Prokaryotic Expression And Transformation Analysis Of PtDRG01 Gene

The Chinese white poplar (Populus tomentosa Carr.), indigenous to China, is a fast growing poplar species, with an excellent wood quality and outstanding resistance to abiotic stress. It has played a key role in forest production and ecological environment construction along the Yellow River. However, it is susceptible to a couple of diseases, particularly the leaf rust caused by Melampsora magnusiana Wagner. It is normally invade into the seedlings and leaves and shoots located in the senior tree, resulted in yellow dot of pathogen and in turn lowered the efficiency of photosynthesis, this consequently severely affected the growth and development of the poplar. But, it is little known about the research of disease resistance in P. tomentosa, especially in the study of disease resistance, including selection of germplasm and breeding, gene mapping and map based cloning of disease resistant gene had not been reported. Therefore, it is indispensable to isolate the gene controlling of leaf rust and analyze the function of gene's product, based on the germplasm of disease resistance, and eventually the transgenic plants with disease resistant genes would be produced. These will have the significance in the theory and practical value for the improvement of disease resistance in P. tomentosa. In this study, the prokaryotic expression of a candidate disease resistant gene PtDRG01 and genetic transformation of RNAi constructed PtDRG01 had been conducted. The major results and conclusions are described as follows.1. The prokaryotic expression vector pGEX-KG with the full-length open reading frame of isolated disease resistance candidate gene PtDRG01 isolated form P. tomentosa ( Accession number:EF157840)) was successfully constructed and transferred into an expression host E. coli strain XA90. The fusion protein of PtDRG01 induced by IPTG treatment was separated by SDS-PAGE electrophoresis, and the result indicated that the size of the fusion protein was about 79 kD, which was consistent with the predicted value. The prokaryotic expression system was also optimized. The result suggested that 1 mmol/L IPTG treatment for 4 h at 37 oC was most effective, and the product was predominately soluble and not extra-cellular secreting. Further, the fusion protein was purified with the affinity chromatography column of Glutathione Sepharose 4B. Catalyses ability of the hydrolysis of ATP with weakly effect by the purified protein with the modified colorimetry of molybdenum blue method was determined.2. The regeneration system of genetic transformation with leave disc was established in P. tomentosa and the plant binary expression vector of Pbi121-PtDRG01 RNAi was constructed and transferred into the triploid poplar through particle bombardment. Many transgenic poplars were obtained and the integration of RNAi sequences into the host genome was confirmed by the PCR detection. The further molecular detection and experiment of disease resistance is ongoing.The above results, therefore, provided the important foundation for function detection of PtDRG01 gene and molecular breeding of new germplasms with disease tolerance in P. tomentosa, have the important practical value in the forest production.