| With the development of plant genetic engineering, the research for introducing multiple heterologous proteins into plants becomes one of the hot spots and the polyprotein strategy for multiple genes transformation is paid more attention to. The polyprotein, encoded by a single open reading frame (ORF) with 2A or LP4 as linker, was dissociated into two or multiple proteins when it was expressed in plants. In this study, the novel Bt cry1Ah gene was connected with the mG2 gene encoding EPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase) by the linker peptide 2A and LP4/2A, and the spliced proteins, Cry1 Ah and mG2, were correctly expressed in transgenic plants.In order to compare the splicing efficiency between the 2A and the LP4/2A, and study the position effect to gene expression, we have constructed four co-ordinated expression vectors: pHAG,pHLAG,pGAH,pGLAH; and two control vectors: pSAh,pSmG2. In vectors pHAG and pHLAG, the cry1Ah was anterior and the mG2 was posterior, with the linker 2A and LP4/2A respectively. And in vectors pGAH,pGLAH, the mG2 was anterior and the cry1Ah was posterior, with the linker 2A and LP4/2A respectively.The six expression vectors were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation and 529 transformants were obtained. GUS histochemical assays showed that there were two hundred and sixty-one GUS-positive plants and the transformation efficiency was 49.3％. GUS-positive plants were further analyzed by PCR amplification and the results showed that the cry1Ah gene and the mG2 gene had integrated into the genome of the tobacco plants. RT-PCR showed that the fusion gene was transcripted correctly in all transgenic plants and the cDNAs of the co-ordinated expression genes were intact during the transcription level. Western blot showed that the foreign proteins Cry1Ah and mG2 were expressed correctly in transgenic tobacco respectively. ELISA results indicated that the mG2 protein in transgenic tobacco leaves transformed with the co-expression vector pGLAH was 0.33％in transgenic tobacco leaf soluble protein and was higher than other transgenic plants transformed with other vectors, and the expression of Cry1Ah in all plants of different vectors was over 0.2％in transgenic tobacco soluble protein. The bioassay results confirmed that the transgenic tobacco plants were toxicity to the larva of Helicoverpa arnzigera Hubner and the relative death rate in transgenic tobacco of all the co-expression vectors displayed higher than 90%. They also displayed glyphosate tolerance when the transgenic tobacco plants were sprayed with 3‰glyphosate in the T0 generation, and the transgenic plants harboring pGLAH vector showed higher tolerance than the transgenic plants harboring other vectors. The experiment of T1 seed germination in MS medium adding to glyphosate showed the transgenic tobacco harboring pGLAH had highest tolerance in all transgenic tobacco.Both the molecular analyses and the bioassay of the transgenic plants showed that the linker LP4/2A was better than the linker 2A, and the expression of the anterior gene is higher than the posterior gene.
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