Enhanced The HCHO Detoxification Capacity Of Plant By Genetic Engineering

In plant genetic engineering,the mostly used promoters are cauliflower mosaic virus (CaMV)35S promoter and ribulose-1,5-bisphosphate carboxylase(Rubisco)small subunit promoter(PrbcS).The gateway technology developed by Invitrogen is very useful for construction of the expression vectors for a target gene.However,the present destination vectors developed by VIB/Gent for expressing a target gene in plants only contain CaMV 35S promoter(35S).The expressed proteins are localized in cytoplasm.In the present study, an entry vector(pENTR~*-Prbcs-~*T-GFP)which contains PrbcS and a reporter gene(green fluorescent protein gene,GFP)was constructed.Thus,a light-inducible plant expression vector for a target gene can be rapidly constructed with this entry vector through the Gateway technology to achieve the high level expression of the taget gene in leaves.The expression of the target gene is induced by light,and the protein encoded by the target gene can locate in chloroplasts or cytoplasm.To test the application of the constructed entry vector,the plant expression vectors,pK2-35S-PrbcS-~*T-GFP and pK2-35S-PrbcS-~*T-GUS were constructed with the entry vector for two reporter genes,GFP and GUS,by the Gateway technology.These two expression vectors contain PrbcS and CaMV 35S promoter. Simultaneously,the constitutive plant expression vectors,pK2-35S-GFP,pK2-35S-GUS and the inducible plant expression vectors,pPZP211-PrbcS-~*T-GFP, pPZP211-PrbcS-~*T-GUS,were constructed for the two reporter genes by ligation enzyme technology.Then tobacco and geranium were transformed with the vectors through Agrobcterium-mediated method.The observation of GFP fluorescence showed that fluorescence appeared in all GFP transgenic calluses.The GFP intensity for the callus produced from the tissues transformed with pK2-35S-PrbcS-*T-GFP was the same as those transformed with pK2-35S-GFP and pPZP211-PrbcS-~*T-GFP.This indicates that GFP gene can expressed normally in plant tissues transformed with the pK2-35S-PrbcS-~*T-GFP.GUS expression level was analyzed in transgenic tobacco leaves by GUS histochemical assay. The results showed that GUS expression level in the pK2-35S-PrbcS-~*T-GUS transgenic leaves are the same as those in the pPZP211-PrbcS-~*T-GUS transgenic leaves.The data also demonstrated that to achieve high expression of the target gene in leave is very difficult by the constitutive promoter.GFP quantitative analysis showed that the intensity of GFP fluorescence in the pK2-35S-PrbcS-~*T-GFP transgenic leaves is similar to that in the pPZP211-PrbcS-~*T-GFP transgenic leaves and is 2-3 times of that in the pK2-35S-GFP transgenic leaves.GUS quantitative analysis showed that GUS activities in the pK2-35S-PrbcS-~*T-GUS transgenic leaves are also the same as that in the pPZP211-PrbcS-~*T-GUS transgenic leaves and are 1-2 times of that in the pK2-35S-GUS transgenic leaves.Thus,light-inducible plant expression vectors constructed by this entry vector can achieve high expression of a target gene in a leaf.Ribulose monophosphate pathway(RuMP)is an important HCHO assimilation pathway in methylotrophic bacteria.HPS(3-hexulose-6-P synthase)and PHI (6-phospho-3-hexuloisomerase)are the two key enzymes in RuMP pathway.Recent studies reveal that HPS and PHI homologous exist in many no methylotrophic bacteria.Homology search show that there HPS and PHI homologous sequences in Escherichia coli,but there is no report about the enzyme activities.In the present study,the HPS and PHI homologous sequences were amplified from E.coli genome by PCR and the prokaryotic expression vectors pDE17-Ehps,pDE17-Ephi were constructed for the two genes respectively.The recombined proteins encoded by the two genes were expressed and purify.Further analysis indicated that the recombinant protein exhibited enzyme activities.The plant expression vectors,pK2-Prices-T-Ehps,pB2-PrbcS-T-Ephi,were constructed.Arabidopsis was transformed with pK2-PrbcS-T-Ehps,pB2-PrbcS-T-Ephi by Agrobacterium-mediate method.The transgenic plants were selected and verified by PCR.RT-PCR analysis demonstrated these two genes were transcripted successfully in transgenic Arabidopsis.Serine Pathway is one of bacterial HCHO assimilation pathways and Serine hydroxymethyltransferase(SHMT)is the key enzyme in the pathway.It is speculated that SHMT may be involved in this phenomenon directly,and many reactions in plant photorespiration pathway are similar to those in bacterial Serine pathway.SHMT is the key enzyme in C1 compound assimilation.Thus the present study attempted to overexpress mitochondrial SHMT from Arabidopsis(shm)and cytoplasmic SHMT from E.coli(smt)in transgenic Arabidopsis to investigate whether SHMT is also the key enzyme responsible for HCHO assimilation in plants.The plant expression vectors,pK2-35S-SMT and pB2-PrbcS-SHM,were constructed.The transgenic plants were selected and verified by PCR.