The Research Of The Multigene Fusion Transformation In Transgenic Maize (Zea Mays L.)

With the increasing application of GMCs (genetically modified crops) and exploration of function genes, genetic manipulation must be qualified not only to complete the transformation of a single gene, but also to achieve the transformation of multi-gene efficiently and conveniently no matter from basic research or application point of view. Therefore, the research of multi-gene transformation becomes a new hot spot.In this study, we connected report gene gfp(green fluorescent protein), gu(sβ-glucuronidase) and the selective marker gene hpt (hygromycin phosphotransferase) by the linker peptide 2A and LP4/2A to construct fusion gene expression vector. The fusion gene expression vectors were transformed into maize in order to analysis the splicing function of linker using maize transicent expression system and stable expression system. Also whether the position of foreign gene linked by linker would impact the expression level of foreign gene was analysed.First of all, we constructed four co-ordinated expression vectors:p35PASAT,p35SAPAT,p35PLASLAT,p35SLAPLAT;and two control vectors:pPT,pST. In vectors p35PASAT and p35PLASLAT, the gfp gene is anterior, the gus gene is posterior and the hpt gene is ultimate, with the linker 2A and LP4/2A respectively. And in vectors p35SAPAT,p35SLAPLAT, the gus gene is anterior , the gfp gene is posterior and the hpt gene is ultimate, with the linker 2A and LP4/2A respectively. In vector pPT, gfp gene is connected with hpt gene directly. In vector pST, gus gene is connect with hpt gene directly.The six expression vectors were transferred into maize calli by microprojectile bombardment for transcient expression analysis. RT-PCR results revealed that the foreign fusion gene can be transcipted correctly and the fusion gene is intact. Western blot showed that the foreign proteins GFP and GUS were expressed correctly in transgenic maize calli respectively. GUS histochemistry assay and GUS fluorescent quatity dectection results showed that the level of GUS expression in maize calli with p35SAPAT is higher than the level of GUS expression in maize calli with p35PASAT; the level of GUS expression in maize calli with p35SLAPLAT is higher than the level of GUS expression in maize calli with p35PLASLAT; the level of GUS expression in maize calli is almost the same between the vector connecting with linker 2A and LP4/2A.The six expression vectors were also transferred into maize by microprojectile bombardment for stable expression analysis. After differentiaton and rooting, we acquired 192 transformants. The plants were analyzed by PCR amplification and the results showed that the gfp gene, the gus gene and hpt gene had integrated into maize genome. RT-PCR showed that the fusion gene was transcripted correctly and the cDNAs of the co-ordinated expression genes were intact during the transcription level. Western blot showed that the foreign proteins GFP and GUS were expressed correctly in transgenic maize plant respectively.According the results of experiment, we have got a conclusion that the linker 2A and LP4/2A can be used for multi-gene transformation in maize: 1) The expression level of the anterior gene is higher than the expression level of the posterior gene; 2) The splicing function of linker is happened during the translation level; 3) The expression level of foreign gene is almost the same between the linker 2A and LP4/2A.