilt. The main results are as follows:1. Constructing the silencing vector of cotton CLA1(Cloroplastos alter ados lgene) gene and using Agrobacterium-mediated virus-induced gene silence(VIGS) assay in virous cotton cultivars with different concentration of Agrobacterium.We find that Agrobacterium concentration between0.1～3.0can all get a good silencing effects. VIGS system has little effect on the genetic background by analysis of30different cotton cultivars. RT-PCR analysis of TRV-RNA2shows that TRV can reach the leaf, stem and root of the plant after infection. The technology can be applied to genes in different sources of tissues and organs.2. MAPK cascade pathway has a role in a variety of biotic and abiotic stress responses. GhMAPK and GhMAPKKK are cloned and constructed to the silencing vector. The function of the resistance to verticillium wilt is verificated by Agrobacterium injection method.VIGS verified that GhMAPKKK have a certain role in cotton resistance to Verticillium wilt. Semi-quantitative RT-PCR found that the GhMAPK and GhMAPKKK were significantly reduced two weeks after injection thus verifying the efficiency of gene silencing in molecular level.