Study On Structure Profile And Metabolic Mechanism Of Pigment In Colored Cotton Fiber

Object: Naturally colored cotton is cotton fiber endowed with natural colors. Compared withthe white fiber cotton, naturally colored cotton is used with little or no processing and dyingsteps during the fabric manufacturing process, which could eliminate dying costs and thedisposal of toxic dye waste. As concerns for human health and the environment increase,naturally colored cotton has become an environmentally friendly option. However, the colorsof naturally colored cotton primarily include green and several shades of brown.Cross-breeding can only generate new shades of brown and green, which are insufficient forclothing versatility. Therefore, the color types have not been altered by conventional breedingdue to the lack of genetic resources for the generation of color. Genetic engineering isrecognized as a way to generate new fiber color types and has just begun. For the geneticengineering approach, it is important to characterize the genes related to pigment biosynthesis.In this study, we will separate and identify the pigment in colored cotton fiber, and analyze itsmetabolic regulation in cotton fiber. This study will help us to better understanding ofpigmentation in color cotton fiber, and ultimately help us to manipulate pigment production incotton fiber by genetic engineering.Method:(1) We‘ll explored the components, molecular weight, polymerization degree, basicstructure unit and space connection mode of the proanthocyanidins in brown and white cottonfiber by MALDI-TOF MS、NMR and HPLC. Meanwhile, we also measured the expression ofGhANR, GhLAR and GhMATE involved in synthesis and transportion of theproanthocyanidin precursor, constructed plant expression vector by Gateway, and transformedcotton use agrobacterium-mediated method.(2) We extracted the pigment in green cottonfiber by fat-soluble and water-soluble extracts and analyzed4CL genes expression by RT-PCR,and constructed plant expression vector by Gateway, and transformed cotton useagrobacterium-mediated method.Results and Conclusion:(1) Proanthocyanidin in brown and white cotton fiber was found tocontain mainly procyanidin (PC) and prodelphidin (PD) units with2,3-cis form(epigallocatechin and epicatechin). But, part of proanthocyanidin in the white cotton fiber wasmodified by acylation and was different from the proanthocyanidin in brown cotton fiber. Therelative ratio of PD was similar to PC in white cotton fiber (1:1.05), while proanthocyanidinin brown cotton fiber consisted mainly of PD units with a90.1%relative ratio. In addition, themolecular weight and DP were all a little changes and reducing in white cotton fiber, but weresignificantly increased after25DPA in brown cotton fiber.Vanillin-HCl staining showed that the content of proanthocyanidin was high in immature brown cotton fiber, but was low in immature white cotton fiber from15DPA. However,proanthocyanidin was not detected in mature brown and white cotton fiber. Borntrager‘s testindicated that the quinone content was gradually increasing during the later developmentalfiber, and high in mature brown cotton fiber. Conversely, that the quinine was not detected inmature brown and white cotton fiber. This can be attributed to the gradual oxidation ofproanthocyanidin to quinone during the late stage of brown fiber development.During the cotton development, the gene expression of GhANR in brown cottons wasmuch high than that of GhLAR, so the transcript level of GhLAR was not measured.GhMATE1a and GhMATE1b contained12transmembrane domains (TMDs), and weregrouped into the same group including Arabidopsis TT12and Medicago MATE1byphylogenetic analysis. This hints that GhMATE1a and GhMATE1b transportedproanthocyanidin precursors without galloylated esters. The expression of GhMATE1a andGhMATE1b were all higher in brown cotton fiber than that in white cotton fiber, and reachedthe peak at21and27DPA.In addition, the over-expression and RNAi vectors of GhANR was structured andtransformed to white cotton and brown cotton respectively. We had obtained7positiveregenerated plants for gene overexpression. And, the T1was immature thought yet to furthervalidated.(2) Dimethyl-2-hydroxysuccinate and dimethyl malate were detected from fat-solubleextract of green cotton fiber, but without chromophoric group. Gallic acid and ellagic acidwere detected from water-soluble extract of green cotton fiber.They were the degradationproducts of the hydrolysable tannin which was light yellow to light brown amorphous powder.This indicated that the biological metabolism of pigment in green cotton fiber was associatedwith phenylpropanoid pathways.Three4CL genes were cloned from green cotton fiber with Motif I—AMP and Motif IIconserved domain. Phylogenetic and expression analysis, speculated that the Gh4CL1may beregulated the biological metabolism of flavonoids in cotton fiber. Gh4CL3may be regulatedthe accumulation of caffeic acid and derivatives in green cotton fiber. And Gh4CL4may beregulated the biological metabolism of lignin (cellulose) in cotton fiber. Therefore, theover-expression and RNAi vectors of Gh4CL3was structured and transformed to white cottonand brown cotton respectively. We had obtained7positive regenerated plants for transformedpGWB17-Gh4CL3. And, the T1was immature thought yet to further validated.