Cloning And Functional Analysis Of Key Enzyme Genes In The Core Pathway Of Flavonoid Synthesis In Gossypium Hirsutum L.

Gossypium spp.is the vatil economic crop globally,the important strategic material related to national economy and people’s livelihood,and provides more than 50% of the fiber sources for the textile industry.Due to the low yield and poor fiber quality,almost all of cotton varieties currently produced and planted are non-pigmented white fiber cottons.Colorful clothing products meet people’s needs,but the chemical auxiliaries and dyes used in the process of chemical printing and dyeing of cotton textiles will have caused harm to the human,and a large amount of sewage discharged brings about great environmental pollution.Fortunately,natural colored cotton can be directly spun into fabric without or less dyeing,which acquires the favor of people because of its green,ecological and environmental-friendly characteristics.The formation of colored cotton fibers is mainly caused by the accumulation of different anthocyanidins and their derivatives in the fiber cell.Anthocyanidin,a water-soluble pigment widely found in plants,is one kind of flavonoid compounds in polyphenolic compounds and is an important class of secondary metabolites.The varieties and germplasm resources of colored cotton which can be used in textile industry are all brown and green series at present,which seriously limits the development of colored cotton industry.Therefore,it is of great significance to study the core pathway of flavonoid synthesis for exploring the mechanism of fiber color formation,improving fiber color and enriching germplasm resources of colored cottons.In this paper,bioinformatics analysis was performed on the four gene families encoded Flavanone 3-hydroxylase(F3H),Flavonoid 3’-hydroxylase(F3’H),Flavonoid 3’,5’-hydroxylase(F3’5’H)and Dihydroflavonol 4-reductase(DFR),respectively.And the gene expression patterns in four different developing fibers of C312,HS2,XC20 and ZX1 were studied.The Gh F3 H,Gh F3’H,Gh F3’5’H and Gh DFR genes interfered in XC20 and ZX1 by VIGS(Virus-induced gene silence)were used to analyze the phenotype,fiber color and the gene expression level.Furthermore,the regenerated cotton plants were obtained through the Agrobacterium-mediated transformation of Gh F3 H,Gh F3’H,Gh F3’5’H and Gh DFR genes.The research results obtained are as follows:(1)The F3 H and DFR gene families in G.hirsutum have four homologous genes,and the F3’H and F3’5’H gene families have two homologous genes respectively.The sequence similarity between the members of each gene family is extremely high,and the structure of motifs and domains are highly conserved.The screened F3H4,F3’H2,F3’5’H2 and DFR1 are all hydrophilic proteins.F3’H2 and F3’5’H2 have transmembrane regions,but the F3’H2 protein is unstable and contains anchoring signal structure.Other proteins are relatively stable and are non-secretory proteins.F3H4 is mainly distributed in the cytoplasm,and another three proteins are basically existed in the nucleus and mitochondria.The secondary structure consists mainly of Alpha helix and Random coil.(2)In the four different varieties(lines)of upland cottons,the expression levels of F3 H,F3’H,F3’5’H and DFR genes in colored cottons are higher than those in white cottons,and there are spatiotemporal differences in expression level among diverse cotton lines.(3)F3H gene is located upstream of anthocyanidin synthesis pathway,mainly expressed in the early stages of fiber development.DFR gene is expressed downstream in the three branches of the synthesis of Pelargonidin,Cyanidin and Delphindin.Moreover,the F3 H and DFR gene families have more copies and more space-time expression,which results in higher expression and plays an important role in anthocyanidin cumulation.(4)Gh F3’H and Gh F3’5’H were expressed in the branches of the synthesis of Cyanidin and Delphindin,respectively.The expression level of the two genes was relatively low,and markedly lower than that of the F3 H gene.(5)In XC20 and ZX1,the transgenic cotton lines interfered by VIGS showed the fiber color change with varying degrees affected by the efficiency of silence and the gene expression pattern.(6)Gh F3 H,Gh F3’H,Gh F3’5’H and Gh DFR genes play an important role in fiber color,among which F3 H gene has the greatest influence on anthocyanin accumulation and fiber color.Functional differentiation of F3’H gene occurred in different varieties(lines).Compared with F3’H gene,F3’5’H gene has higher expression level,longer expression time,and lighter fiber color after interference.And the expression of DFR gene in late stage of fiber development takes a greater effect on fiber color.(7)Regenerated cotton lines of over-expressed F3’5’H gene and interfered F3’H gene were obtained through Agrobacterium-mediated transformation.The expression of Gh F3’5’H gene was significantly highter in transgenic Gh F3’5’H-OE plants than that of in WT(S1).In this paper,we found that the content,species and fiber color of anthocyanidins in cottons are closely related to the F3 H,F3’H,F3’5’H and DFR genes in the core pathway of flavonoid synthesis.On this basis,transgenic regenerated plants over-expressed F3’5’H gene and silenced F3’H gene have been obtained.Consequently,the anthocyanidin synthesis and fiber color change in colored cotton fibers can be studied further.It also lays the foundation for exploring the function of transcription factors in anthocyanidin synthesis regulatory network,the differences of expression patterns and pigment formation processes among different colored cottons.In addition,it provides a more comprehensive theoretical basis for the color improvement of colored cotton fibers.