Research On The Expression Of Human Insulin In Transgenic Tremella Fuciformis

Since Agrobacterium tumefaciens-mediated transformation system wassuccessfully for the first time applied to transfer the human insulin BCA gene intoyeast-like conidia of Tremella fuciformis in our lab, five randomly transgenic Tremellafuciformis strains have been selected to carry out research on the fruiting bodies andoptimization of fermentation conditions of transgenic Tremella fuciformis as well asanalysis of expression of the human insulin BCA gene with Real-timefluorescence-based quantitative PCR according to the results of hygromycin Bresistance and PCR assays. The experimental conclusions were as follows:Before using the absolute method to estimate the human insulin BCA gene copynumbers from five transformants of transgenic Tremella fuciformis, plasmid standardsand standard curves were prepared for the transgene (BCA) and the endogenous GPDgene. The standard curves for the GPD and BCA genes, were y=-3.349x+40.25andy=-3.379x+37.75, respectively. Both curves were highly linear (R²=0.998andR²=0.995) in the range. From the slopes, amplification efficiencies of98.89%and97.67%were determined for the GPD and BCA genes, respectively, in theinvestigated range, indicating that our method was applicable to estimation oftransgene copy number. According to the results of standard curves and the Ct values,five transgenic Tremella fuciformis DNA samples were tested, and the resultsindicated that T26, T56and T65had single copy, T53had two copies of the transgene,while T20was a non-transgenic strain.Moreover, the transformant strain T26was used to do further research on theformation of fruiting bodies cultured with various non-transgenic single-spore strainsfor the production of Tremella fuciformis. The results demonstrated that T26+T₄₍₃₎andT26+T₁₄could form transgenic fruiting bodies of Tremella fuciformis. RT-PCR andELISA experiments verified that the human insulin BCA gene had been expressed infruiting bodies of transgenic Tremella fuciformis.Subsequently, the expression of the human insulin BCA gene at the level of transcription was determined by the relative method. As the absolute value of theslope was close to zero, the efficiencies of the GPD and BCA genes were similar, and2-(Ct)method calculation for the relative quantification of the human insulin BCAgene can be used. The results showed that the expression of the BCA gene had variedgreatly among the four transformants of transgenic Tremella fuciformis (T53, T26,T56and T65). However,the expression level of the human insulin gene in yeast-likeconidia was higher than in the fruiting bodies.Based on the results of Real-time fluorescence-based quantitative PCR andenzyme-linked immunosorbent assay (ELISA), the human insulin BCA gene wassuccessfully expressed in both yeast-like conidia and fruiting bodies of transgenicTremella fuciformis. By using the dimorphism character (yeast-like conidia andmycelium; both are edible, nontoxic, and harmless) of Tremella fuciformis, thetransgene product can be produced by both conidia fermentation and large-scaleartificial cultivation of fruiting bodies. Considering the optical density and wet weightof fermentation culture in shaking flask, as well as the results of protein products ofthe human insulin BCA gene detected by sandwich ELISA as screening markers offermentation conditions for yeast-like conidia of transgenic Tremella fuciformis(taking the transformant strain T65for example), the optimal fermentation conditionswere as follows: cultured with mannitol as carbon sources, beef extract and yeastextract as sources of nitrogen, pH6.0and7.0as the optimum pH for growth. Thesefindings will lay a good foundation for further development of Tremella fuciformisbioreactor to be a new efficient expression system for human insulin.