Study On Obtaining Fibroblast Growth Factor FGF21 In Tobacco By Transgenic Technology

Fibroblast growth factor 21(FGF21) is a stress-induced hormone that plays an important role in metabolic regulation.Because of its substantial improvement of insulin sensitivity,blood glucose control and blood lipid profile,it is considered to be a new candidate medicinal protein for the treatment of metabolic diseases such as type 2 diabetes.In China,the proportion of type 2 diabetes mellitus is 90-95%,the proportion of type 1 diabetes mellitus is 5-10%,and the other few types are gestational diabetes mellitus or special type diabetes mellitus,accounting for about 1%.Diabetes has become the third serious chronic disease in the world after cancer and cardiovascular disease.However,the production pathway of FGF21 in Escherichia coli and Pichia pastoris cells is easy to degrade.Therefore,transforming the target into plant expression system provides a new way to obtain FGF21 protein as a medicinal protein.In order to obtain FGF21 in plant expression system,FGF21 gene expression vectors pcambia3301-new FGF21 driven by camv35 s promoter were constructed.The expression vector pcambia3301-new FGF21 was transformed into Agrobacterium tumefaciens eha105 and transformed into tobacco by leaf disc method.Transgenic plants were obtained by hygromycin resistance screening,and their molecular detection was carried out.The genomic DNA of transgenic tobacco plants was extracted and detected by PCR.Semi quantitative RT-PCR and Western blot were used to detect the expression of foreign genes at transcriptional level.In this study,the FGF21 gene sequence was optimized and chemically synthesized according to the codon preference of tobacco plants.The plant expression vector pcambia 3301-new FGF21 was constructed by optimizing the driving codon of camv35s promoter.Agrobacterium tumefaciens mediated tobacco leaf disk method was used to infect the small vein of transformed tobacco.Five resistant plants were obtained after screening with 1% basta herbicide.These plants were named 2021ntf21s1 ?2021ntf21s5.The results of RT-PCR and Western blot showed that the expression of FGF21protein was the highest in the mature leaves of transformation event 2021 ntFGF21s3,which confirmed the successful expression of foreign gene FGF21.