| There is a close relationship between Brassica napus and Arabidopsis thaliana. They both belong to genous Brassica of Brassicaceae. The seeds of Brassica napus can be black and yellow. There is significant difference between yellow seeds and black seeds. Compared to black seeds, yellow seeds are more advantageous, such as thinner seed coats, lower cellulose and higher contents of oil and protein. In addition, because of the declining proancyanidins, in yellow-seeded rapeseed the pigment and polyphenols antinutrients are lower in oil or cake, and the economic value of canola and cakes is increased. Patental species B. oleracea and B. rapa both have stable natural yellow seed stocks, but B. napus has not. Because of limitation of yellow seed resource and hard breeding procedure, creation of novel yellow-seeded B. napus stocks is one of the important rapeseed breeding goals.Flavonoid pathway is an important secondary metabolic pathway of various metabolic pathways in plants. Meanwhile it is a core branch pathway of common phenylpropanoid pathway. Flavonoids are products of flavonoid pathway. They have various biologic functions. In Brassicaceae, the surface color of plant organs mainly contains anthocyanidins, PA and flavonols. Researches on Arabidopsis shows that the core component of seed coat pigments is PA, which accumulates in endothelium. Some researches on gene mutations of Arabidopsis reveal that restraining or blocking flavonoid pathway can make the color of Arabidopsis seed coat change from dark brown to yellow. Such mutants are named transparent testa (tt) mutation.Arabidopsis thaliana transparent testa 8 (AtTT8) gene codes a basic helix-loop-helix (bHLH) positive regulation transcription factor, which mainly regulates the accumulating of flavonoids. The regulation of TT8 is necessary for accumulating PA in seed coat. Regularly, there is inducible expression in cells of cyanine or where PA accumulates. TT8 can joint with TT2 and TTG1, and form a transcription factor complex. TT8 can mediate between TT2 and TTG1. and regulates the structural genetic expressions of flavonoid pathway, such as DFR and BAN, which are in "later period". Furthermore, TT8 can regulate the accumulating of PA, cyanine and mucilage in seed coat.This reseach constructed plant expression vectors of Brassica TT8 gene family using two different gene silencing technologies, RNA interference (RNAi) and chimeric repressor gene-silencing technology (CRES-T). Through Agrobacterium tumefaciens, they were used to transform hypocotyl segments of typical black-seeded B. napus cultivar Zhongshuang 10, the transgenic effects were analysed and comparison was made between the two methods.1. RNAi fragment of Brassica TT8 gene family was cloned. Its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites of an improved plant RNAi basic vector pFGC5941M using NcoI+SwaI and BamHI+XbaI double digestions to form recombinant vector pFGC5941M-BTT8I. Finally,11380bp pFGC5941M-TT8I is formed. Through multiple PCR identifications, pFGC5941M-TT8I is successful. Then, through transformation of Agrobacterium tumefaciens LBA4404 strain to generate engineering strains, which can be used to research the molecular mechanism of Brassica plants and genetic modification.2. We cloned CRES-T fragments of Brassica TT8 family. NT8-1CRE (1569bp) and OT8-2CRE (1569bp) were subcloned into CRES-T platform vector pC2301M1SDPB through double digestion with XbaI and SacI, and CRES-T expression vectors pC2301M1SDPB-NT8-1CRE and pC2301M1SDPB-OT8-2CRE were formed. NT8-2CRE (1818bp) and RT8-2CRE (1447bp) were subcloned into CRES-T platform vector pC2301M1SMPB through double digestion with XbaI and SacI, and the CRES-T expression vectors pC2301M1SMPB-NT8-2CRE and pC2301M1SMPB-RT8-2CRE were obtained. Through multiple PCR identifications,4 vectors we have reconstructed were successful. Then they were transformed into Agrobacterium tumefaciens LBA4404 strain to generate engineering strains.3. Through Agrobacterium-mediated transformation of pFGC5941M-TT8I into the typical black-seeded B. napus cultivar Zhongshuang 10, we got 20 Basta-resistant regenerated plants. The results of multiple PCR identifications showed that there were 18 transgenic positive plants.4. With Agrobacterium-mediated technique, we transform Zhongshuang 10 with pC2301M1SDPB-NT8-1CRE, pC2301M1SMPB-NT8-2CRE and pC2301M1SMPB-RT8-2CRE, and we got 32 Basta-resistant regenerated plants. The results of multiple PCR identifications showed that there were 28 transgenic positive plants.5. We systematically observed the traits in the growth process of transgenic plants we got through the transformation of RNAi vectors into Zhongshuang 10. The result showed that there was no significant change on transgenic B. napus in major morphology and growth traits. But, compared to typical black-seeded CK plants, seed coat pigments were significantly lower, color of seeds was lighter, and the appearance was light red and reddy-brown. Seed plumpness declined at large, grain weight declined from 3.25 g to about 2.64 g. and grain shape drifted from sphere. Husk ratio increased from 18.9% to 20.4%. Seed seting percentage declined from 85.3% to about 71.7%. Pod length declined from 7.6 cm to about 6.1 cm. 6. We systematically observed the traits in the growth process of transgenic plants we got through the transformation of the 3 CRES-T vectors into Zhongshuang 10. The result showed that there was no significant change on transgenic B. napus in major morphology and growth traits. But, compared to typical black-seeded CK plants, seed coat pigments were significantly lower, color of seeds was lighter, and the appearance was light red and reddy-brown. Seed plumpness declined at large, grain weight declined from 3.25 g to about 2.58 g, and grain shape drifted from sphere. Husk percentage increased from 18.9% to 22.4%. Seed seting percentage declined from 85.3% to about 79.3%. Pod length declined from 7.6 cm to about 7.2 cm.Transgenic effects by both RNAi and CRES-T methods indicated that Brassica TT8 genes might participate in regulating multiple traits such as seed coat pigment deposition, seed development and embryo pigment deposition. Suppression of their expression can create transgenic stock with large decrease of seed coat pigments, but can simultaneously generate some side effects such as reduction of seed seting percentgage and 1000-seed weight.7. RT-PCR detection indicated that both overall and member expressions of BnTT8 gene family in developing seed of 20 d after flowering of RNAi-transgenic plants were largely suppressed as compared with CK, and in leaves the suppression was complete. Suppression of BnTT8-2 was a little weaker than of BnTT8-1, which was conforming to their homologies to the RNAi fragment. RT-PCR detection indicated that both overall and member expressions of BnTT8 gene family in developing seed of 20 d after flowering of CRES-T-transgenic plants were suppressed to certain extent as compared with CK, which was conforming to the Arabidopsis report that TT8 can regulate the expression of itself by feedback. |
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