| Folate (Vitamin B9) content is one of the important rice nutrition qualities. Folate fortification of rice based on QTL analysis and metabolic engineering were carried out in this research. Rice cultivars with high folate content were identified and the impact on rice folatent content causing by storage time and cooking was examined. Furthermore, a recombinant inbred lines population derived from the cross of Lemont and Teqing and a backcross inbred lines (BIL) population derived from the crosss of Koshihikari and Kasalath were used to detect QTL relative to rice folate content. Metabolic engineering of folate biosynthetic pathway of rice leaves and seeds have been achieved by overexpression seven Arabdopsis genes of folate biosynthesis pathway. Expression profiling of rice genes of folate biosynthetic pathway in wild-type and engineered AtGCHI transgenic lines was detected by Realtime PCR analysis. The main conclusions are as follows:1. The tri-enzyme method and microbiological assays were used to investigate folate variation in 78 rice cultivars. Folate contents of brown rice varied substantially from 13.3 to 111.4μg/100g, whereas milled rice varied from 10.3 to 77.7μg/100g. Folate content of brown rice of four cultivars, viz. Chaoyangzao 18, 111.4μg/100g , Teqing (107.9μg/100g), Dabaigu 13 (106.2μg/100g) and Qingfengai (101.4μg/100g), were over 100μg/100g. The average folate losses caused by storage and cooking were 23% and 48.3%, respectively. The highest folate content in cv. Laoshuya when cooked was 26.3μg/100 g, about 25% of the recommended dietary intake (RDI) (400μg/day), assuming a daily per capita consumption of 400g/day of cooked rice. The results suggested that it is potentially possible to screen for higher folate content varieties in germplasm and in breeding.2. Two QTLs relative to seed folate content were identified using Lemont/Teqing RIL population in 2009 and 2010, named qFC-3a and qFC-3b. Meanwhile, qFC-3b was detected in both of environments, and the mean of PVE is 13.4%. One QTL qFC-3c having a PVE of 24.8% was identified using Koshihikari×Kasalath BIL population in 2010, and epistatic interactions in this population were detected.3. Folate contents were determined in 27 T2 populations of transgenic plants of rice, and the effect of genes of folate biosynthetic pathway from Arabdopsis were analyzed syntematically, they were reduce to three types: (1) Seed folate contents were raised by 265.8%~505.4%, 8.0%~17.0% and 1.2%~21.2% in AtGCHI,AtDHFS and AtFPGS transformants, respectively, relative to wild type. While, the mean of leaf folate content in AtGCHI,AtDHFS and AtFPGS transenic lines decreased by 32.3%, 20.2% and 0.8 % relative to wild type, respectively. (2) Folate content decreased by 10.5% and 12.9% in AtDHNA and AtADCL transgenic seeds relative to wild type, respectively, and leaf folate content in AtADCL transgenic lines decreased by 21.8% relative to wild type. (3) There were no significant difference of folate contents of seeds between AtHPPK transgenic lines and wild type except the line A263-10, which averagely raised seed folate content by 9.4% relative to wild type. Folate content of seeds of AtDHFR transgenic lines also showed no significant difference relative to wild type. However, average folate content of leaves of AtHPPK and AtDHFR transgenic lines decreased by 25.5% and 27.0% relative wild types, respectively. Therefore, AtGCHI,AtDHFS and AtFPGS are benefit for folate enhancement in rice seeds, while others genes has no effect or negative effect.4. De novo folate synthesis is occurring in many rice tissues, including radicle, young stem, young leaf, mature root, mature stem, mature leaf, endosperm and embryo of 25DPA. Endogenous folate content of these tissues organized from high to low levels were young leaves> mature leaves> embryos of 25 DPA > Root> young stem> mature root> mature stems>endosperm of 25 DPA. Total folate content descended during development of rice seeds at 10 to 30 DPA. But gene expression patterns of folate biosynthesis genes differed from their trend of folate content in rice seeds. Single-copy genes OsGCHI, OsDHFS and one of seed preponderant genes OsDHNAⅢgradually increased during 10 to 25 DPA, and descended afterward. The expression of the other single-copy genes and seed preponderant genes, including OsADCL I, OsFPGS I, OsADCS and OsHPPK gradually increased during seed development, and maximizing in seeds of 30 DPA. OsDHFR I constitutively expressed during seed development, whereas the expression of OsDHFRⅡdeclined steeply from 10DPA to 30DPA. Expression of OsDHNA I,OsDHNAⅡ和OsFPGSⅡgenes did not vary greatly during seed development. Therefore, there are probably a suit of genes for de novo synthesis of rice seeds, including sigle-copy genes OsGCHI,OsDHFS,OsADCS, OsHPPK, high abundance genes of rice seeds OsDHNAⅢ,OsADCL I, OsFPGS I and constitutative expression gene OsDHFR I.5. Expression of folate synthesis genes of rice were impacted by folate levels. Transcript levels of endogenous genes, OsDHNAⅡ,ADCS and FPGS I,Ⅱwere significantly (P<0.05) decreased in seeds of all transgenic lines than in the wild-type. The endogenous genes, OsGCHI, OsADCS, OsADCLⅡ, OsHPPK, OsFPGS I and OsFPGSⅡ, increased significantly (P<0.05) in transgenic lines , of which showed lower folate contents in leaves. Therefore, there appear likely to be instances of feedback regulation in rice folate biosynthetic pathway, and genes mentioned above maybe the potential regulatory loops. |
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