Expression Of Cholera Toxin B Subunit And Proinsulin As A Fusion Protein In Transgenic Cucumber Plants

Objective:Genetic engineering is to process and re-mix the genetic material under the appropriate conditions according to people's needs, and change certain traits and functions, and then transfer the newly-combined genetic information through the proper vector to the host in vivo to make it express; thus gain the new biological function. Plants gained by this approach are known as "genetic engineering plants." Transgenic plants were first gained in 1983, since then transgenic technology has developed very rapidly, some GM products have entered the market.To assist the research and development of transgenic plants, biotechnology is taken into the high-tech research and development programs and national scientific and technological research programs of the "863". With the support of national scientific and technological research programs of the "863", China's transgenic biotechnology research and application have much more progress in the recent years. The biotechnology section in "863" program includes three topics:new varieties of high-yield, high-quality and stress tolerance plants and animals; new vaccines, drugs and gene therapy; protein engineering。The purpose of this study is to explore the feasibility of trafficking fusion protein, in the cholera toxin B subunit-human proinsulin gene expression in cucumber callus; using plasmid digestion method to combine the artificially synthesized CTB subunit genes and human proinsulin genes to get CTB+fusion gene of human proinsulin; to connect this target gene to the vector transgenic plant PBI 121, and clone the plasmid into the Agrobacterium LBA4404.The study is also to transfect the cultured cucumber callus through Agrobacterium mediated transformation method, to gain the resistant callus by a series of screenings, to test whether the target genes transfected the plants callus and whether fusion proteins were expressed by ways of DNA, RT-PCR, Western blo and so on.Methods:the test has a rational design based on the latest results of transgenic research.a. Obtained the CTB gene and the human proinsulin gene (SPBCA) by multiplex PCR;b. Putting the GPGPGP hinge between the CTB gene and SPBCA in favor of the produced protein folding to the correct spatial structure;c. In order to avoid protein hydrolysis by the host body, joined KDEL sequence in the SPBCA sequence later,the fusion gene encoding the protein retention in the endoplasmic reticulum;d. In order to increase the expression of fusion protein, in front of CTB gene and behind the KDEL sequence were added a MAR sequences,, constitute the structure of MAR-Gene-MAR;e. According to codon preference of cucumber on the MAR-Gene-MAR in the amino acid codon was properly optimized, removing the use of low amino acid codon;f. The purpose of the final fusion gene by MAR+CTB+gpgpgp+SPBCA+ KDEL+MAR.g. Two restriction sites BamH 1/Sac 1 were added at both ends of the target gene, while designed of the primers, to facilitate insertion into the plant expression vector.;h. Choose to efficient expression of the plant-specific transgenic vector。Plasmid PBI121 been transformed vector, and is a transgenic plant expression systems commonly used in an expression vector, with CaMV 35S promoter, the promoter does not show temporal and spatial specificity, and not subject to external factors, induction, can control the foreign gene high expression in the plants。 i. According to the method reported about the literature cucumber callus culture and after infection by Agrobacterium calli, has been well-developed callus blocks were used for follow-up tests.j. Using known methods of molecular biology DNA, RT-PCR, Western blot detection of fusion gene is transfected into callus, and whether expressed fusion protein.Results:According to the results of experimental design, two optimized gene sequences have been got. Many tests show that synthetic gene order is included in the Construction of plasmid PBCSPBCA.It has been testified that artificially synthesized gene order has been transferred to the callus of cucumber and fusion proteins were expressed by the way of extracting the DNA, mRNA and protein in screening callus by the corresponding kit, and through the tests of DNA, RT-PCR, Western blot and so on.