Gene Cloning And Functional Analysis Of Colored Cotton Flavonoids Metabolism-related Genes GhANS And GhCHS

Cotton is one of the world’s most important economic crops, cotton fibers arehuman necessities of daily life and an important raw material of the textile industry.With people constantly enhance environmental awareness, colored cotton productsbecoming the new fashion of international fashion trends and healthy.But naturallycolored cotton research existing some problems：Firstly, the production is not highand the poor fiber quality; Secondly, monotone color, color fastness and colorsaturation is not enough, pigment genetic instability.These problems greatly limits thedevelopment and application of colored cotton products. Therefore, in order to furtherdefine the natural pigment colored cotton fiber material composition and sedimentarymechanism, expounds natural colored cotton fiber color formation, molecularmechanism of natural colored cotton breeding and germplasm innovation is veryimportant.Since most studies suggest that colored cotton pigment substances may beflavonoids, therefore, in order to clear the material composition of the pigmentcolored cotton fiber, In this study, two important enzymes gene involved in theflavonoids biosynthetic pathway was cloned from colored cotton. Usingbioinformatics, real-time fluorescent quantitative PCR and subcellular localizationmethod is systematically analyzed their molecular characteristics and expressioncharacteristics and functional activity, etc, the results are as follows:1. Materials is12DPA brown cotton white near-isogenic lines fiber, proteintwo-dimensional electrophoresis and mass spectrometry analysis identified adifferentially expressed proteins the anthocyanin synthesis enzyme(ANS). In order to15DPA brown cotton fiber cell cDNA template, in silico cloning full-length cDNAsequence of the gene encoding the protein sequence analysis found that the gene openreading frame of1062bp, encoding354amino acids, the anthocyanidin reductase2-ketoglutarate dioxygenase subfamily named the gene GhANS. Real-time PCRanalysis showed GhANS gene in cotton constitutive expression of the gene expressionin brown cotton fiber cell development period were higher than their white cottonnear-isogenic lines. Such as the amount of expression in the12DPA brown fiberrelative value of0.26, more than three times higher than the expression level of thesame period,white fiber, a significant difference;27DPA brown fiber in gene stillexpression was detected in the white fibersless than it, the above results show thatGhANS gene may play some role in the process of pigment formation in brown cottonfibers. Build GhANS gene GFP fusion plant expression vector was transformed intotobacco, fluorescence confocal microscopy found GhANS gene is located on the celledge.2. The amino acid sequence（EU921263）to the brown cotton CHS gene as a probe,the cotton EST sequences in GenBank tBLASTn contrast selected high homologyEST sequences using the software for splicing, to get a chalcone synthase homologouscDNA sequences. This cDNA primers were designed to3DPA after flowering greencotton fiber cDNA as a template for PCR amplification, sequence analysis showedthat GhCHS length cDNA1524bp, contains a complete open reading frame of 1221bp,encoding407amino acids with the polyA tail, GhCHS the interference lengthcDNA204bp, encoding68amino acids.3. In order to further research GhANS and GhCHS gene function, respectivelybuilding GhCHS gene expression vector GhCHS gene RNAi interference vector andGhANS gene expression vector and GhANS gene RNAi interference vector. And greencotton infected by Agrobacterium tumefaciens-mediated hypocoty, The obtainedregeneration of transgenic plants, and laid the foundation for the analysis of genefunction in green cotton fibers.