Recloning And RNAi Study Of DFR Gene Families From Brassica Napus, B. Rapa And B. Oleracea

Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. Besides providing beautiful pigmentation in flowers, fruits, seeds, and leaves, flavonoids also have key roles in signaling between plants and microbes, in male fertility of some species, in defense as antimicrobial agents and feeding deterrents, and in UV protection. The main seed coat pigments of plants like Arabidopsis are polymers of proanthocyanin, which is synthesized via the phenylpropanoid-flavonoid-proanthocyanidin pathway. The transparent testa of Arabidopsis is due to kinds of deficient steps in flavonoid synthesis pathway, which makes identification of single gene function possible in flavonoid pathway. Over 20 loci have been characterized. Dihydroflavonol 4-reductase (DFR) is a key enzyme in the synthesis of anthocyanin and proanthocyanidin in flavonoid pathway. In A. thaliana, when the single copy DFR gene was inactivated by X-ray irradiation, the seed color of mutant appears yellow; it is because that the synthesis of proanthocyanidin was blocked. The comparison study in near-isogenic lines of yellow-seeded and black-seeded B. carinata suggested that the synthesis of pigments in the developing seed and the laminae of seedling were controlled by DFR locus. It also revealed that there was a temperature-sensitive DFR regulator in yellow-seeded mustard, causing the lack of pigment. The comparison study of yellow-seeded and black-seeded B. napus show very low gene expression levels and DFR enzyme activity in black-seeded seed coats, the PA synthesis is severely diminished in the seed coat of the yellow seed. Sugest that low levels of DFR gene expression is main reason of yellow-seeded in PA metabolism pathway.B. rapa, B. oleracea and Brassica napus and Arabidopsis all belong to Brassicaceae (previous known as the Cruciferae) in which there is a considerable number of vegetables for daily tables, crops, for edible oil, gardening, forage dye and medicinal. The yellow seed characteristic in B. napus is desirable because of its association with higher oil content and better quality of oil-extracted meal. So cloning and identification function of Brassica DFR genes are important for mechanism characterization of seed coat development and seed coat pigmentation as well as molecular breeding of yellow seed trait in Brassica.In this reserch, the genomic sequences of DFR gene families from B. napus, B. rapa and B. oleracea were recloned and analyzed. The numbers of DFR gene families in the three species were identified. The RNAi plant expression vectors of Brassica DFR genes were constructed, and the RNAi vector was transformed into B. napus black-seeded cultivar Zhongshuang 10. A batch of transgenic plants was obtained and the agricultural traits and content of biological with inhibition of DFR gene families were investigated. The expression levels of DFR and its members in flowers and 20DAF seeds were detected in transgenic plants using semi-quantity RT-PCR. The main results are as follows:1. Recloning and identification of DFR gene families from B. rapa, B. oleracea and B. napus.Based on our prevours study and electronic cloning, we designed 3 pairs of primers to amplify genomic sequences of DFR gene families from B. rapa, B. oleracea and B. napus with gradient PCR and total gDNA templates in highest Tm and amplification with 2 paris of peculiar primers using PCR. BrDFR1 and BrDFR2 were derived from B. rapa; the full length of genomic sequence of BrDFR1 is 1781 bp and BrDFR2 is 1806 bp. BoDFR1,BoDFR2 and BoDFRpse were derived from B. oleracea; the full length of genomic sequence of BoDFR1 is 1794 bp and BoDFR2 is 1814 bp. BnDFR1,BnDFR2 and BnDFRpse were derived from B. napus, the full length of genomic sequence of of BnDFRl is 1794 bp and BnDFR2 is 1781 bp. Both pseudogenes BoDFRpse and BnDFRpse are 1920bp in length. 2. Analysis of the similarities and evolution of DFR gene family from B. oleracea, B. rapa and B. napusThe sequence of BnDFRl from B. napus has highest similarity with BoDFR1 from B. oleracea; the sequence of BnDFR2 from B. napus has highest similarity with BrDFR2 from B. rapa. Sequence alignment indicate the genomic identities of 100%, 100%,99.5% between BnDFRl and BoDFRl, BnDFR2 and BrDFR2, and BnDFRpse and BrDFRpse. It can be concluded that BnDFRl is derived from BoDFRl, BnDFR2 from BrDFRl and BnDFRpse from BoDFRpse. Clues from DFR loci support that B. napus is a tetraploid species of B. rapa and B. oleracea. These clues also indicated that the "Triplicated" DFR genes in Brassica ancestor experienced gene loss and pseudogenization, and current Brassica diploid species keeps only one functional DFR gene. However it is a heterozygous allele perhaps for the functional requirements of strengthening and diversification.3. Construction of RNAi and 4 antise expression vectors of Brassica DFR genesA 600-bp RNAi fragment of Brassica DFR gene family was cloned. Its antisense fragment was subcloned between the promoter and the spacer of pFGC5941M by Swaâ… and Aatâ…¡double digestion, and the sense fragment was subcloned between the spacer and the terminator of pFGC5941M by BamRâ… and Xbaâ… double digestion, forming the 11400-bp RNAi vector pFGC5941M-BDFRI. It was successfully verified by multiple PCR checking and then transformed into Agrobacterium tumefaciens strain LBA4404 to form engineering strain.Using BamHâ… and Xbaâ… double digest gene fragments for the 4 members of DFR gene family, BnDFR1/BoDFR1, BoDFR2, BnDFR2/BrDFR1 and BrDFR2 were subloned into platform vector pC2301M1DPB to generate plant sence vectors which was modified by our research team recently. Plant sence vectors pC2301M1DPB-BnDFR1/BoDFR1,pC2301M1DPB-BoDFR2,pC2301M1DPB-BnDFR 2/BrDFRl, pC2301M1DPB-BrDFR2 were constructed.4. RNAi transformation results indicate that DFR is an important locus of seed pigmentation in BrassicaUsing improved leaf-disc method, an engineering strain of RNAi vector pFGC5941M-BDFRI was used to transform the hypocotyl segments of typical black-seeded cultivar Zhongshuang 10. Among the 36 regenerated Basta-tolerant plants of the first batch, multiple PCR check identified at least 14 transgenic positive plants.During the growth and development, the traits were observed systemically. The results indicated that after RNAi inhibition of DFR Brassica gene family, the transgenic plants have little diffence in colour of the stem and leaves, developmental and morphological traits compared with non-transgenic plants.Compared transgenic seeds and non-transgenic, the color of transgenic seeds turn light, they were light brown or brown in contrast with the pure black seeds from the non-transgenic CK. Seed color analysis showed that transgenic seeds showed a 49.92% increase in red-light value compared with CK, The 1000-seed weight of seeds decrease little. The results of this study indicated that in Brassica plants, such as B. napus, DFR genes mianly participate in the course of formation seed coat pigments.5. The expression of DFR gene family and its members in flowers and 20DAF seeds in transgenic plantsRNA was isolated from flowers and 20DAF seeds of non-transgenic and transgenic plants, detected their expression by semi-quantity RT-PCR and compared.The expression of BnDFR gene family and each member have clear decrease in transgenic plants compared to non-transgenic plants.The expression of BnDFR gene family decreases distinctly in different plant, show different modifications in different transgenic plants.The expression of BnDFR gene family and each member is higher in 20DAF seed than in flower, which is consistant with organ-specificity results of BnDFR revealed by our team.