Study On Genetically Modified Forage Applied Co-transformation Of Photorespiration Bypass Genes

Photorespiration has long been thought to be an energy wasteful process.Studys on decreasing photorespiration to improve crops is continuous.However,the methods of taking advantage of photorespiration mutants,chemical regulation and altering C3 plants by C4 pathway were not effective.Kebeish established a photorespiration bypass by introducing the E.coli glycolate catabolic pathway to plant chloroplasts to supply a feasible method to these kinds of research.Stylosanthes guianensis is a good leguminous forage.With some quality such as high forge quality,easy cultivation and high protein level,it is an excellent feeding forge.Alfalfa,often called “the queen of forage”,is an important grass supporting our animal husbandry development.It is one of the highest content of crude protein in forges and has very high yield and excellent forge quality.Our pratacultural industry,which needs a prodigious amount of forages is now developing.Therefore,the significance obviously is there for popularizing the biomass of S.guianensis and alfalfa,and then promoting the development of our pratacultural industry by decreasing the photorespiration of S.guianensis and alfalfa.On the other hand,it will promote the thriving development of our animal husbandry too.Both the plasmid vector DEF2 in pCAMBIA-mCherry and TG1 in pEarleyGate100 are presented by Dr.Qu Rongda.DEF2 contains gene GDH(glycolate dehydrogenase),which has three subunits encoded by three genes,GLcD,GLcE,and GLcF,and gene Bar that is the selective marker of plants.TG1 contains gene GCL(glyoxylate carboligase),TSR(tartronic semialdehyde),and gene mCherry that is the selective marker of plants.All of these five genes make up the Escherichia coli glycolate catabolic.Transgenic S.guianensis plants co-expressing TG1 and DEF2 were generated by Agrobacterium-mediated transformation in our study.The explants of S.guianensis has passed through callus stage to become new regenerated plants.At last,we got thirteen transgenic S.guianensis plants with TG1,and three S.guianensis plants with both TG1 and DEF2.TG1 transgenic Stylosanthes guianensis plants were found out by sprinkling 60mg/L Basta on their leaves and stems.PCR amplification illustrates that six genes(Bar,GLcD,GLcE,GLcF)of TG1 and DEF2 were integrated into the genome of S.guianensis plants.In order to testifying the existence of DEF2 in transgenic S.guianensis plants,we observed the mcherry fluorescence in their leaves and root tips under fluorescence microscope.In the end,we obtained 6 transgenic Stylosanthes guianensis plants with TG1,and 3 with DEF2 and TG1.The T1 generation transgenic S.guianensis plants with TG1 were sprinkled by Basta for selection.There were nothing alive for wild S.guianensis CITA 184,but part of them alive for transgenic S.guianensis plants.The T1 generation transgenic S.guianensis plants with TG1 and DEF2 were tested for the relative expression of gene GLcE and GCL by real-time PCR.It turned out that gene GLcE and GCL were expressed for high level.Transgenic alfalfa plants that co-expressed TG1 and DEF2 were generated by Agrobacterium-mediated transformation technique in our study.The explants of alfalfa plants has passed through callus and tissue differentiation stage to eventually become new regenerated plants.We ultimately got twenty-two transgenic alfalfa plants with TG1,and four alfalfa plants with both TG1 and DEF2.TG1 transgenic alfalfa plants were found out by sprinkling 30mg/L Basta on their leaves and stems.PCR amplification illustrates that six genes of TG1 and DEF2 were integrated into the genome of alfalfa plants.We observed the mcherry fluorescence in their leaves and root tips under fluorescence microscope for testifying the existence of DEF2 in transgenic alfalfa plants.We obtained 19 transgenic alfalfa plants with TG1,and 4 with DEF2 and TG1.