Construction Of Plant-insulin Expression Vector And Genetic Transformation Of Tobacco And Maize Using Cre/Loxp Recombination System

Exciting researches and significant strides have been published in the area of the plant-based bioreactor study in recent years. Low energy consumption, high-security and easy collection have provided the powerful driving force for its rapid development. Now, Plant-based bioreactor can be used to produce various of products including vaccine antigens, medical therapeutic proteins, industrial proteins, nutritional supplements (inhibiting substances, vitamins, carbohydrates and biopolymers).Insulin is a certain medicine for the treatment of type Ⅰ diabetes and type Ⅱ diabetes, which can lower the blood glucose by regulating the glycometabolism including reducing the gluconeogenesis and glycogen synthesis, inhibiting glycogen decomposition, accelerating aerobic oxidation and anaerobic glycolysis of glucose. Diabetes and its complications have taken a toll on the patients, and also have financial burdens on individuals, families, and countries. The number of patients with insulin has grown more than tenfold in recent 20 years, and the insulin has become one of the most severe health illnesses affecting humanity. The fast rise of diabetics will eventually lead to the large production of Insulin.In this study, proinsulin was optimized according to the codon preference of the Plant Bioreactor, and the proinsulin turned into the long-acting and fast-acting proinsulin by changing some amino acids of the proinsulin. The flag tags were added to the C-terminus of the modified and original proinsulin sequences for the convenience of protein detection and purification.We use pCAMBIA2301-pRUBP-proinsulin and pCAMBI A2301 RD29A - Cre,which build by Dr Zheng Ling, and apply with double bacterium infection tobacco by co-transformation method. The characteristics of the plant expression vector is base on the pCAMBIA2301 skeleton, insertion of arabidopsis thaliana pRUBP proinsulin gene promoter, the resistance genes NptⅡ added the Cre recombinant enzyme recognition sites loxp sequence in the polyclonal loci.Arabidopsis thaliana induced RD29A promoter sequences and Cre recombinant enzyme inserted into a plant expression vector pCAMBIA2301, constructed pCAMBI A2301 RD29A - Cre plant expression vector.After the PCR detection and GUS reporter gene,there are 4 transgenic tobacco plants.Three kinds of pCAMBIA3301-35S-proinsulin plant expression vectors based on the plant expression vector skeleton of pCAMBIA3301 were constructed to transform the tobacco and corn. Transgenic tobacco was obtained by the Agrobacterium-mediated transformation. It had been proved that the above of expression vectors had been integrated into the tobacco genomes and expressed at high level by the methods of PCR, and RT-PCR. The study of the proinsulin gene expression in the transgenic tobacco provided fundamental basis for the study of the proinsulin gene expression in the corn. The transform study of the corn was carried out with Agrobacterium-mediated method and gene gun bombardment, and the primary PCR detection was finished.