Agrobacterium-mediated Transformation And Regeneration Of Transgenic Plants With FOC1 Gene In Brassica Oleracea

Brassica oleracea fusarium wilt is a devastating disease leading to decrease of the quality and yield of the cabbage and all over the world for many years. By Cooperation with Institute of Vegetables and Flowers; Chinese Academy of Agricultural Sciences we identified FOC1 a candidate gene resistance to the disease, but its function has not yet been validated.We studied the regeneration system of cabbage through the seedling age, the different phytohormones combination and concentration of Ag NO3. Then by studying the optimal concentrations of the selection agent and the agrobacterium inhibitors and the bacterium solution and the infection time, the cabbage genetic transformation system was established. In order to study the function of FOC1 in transgenic cabbage, the plant expression vector p BI121-35S-FOC1 was constructed by homologous recombination method and was then transformed into Agrobacterium tumefaciens strain LBA4404 by freeze-thawing method. And the high-susceptible cabbage inbred line 213 was infected through Agrobacterium tumefaciens-mediated transformation and the transgenic plants were detected by PCR method. Results were as follows:1. Establishment of the regeneration system and genetic transformation system in cabbageFour day-age of seedling was selected as receptor materials; When hormone combination of 6-BA 3 mg/L and 0.1 mg/L NAA, the explant regeneration rate was as high as 31.4%; and it showed that Ag NO3 had no effects on polarization for the tested cabbage 213. The screening agent kan with concentration of 10 mg/L, the agrobacterium inhibition timentin with concentration of 150 mg/L, agrobacterium solution concentration with OD600 = 0.5 and infection time for 5 min were the most suitable.2. Construction of FOC1 sense expression vectorBased on the total length of sequence information of FOC1 cloned by RACE, the primers in-fusion ZY-FOCF/R were designed with 15 bp homologous to the the end of the expression vector p BI121. And we amplified the CDS of FOC1 using the template of c DNA obtained by reverse transcription. Then the restriction enzyme digestion was performed using endonuclease Xbaâ… and Sacâ… and p BI121-35S-FOC1 was constructed by infusion method. And the constructed expression vector was transformed into the competent escherichia coli TOP10 by thermal activation.3. Genetic transformation of plant expression vector and identification of transgenic cabbageAfter the agrobacterium-mediated transformation and co-culture and screening process, we finally obtained a total of 40 plants resistance to kanamycin resistance. Among them there were 10 positive plants by PCR showing that the gene FOC1 had been successfully integrated into the genome of receptor cabbage. But its copy number, if it is normal expression and if it can translate the functional protein were still need further validation through the Southern, Northern and Western.