Analysis Of The Linker Peptides2A And LP4/2A In Maize And Generation Of Transgenic Maize With Insect Resistance And Glyphosate Tolerance

Multiple genes transformation is one of the hot spots in modern genetic transformationresearch. Linker peptide has overcome the drawbacks of traditional methods to some extent andbecome a new way to construct gene-stacking vectors. Using2A from foot and mouth virus andLP4from Raphanus sativus seeds as linkers for multiple genes transformation is a new strategywhich can be substitute traditional methods in plant genetic engineering. The linker peptide can beused for connecting multiple genes and genes are expressed coordinately. The hybrid peptideLP4/2A has the both advantages of LP4and2A which is a more effective peptide for multiplegenes transformation.We used linker to connect two reporter genes gfp and gus and have constructed four fusiongene expression vectors: pSgAs, pSgLs, pSgLAs and pSsLAg. Our aims are studying the thefunction of linker in maize and comparing the splicing efficiency among2A, LP4and LP4/2A.The results showed that the cleavage efficiency of LP4/2A was the highest among the linkers. Thecleavage efficiency of LP4/2A was about80%, the cleavage efficiency of2A was about60%andthe cleavage efficiency of LP4was about30%.The crisis of pests and weeds are important factors for maize production reduction. Thedeployment of stacked traits of insect resistance and herbicide tolerance is becoming increasingimportant. The stacked maize can meet famers' requirement and is very prevalent in manycountries. The Bacillus thuringiensis (Bt) cry gene and the epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistance andglyphosate-tolerance crops. The cry1Ah gene is a novel insecticidal gene cloned from the BT8isolate. This gene shows high toxicity to the Lepidoptera insect. G2-epsps is a new epsps geneisolated from glyphosate-contaminated soil and was designed as2mG2-epsps after it wasoptimized with a plant codon preference.The manA gene as the positive selectable marker codes phosphomannose isomerase (PMI),and it will avoid the affect of natural environment and food-safety caused by antibiotic--resistantgenes. The manA gene from Escherichia coli has been used to successfully produce transgenicsugar beet, cassava, maize and rice. Furthermore, the database search revealed no significanthomology of the E. colimanA gene product to any known toxin or allergen. So the PMI/mannoseselection system is an ideal method for genetic transformation and will have a board implication.In our experiment, the Bt cry1Ah gene and the2mG2-epsps gene were connected by linkerLP4/2A. The two genes transferred into maize calli by Agrobacterium tumefaciens-mediatedtransformation. The mannose as selective material and the screening concentration is10g/L. Sixtytransformation plants were gotten and the transgenic plants were24, which belong to16events.Then T1transgenic plants were detected. Molecular detection and bioassay showed the two genesin the fusion vector were expressed simultaneously and bioassay detection showed that thetransgenic maize plants had good pest resistance and glyphosate tolerance. Meantime, we modified the linker2A and acquired two new2As. We used linker2Am1and2Am2to connect double reporter genes gfp and have constructed two fusion gene expressionvectors: pSg2Am1g and pSg2Am2g. The two2As vectors were used to check the cleavageefficiency of the new2As. We also wanted to try a novel strategy for increasing gene expressionwith this method. The results of this part showed that the gene expression increased with thismethod and the cleavage efficiency of the new2As is higher than the original2A.