Studies On Construction Of Orf25 Gene Expression Vectors And Their Genetic Transformation

In this study, a tobacco variety "MS. 90" and "MS innovation 3" was used as receptor materials, by Agrobacterium tumefaciens mediated genetic transformation method, To further explore and optimize transformation system of Agrobacterium mediated transgenic tobacco. To provide material basis and method for the research of Orf25 gene function and the difference of tobacco male sterile line and fertile line, in order to investigate the genes related to male sterility in tobacco Orf25 was cloned and its role in the formation of the male sterility in tobacco, and lay the foundation for the theory and application research, to reveal the molecular mechanism of male sterility in tobacco the deepening of tobacco male sterility and Heterosis utilization. The main results are as follows:1.The preservation of p UC57-Orf25 in aboratory as template, enzyme recovery gene Orf25 linked with the double digested expression vector p BI121, get a new expression vector pBI121-Orf25, and at the same time, the CaMV35 S promoter in the expression vectors of pBI121-Orf25 into the flower specific promoter PchsA, constructing the expression vector p BI121-PchsA-Orf25, and the expression vector pBI121-Orf25(containing CaMV35 S promoter) and pBI121-PchsA-Orf25 were transferred into Agrobacterium tumefaciens LBA4404, lay the foundation for the next step of Agrobacterium mediated genetic transformation of Orf25 gene.2.Optimization of the culture system of tobacco: the best differentiation or adventitious bud tobacco differentiation medium is MS+1.0mg/L6-BA+0.1NAA; the best rooting medium of tobacco adventitious buds is MS medium.3.the optimization of the Agrobacterium tumefaciens mediated genetic transformation system of tobacco Orf25 gene: By Agrobacterium bacterium liquid diluted 100 times(OD600 = 0.5) infect the after 3 days of pre culture of explants 8 minutes, the best medium for differentiation of the cultured for 3 days, And then transferred to the 80mg/L containing kanamycin and ampicillin selection differentiation 500mg/L medium to induce differentiation of adventitious buds, After adventitious buds grow to about 2cm,switching to 80mg/L containing kanamycin and ampicillin screening 500mg/L rooting medium.4.Screening and PCR detection of transgenic plants: pBI121-CaMV35S-Orf25 transgenic tobacco plants of DNA and pBI121-CaMV35S-Orf25 positive plasmid DNA amplified fragments is about 494 bp, the specific bands of pBI121-Pchs A-Orf25 transgenic tobacco plants, the total DNA and pBI121-PchsA-Orf25 positive plasmid DNA amplification fragment was the same size, about 480 bp specific band the non transgenic plants, negative control without amplified bands produced. A total of 52 strains of pBI121-CaMV35S-Orf25 transgenic tobacco plants and 76 transgenic pBI121-PchsA-Orf25 tobacco plants, and identified: pBI121-CaMV35S-Orf25 transgenic positive plants was 14, the conversion rate was 26.9%; pBI121-PchsA-Orf25 transgenic positive plants was 18, the conversion rate was 23.7%. The experimental results indicated that foreign gene had been integrated into tobacco genome.