Co-transformation Of MsADCS And MsGCHI Genes Encoding The Key Enzymes For Folate Synthesis Into Lotus Corniculatus

Folate is a water-soluble B vitamin(vitamin B9),which plays an important role in the metabolism,growth and development of animals and plants.However,insufficient folate intake is common in humans and other animals.Increasing the content of folate in crops by biofortification has became an effective way to solve the problem of general lack of folate in food.Numerous previous studies have demonstrated that GTP cyclohydrolase I(GCHI)and aminodeoxychorismate synthase(ADCS)are the two most important rate-limiting enzymes regulating folate biosynthesis in plants.The coexpression of both GCHI and ADCS genes can result in significant increase of folate content in many transgenic crops.However,there is no report related to folate biofortification in forages so far.In the previous study,we have cloned the genes encoding the key enzymes GCHI and ADCS in folate biosynthesis from alfalfa(Medicago sativa),which was rich in folate.In the present study,a bivalent plant expression vector carrying MsGCHI and MsADCS genes was constructed and introduced into Lotus corniculatus,an excellent legume forage.The transgenic lines with relatively consistent and high expression levels of both target genes were screened by molecular identification.Finally,the contents of folate and its components as well as the growth characteristics of these transgenic lines were analyzed and evaluated under greenhouse and field conditions.The main results were as follows:1.The MsGCHI and MsADCS bivalent plant expression vector carrying the herbicide resistance selectable marker gene Bar was successfully constructed and introduced into L.corniculatus via Agrobacterium-mediated transformation.A total of13 positive plants were identified by PCR,and among them,five transgenic lines with relatively high expression levels of both MsGCHI and MsADCS genes were screened by RT-PCR and RT-qPCR analysis.2.The co-expression of MsGCHI and MsADCS genes up-regulated the transcription abundances of several genes encoding folate biosynthesis-and regulationrelated enzymes in transgenic L.corniculatus.Compared with the wild-type,the expression levels of genes that encode aminodeoxy chorismate lyase(ADCL),hydroxymethyldihydropterin pyrophospho kinase/dihydropteroate synthase(HPPK/DHPS),dihydrofolate reductase(DHFR)and dihydroneopterin aldolase(DHNA)showed significant increase in transgenic plants.3.The co-expression of MsGCHI and MsADCS genes significantly increased the contents of folate and its precursors(pterin and p-aminobenzoic acid)in transgenic L.corniculatus.HPLC-MS/MS analysis showed that the contents of folate,pterin and paminobenzoic acid in transgenic lines were significantly higher by up to 3.3,6.1 and3.7 times,than those in wild-type,respectively,under either greenhouse or field conditions.Moreover,in the transgenic lines,the folate content in leaves showed significantly higher compared to that in stems,while the contents of pterin and p-ABA in stems were significantly higher than for leaves except GA-5 line.4.The contents of folate derivatives in transgenic L.corniculatus were significantly higher than that in wild-type plants.Under greenhouse conditions,the contents of 5-methyl-tetrahydrofolate(5-M-THF)and 5-formyl-tetrahydrofolate(5-FTHF)in leaves of transgenic lines increased the most,which was 1.9 times and 2.1times of that of wild-type,respectively.Under field conditions,the 5-F-THF showedmost in leaves of transgenic lines increased the most(higher by 1.1 times than for wild-type),and the contents of 5-M-THF and 5-F-THF in stems of transgenic lines increased by 0.5 times and 0.8 times,respectively,compared with wild-type.In addition,the contents of 5-M-THF,5,10-methylene-tetrahydrofolate(5,10-CH=THF)and tetrahydrofolate(THF)in leaves of transgenic lines were significantly higher than those in stems,and the content of 5-F-THF in stems of transgenic lines was significantly higher than that in leaves except GA-11 line.5.The co-expression of MsGCHI and MsADCS genes promoted the growth performance of transgenic L.corniculatus.Under greenhouse condition,the growth rate and biomass of transgenic line GA-3,as well as the branch number and root length of transgenic line GA-11,were significantly better than those of the wild-type,respectively.Compared to wild-type plants,after 42 days of field transplanting,the leaves of transgenic L.corniculatus showed larger with 61.2% increase of leaf area than those of wild-type plants.Correspondingly,the branch number and plant height of transgenic plants significantly increased by 18.0% and 6.8% than for wild-type plants after transplanting for 68 and 82 days,respectively.Interestingly,transgenic L.corniculatus showed longer vegetative growth period and later flowering compared to wild-type plants,and its leaf/stem ratio at the initial flowering stage was significantly increased by 18.9% than for wild-type plants.In addition,the transgenic L.corniculatus also showed herbicide(glufosinate-ammonium)resistance.The above results indicated that the co-expression of MsGCHI and MsADCS can significantly promote the growth of L.corniculatus increased the contents of folate as well as its precursors and components in transgenic lines.The results laid a solid theoretical and practical foundation not only for generating the L.corniculatus cultivar with high folate content,but also for producing the folate through bioreactor.