Folate And Anthocyanin Fortification Of Wheat By Genetic Engineering Approach

World’s large population is suffering from the malnutrition.Cereals and tubers are the major food groups which are fail to supply all types of essential nutrients required for nourishment.Several attempts have been made to solve this problem like multinutrient supplementation,industrial food fortification and biofortification of food crops.Wheat（Triticum aestivum L.）as“king of cereals”is very important food crop among cereals.It is considered a key crop for global food security.Therefore,it has great potential for biofortification.Folates are an essential requirement for the maintenance of normal cellular function.Many organisms including plants obtain folate coenzymes through de novo synthesis.Humans in common with all other vertebrates depend solely on nutritional sources and suffer from a number of disease states arising from inadequate folate consumption.The anthocyanins represent a class of secondary metabolites synthesized by higher plants.They are responsible for the pigmentation of flowers and fruits.They are also used as visible markers in conventional and molecular plant breeding programs.The presence of anthocyanins in plant-derived food is also important because their intake in the human diet is associated with protection against coronary heart disease and due to their anti-inflammatory and anticarcinogenic activities.In anthocyanin biosynthesis pathway,MYB and bHLH-type transcriptional factors are involved they interact with each other and made a ternary complex with WD40 protein,which regulate the structural genes of this pathway.In the present study,we evaluated the Chinese wheat germplasm to explore the natural variations for folate contents present among wheat accessions.Moreover,wheat grains were biofortified for high folate contents through metabolic engineering.Firstly,soybean genes GmGCHI（GTP cyclohydrolase I）and GmADCS（aminodeoxychorismate synthase）which are involved in the synthesis of folate precursors pterin and p-aminobenzoate,respectively were co-transformed and expressed in wheat plants under the regulation of the endospermic promoters of rice GluC and maize Leg1A,respectively.While,in the second strategy,soybean codon-optimized GmGCHI（GTP cyclohydrolase I）and tomato LeADCS were co-transformed under wheat endosperm specific glutenin（1Dx5）promoter to optimized the folate enhancement.At the same time,we expressed the maize MYB and bHLH TFs in the wheat plants together and individually,to evaluate the expression of these genes in wheat plants.Comprehensively,the main results of the present study are as follows:1.We determined the folate contents of 360 wheat samples consisting on 315 wheat genotypes through HPLC-MS/MS and found a significant variation among wheat genotypes for folate contents ranged from 10.15±2.86 to 91.44±5.64μg per 100 g grains.Fifty-two wheat cultivars such as Henong58-3 were identified as good source with more than 50μg per 100 g grains folates.It was also observed that environmental factors also have great influence on folate production in wheat e.g.genotypes Liangxing66 and Shi4185 were grown at three different locations and their folate contents were varied among locations as 36.9（Shijiazhuang）,29.4（Beijing）and 57.5（Zhengzhou）μg per 100 g grains;and 41.5（Shijiazhuang）,20.4（Beijing）,and 28.3（Zhengzhou）μg per 100 g grains,respectively.2.It was found that 5-formyltetrahydrate（5-CHO-THF）and 5-methyltetrahydrate（5-CH₃-THF）were the two major folate vitamers present in wheat accessions.They contributed to more than 50%of the total folates quantified in this study.3.To enhance the folate contents in wheat plants through metabolic engineering,two soybean genes GmGCHI and GmADCS related to folate biosynthesis pathway,were expressed in wheat plants by Agrobacterium-mediated transformation and 2.3 fold folate contents were increased in transgenic wheat grains than the wild-type control（⁶5μg/100g versus²8μg/100g）.4.For further investigation and enhancement,soybean codon-optimized GmGCHI and tomato LeADCS genes were expressed in wheat by Agrobacterium-mediated transformation and found 5.6-fold increase in folate contents in the transgenic wheat grains than the wild-type control（¹27μg/100g versus²3μg/100g）.5.We transferred maize TFs ZmC1（MYB-type C1）and ZmR（bHLH R）genes into wheat by Agrobacterium-mediated transformation and found that the expression of both genes in the transgenic plants regulate the anthocyanin biosynthesis in tissue-specific manner.6.It was observed that wheat transgenic plants with both transgenes showed the highest pigmentation in almost all tissues e.g.leaves,stems,auricles,spikelets and seeds.It was found that ZmC1 controls the pigmentation in vegetative tissues like coleoptiles,auricles and stems while ZmR regulates the pigmentation in reproductive tissues such as spikelets and seeds.The anthocyanin content in the seeds of the transgenic plants containing the both transgenes was 6.51 mg/kg.7.Quantitative real-time PCR（qRT-PCR）analysis revealed that expression of two transgenes ZmC1 and ZmR,two conserved homologous wheat genes TaC1 and TaR,and six associated structural wheat genes involving in anthocyanin biosynthesis TaCHI,TaCHS,TaF3H,TaF3’H,TaDFR and TaANS were greatly up-regulated in the transgenic wheat plants.