Construction Of RNAi Vector Of Poplar MXC3 Geneand Its Transformation

In recent years, Poplar rust occurs very serious, the growth of poplar plantation has been restricted. Therefore cultivate fast-growing disease-resistant varieties is one of the most urgent problems that we’re facing. Poplar is perennial plant, growing season is quite long, In a short period of time, it’s difficult to use conventional breeding method to produced good varieties with target traits. Nowadays, genetic engineering methods can reform the disease resistance of existing good varieties specially and produce the disease-resistant varieties in a very short time. The gene of MXC3 have two function domain, which are Thaumatin family and Protein kinase domain. These funcitions may play very important roles in poplar leaf rust resistance. RNAi(RNA interference) is an important technology of gene silencing and widely used in Function of plant genes and genetic improvement research. This article chose MXC3 genes of poplar rust resistance as the research object, then build the RNAi carrier of MXC3, the receptor material is Populus×euramericana cv. ‘Nanlin895’. And at the same time, in this article, I carry out the Poplar genetic transformation research of Agrobacterium mediated to lay the foundation of MXC3 gene function research. The main results of this research are as follows: 1. Building the RNAi expression carrier of poplar MXC3 genes by Gateway technology, and successfully import it into agrobacterium C58; 2. Optimizing the early generation of medium of ‘Nanlin895’ poplar stem section:MS+6-BA1.0mg/L+NAA0.05mg/L+0.25%MES+0.2% inositol; 3. Building a high efficiency, stable genetic transformation system by using the somaclone blade of ‘Nanlin895’ poplar as the conversion receptor material of Rust resistance genes MXC3.(1) This experiment using kanamycin(Kan) as a screening of antibiotics to screen the proper concentration of kanamycin(Kan) is 20mg/L;(2) By the method of agrobacterium mediated transformation MXC3 genes, doing the research about the factor of affecting the conversion efficiency of microbial concentration, infection time and the way of screening factor, then getting the optimal conditions: Remove the blade edge and the main vein, By the method of far shaft surface contact medium, put the leaf on differentiation medium in the dark for 3 days, then put the leaf into the OD600 0.8 bacteria liquid and let it be infected for 20 min, cultivate in the dark for 4 days, delay screening for 10 days, Using this conversion system, resistance bud induction rate of ‘Nanlin895’ poplar can be up to 19.39%;(3) CTAB method was used to extract the DNA of 28 strains resistant plant in this experiment, and the PCR detection, the result to be that there are 22 strains was positive, preliminary evidence that Poplar MXC3 RNAi has been intergated into ‘Nanlin895’ poplar genome. The positive rate was 78.57%;(4) The transgenic plants m RNA which were extracted by the CATB methord, RT-PCR test results show that the expression of MXC3 gene in 22 transgenic plants are reduced compare to the contrast(non-transformed plants). Showed that exogenous genes affect the expression of endogenous MXC3 gene in poplar; 4. Use the method of refined seedling transplanting to transplant the transgenosis positive somaclone, totally transplant 22 transgenosis positive plants and 19 plants survived, the survival rate is 86.36%.