Transcriptome Analysis Of Green Cotton Fiber And Identification Of 4CL Gene Family

Objective: Naturally colored cottons(NCCs)refer to the varieties of cotton that produce fiber in color.As an environmentally friendly material,NCCs have excellent properties such as no chemical residue,skin friendly,antibacterial,etc.,and with high market potential and economic benefits.Therefore,it is of great significance for the improvement of NCC varieties to clarify the metabolism-transcriptional regulatory network of fiber pigments synthesis and to excavate the genes related to the synthesis of fiber pigments.Methods: In this study,transcriptome sequencing and metabolome analysis were used to compare the difference transcripts and metabolites in 12 DPA,18 DPA and 24 DPA fibers of green colored fiber cotton C7 and its near-isogenic white cotton C7-NIL.The genome-wide identification and bioinformatics analysis of the Gh4 CL gene family members,the core gene of green cotton fiber pigment synthesis,and the enzyme kinetics analysis of Gh4 CL in class I and class II were carried out.In addition,the CRISPR/Cas9 gene editing and overexpression vectors of Gh4CL20 and Gh4CL30 genes were constructed to genetically transform green cotton,and the obtained regenerated plants were identified and for further analyzed.from greenResults and conclusions:(1)Comparing the metabolites of 12,18,and 24 DPA fibers from C7 and C7-NIL.We found a total of 2047 non-redundant metabolites in GCF and WCF that were enriched in 80 pathways,including those of biosynthesis of phenylpropanoid,cutin,suberin and wax.most metabolites of the phenylpropane pathway were higher accumulation in green colored fiber(GCF)than that of white colored fiber(WCF),For example,sinapaldehyde,caffeic acid,ferulic acid,colorless anthocyanin,flavonol,etc,and the expression of genes related to the biosynthesis of these metabolites were significantly up-regulated.The results showed that the main pigments of GCF included cinnamic acid and its derivatives and flavonol.(2)Transcriptome sequencing was used to compare the differentially expressed genes(DEGs)of GCF and WCF in three developmental stages.Weighted gene co-expression network analysis(WGCNA)of DEGs between GCF and WCF was used to uncover gene-modules co-expressed or associated with the accumulation of green pigments.Of the 16 gene-modules co-expressed with fiber color or time points,the blue module associated with G24(i.e.,GCF at 24 DPA)was of particular importance because a large proportion of its genes were significantly up-regulated at 24 DPA when fiber color was visually distinguishable between GCF and WCF.A total of 56 hub genes,including the two Gh4CLs(Gh4CL12 and Gh4CL30)that could act in green pigment biosynthesis,were identified among the genes of the blue module.Our results provide novel insights into the mechanisms underlying pigmentation ofgreen fibers and clues for developing cottons with stable green colored fibers.(3)A total of 34 Gh4 CL gene members were identified in the Gossypium hirsutum genome and they are randomly distributed on 22 chromosomes.Phylogenetic analysis shows that Gh4 CL can be divided into three groups: Class Ⅰ,Class Ⅱ,and Class Ⅲ(4CL-like).Gene structure analysis showed that Gh4 CLs have multiple introns and multiple exons structures,and members in the same group have similar motif and exon-intron arrangements,indicating that members of the same group may have similarly function.(4)RNA sequencing analysis of the expression of Gh4 CL genes in class I and class II showed that Gh4CL5 and Gh4CL18 were not expressed in fibers,Gh4CL6 and GhCL24,Gh4CL12 and Gh4CL30 were predominantly expressed in 24 DPA fibers of green colored cotton,while Gh4CL7 and Gh4CL25 were mainly expressed in green cotton 18 DPA fibers,Gh4CL20 was mainly expressed in 12 DPA fibers.Enzyme kinetic analysis indicated that Gh4CL20 showed a substrate preference for 4-coumaric acid,but with a low conversion rate;Gh4CL24 showed a substrate preference for ferulic acid;Gh4CL25 showed a substrate preference for ferulic acid and caffeic acid,but a lower catalytic activity for 4-coumaric acid and cinnamic acid;Gh4CL30 showed a substrate preference for ferulic acid and caffeic acid.None of the four Gh4 CL was able to convert sinapic acid into its corresponding CoA thioester.It is suggested that Gh4CL30 may be involved in the metabolism of caffeic acid and ferulic acid residues and related to the synthesis of GCF pigments,Gh4CL20 may be involved in the synthesis of flavonoids,and four Gh4 CL genes may have different biological functions.(5)The gene editing knockout plants of Gh4CL20,Gh4CL24,Gh4CL25 and Gh4CL30 were obtained through agrobacterium mediated genetic transformation of cotton.Gh4CL20 gene editing cotton plants showed that the petals color turned into white,the stigma elongated,the number of pollen decreased and the plant was sterile.Gh4CL20 overexpression cotton plants showed that the anthers color turned into red and the fibers color unchanged.Transcriptome sequencing and metabolome analysis results showed that the content of flavonoids in the petals of Gh4CL20 gene editing plants was lower than that of wild-type plants,and the expression of flavonoids biosynthesis genes(PAL,C4 H,CHS,F3 H,DFR,ANR,FLS,ANS and UFGT)were downregulated.These results indicate that Gh4CL20 gene is involved in the biosynthesis of flavonoids in cotton flowers and related to the fertility of cotton.Gh4CL30 gene editing plants had no significant phenotypic difference compared with wild type plants,and there was no visible color change in fibers,indicating that Gh4CL30 might have functional redundancy gene in green cotton.