| Cotton is one of the most globally important cash crops, and cotton fiber is the main natural fiber in textile industry. Color cotton fiber is a kind of cotton with natural color in its fiber. Color cotton is healthy and profitable for mankind for it no dye printing in processing. Due to the advantage, it has caught tremendous attention and named "ecological fiber" highly praised by consumers. However, there are still some weak points that block its further development, only a few color series, instability of pigment, etc. which hinde the development of cotton industry.This research regarded colored cotton as materials and focused on fiber biochemistry and molecular biolosy. Cloning of genes related to color formation study will help thoroughly understand pigment synthesis pathway and molecular mechanism, and perform genetic engineering. Two important genes related to flavonoids bio-synthesis were cloned, and their characteristics, expression profiles and functional activity were studied by bioinformatics analysis, blot, Real Time-PCR, and expression in tobacco. The results are as follow:1. The study was carried out to search the different expressing genes in the fiber of brown, green and white color cotton though complementary DNA amplified fragment lenth polymorphism-(cDNA-AFLP) analysis. The result showed that sixty-four pairs of primers were used to amplify products for analysis. About 3800 bands were detected and the average number of bands per pair of primers amplified was 25. Using cDNA as probe,60 special framents were analyzed by reverse Northern blot, and got 17. BLASTx analysis showed that 6 TDFs had the homologous sequences with other sequences in GenBank, we got two framents which relatived to pigment formation. They are flavoionds 3'hydroxylase in brown color cotton fiber and flavoionds 3'5'hydroxylase in green color cotton fiber.2. Using rapid amplification of cDNA ends (RACE), the complete cDNA sequence of GhF3'5'H gene was isolated from green cotton fiber. The full-length of this cDNA colne was 1876bp, and its open reading frame encoded 509 amino acids. The gene was designated as GhF3'5'H (GenBank accession:GU062184). By the same way, we also got the complete cDNA sequence from brown cotton fiber and named GhF3'H (GenBank accession:GU062185). The full-length of this gene have 1873bp, and its open reading frame encoded 510 amino acids. Both GhF3'5'H and GhF3'H genes are belong to P450 family by the analysis of their conservation structure region.3. RT-PCR and Fluorescence real time quantitative PCR technology were used to study the expression of GhF3'5'H and GhF3'H genes in different development stages of fiber and organs. Real-time PCR analysis showed that GhF3'5'H and GhF3'5'H specially expressed in naturally color cotton fibers prior to white cotton fibers, and there expression levels are much higher in color cotton than in white cotton. They shrink in proportion to fiber development. Suggesting that GhF3'5'H and GhF3'H genes may involve in the precursor sythesis and play an important role in pigment formation during natural green and brown cotton fibers development.4. In the flavnoid sythesis pathway, studying on constructed enzyme choosing substrate is most important to reveal flower pigment formation. We constructed pET28a-GhF3'H and pET28a-GhF3'5'H protein vector at same time, which were induced by the IPTG in different conditions. We could found induced aim albume in the gel. Both pET28a-GhF3'H and pET28a-GhF3'5'H albume were presence in inclusion body which is had to pure.5. We constructed GhCHS gene expression vector and GhCHS RNAi vector repectively, for the tobacco and cotton transformation research. Got ten transformed tobacco plants which detected by PCR technique. By the assay, we found that tranformed plants grow more slowly and smaller than wild plants. With sucrose streesing, total flavonoid in transformed and wild tobacco planta is increasing as the time goes by, moreover, transformed plants have much more flavonoid than wilds'.we have a conclusion that sucrose might promote flavonoid proucts sythesis.6. We established a determination method which could detect four effective flavonoids in cotton fiber at the same time. During the fiber developing, we detected myricetin,quercetin,naringenin and kaempferol four flavonoids in the fiber through HPLC. We got following results:(1) There are many different flavonoids in both white and colour cotton fiber; (2) Naringenin accumulated in the early time and decreased by the time; (3) Myricetin and quercetin accumulated much more in colour cotton than white cotton fibers, and difference remarkable; (4) Kaempferol accumulated little in the fast elongation stage. These results demonsted that numerous actively and complicated flavonoids sythesis and transtfored in the process of cotton fiber development. Myricetin,quercetin naringenin and kaempferol are useful to precursor pigment in the color fiber.
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