Construction Of Plant Expression Vectors With Bivalent Genes Harbouring Synthetically-modified VHb Gene And Bt,Bt-CPTi Gene Respectively And Their Expressions In Tobacco Plants

One of the key objectives that breeding faces is to manage to increase the yield anof the crops,meanwhile,for traditional breeding technology, the potentiality to increase yield of crop is rather limited.Pest is one of the main factors which endanger the high yield of crops.Genes can be transferred from one species to another by applying transgenic engineering technology ,which enable the charactersitics including high-yield and pest-resistance to be transferred to one species. In this article, the highly-efficient plant vectors with bivalent genes have been constructed by combining the gene of VHb with Bt gene and CFMCryla-CPTi gene respectively, studying the influence over the plants metabolic level and growth .We manage to reach the goal of increasing the yield of transgenic plants and cultivate new plant varities with pest-resistance by applying transgenic approaches, and eventually in the hope of cultivating new varities of crops with hoth highly-steady-yield and pest-resistance. For these goals ,the following jobs have been done and some results have been obtained.1 According to the Vitreoscilla hemoglobin (VHb) amino acid sequence and plajnt preference codon usage, vgbM gene was designed and synthesized by annealing 22 synthetic fragments respectively, the modified vgbM gene of full length of 450 base pairs was synthesized .2 The transformants were obtained after pBV221SVHB was introduced into E. coli. BL21 strain. The temperature induction expression of the recombinant E. coli. was carried out and VHb protein of 16k-D was expressed.3 We demonstrated the VHb owns biological activity by CO difference spectrum. The growing graphs of E. coli BL21 containing pBV221 and pBV221SVHB according to the OD600 values showed that the E. coli vgb+ grew faster4 the highly-efficient bivalent vectors which are named PGBIF4ABCVHB and PGBI4ASVHBBt respectively ,havfs been.suî–©essful]y constructed .The tobacco leave discs were transformed with Agrobacterium tumefaciens carrying the plant expression vectors PGBIF4ABCVHB and PGBI4ASVHBBt .38 and 36 transgenic tobacco plants have been obtained respectively after kanamycin screening, PCR detection, Southern blot analysiss showed that the foreign genes have been inserted into the tocacco plant genome .The expression of 16kD VHb protein in transgenic plants was proved by Western blot. After 60 days of growth the leaf net weight of transgenic tobacco plants has increased by108.4%.112.7% respectively compared to wild-type controls. Furthermore, chlorophyll in transgenic plants has risen by 34%.38%compared to wild-type controls. Toxicity assay was performed by feeding cotton boll worm larvae with the leaves of the transgenic tobacco, transgenic plants showed highly pesticadal toxin ,the result showed that the pesticadal genes have been expressed.