Different Agrobacterium Tumefaciens-mediated Transformation Of Human Insulin Gene To Tremella Fuciformis And Detection Expression Products By ELISA

In accordance with the transgenic methods of Tremella fuciformis mediated by Agrobacterium EHA105 that previously established in our laboratory, our experiments used one hygromycin sensitive strain of Tremella fuciformis named Tr21-01 as a original strain. In our research, with the method of Freeze-thaw, the plasmid pBHg-BCA which consisted of the human insulin BCA gene as objective gene and hygromycin phosphotransferase gene as genetic markers transformed Tremella fuciformis mediated by Agrobacterium GV3101, LBA4404 and AGL-1. As a result, we found AGL-1 had higher conversion rates; while GV3101 had lower conversion rates and higher false positive rates; LBA4404 did not grow any resistant colonies. The results indicated that different Agrobacteriums had different abilities of infection with Yeast-like conidia of Tremella fuciformis, and the receptors of infection were peculiar.The experiments applied EHA105 as a reference strain to investigate transformation of Tremella fuciformis mediated by Agrobactrium AGL-1. Since Agrobacterium tumefaciens-mediated transformation can be affected by numerous factors, our tests optimized some influencing factors such as the coculture time of Agrobacterium with Tremella, the concentration of Yeast-like conidia of Tremella fuciformis and Agrobacterium, the concentration of acetosyringone used as inducer, as well as the positive rates of the transformed strains. The optimization results showed that the OD value of Agrobacterium AGL-1 was 0.4~0.5, the induced concentration of AS was 250μg/mL, while the concentration of AS in the coculture was 150μg/mL, the concentration of Yeast-like conidia of Agrobactrium tumefaciens was 10~8/mL,when the coculture time came up to 72h, the transformation rate reached 500/108 and the positive rate of the colonies was 89%. While with regard to Agrobacterium EHA105, the OD value was 0.4~0.5, the induced concentration of AS was 200μg/mL, while the concentration of AS in the coculture was 200μg/mL, the concentration of Yeast-like conidia of Agrobactrium tumefaciens was 108/mL,when the coculture time reached 48h,the highest transformation rate was up to 350/10~8 and the positive rate of the colonies was 83% . In a word, Agrobacterium AGL-1 had higher transformation rates than Agrobacterium EHA105.With the transgenic methods above, we screened a large number of transformants.Then we randomLy selected 30 transformants to validate the level of DNA with ITS,Hpt and BCA gene, while at the same time we randomLy selected 6 transformants to validate the level of cDNA with Hpt and BCA gene. The results demonstrated that all the transformants we selected were positive strains. Moreover we detected the level of mRNA of Hpt and BCA gene by RT-PCR.We applied the method of ELISA to detect the expression products of BCA gene from 60 transformants. The results showed that some transformants could express our BCA objective gene. Furthermore,the analysis found the transformants that expressed BCA gene had a high hygromycin resistance, which were up to 350μg/mL. In conclusion, we verified that it had a certain relationship between the expression of the Human insulin gene and Hpt resistance gene.