Regulating PSY And CCO Genes To Increase Carotenoid Levels In Cotton Fibers

Carotenoids are an important group of natural pigments.These lipid-soluble pigments are ubiquitous in animals,higher plants,fungi,and algae.Carotenoids are photosynthetic pigments,and also coloring factors for plant flowers and fruits.The phytoene synthase(PSY)is the first rate-limiting enzyme in the carotenoid biosynthesis pathway,which catalyzes the conversion of geranylgeranyl pyrophosphate(GGPP)to phytoene.Elevating expression of PSY gene in maize endosperm,rice endosperm,potato tubers,tomato fruit and rapeseed can increase the content of carotenoids significantly and improve the nutritional quality of agricultural products.On the other hand,carotenoid cleavage dioxygenase(CCO),catalyzing the degradation of carotenoid,is a key factor affecting carotenoid content.Cotton(Gossypium)is an important economic crop in the world.Cotton fiber is the most widely-used natural fiber in the textile industry and plays an important role in the national economy.Natural colored cotton bypasses dying and pollution in processes.However,the limitation in color diversity and stability have restricted the development of colored cotton industry.It is of great theoretical significance and good application prospect to cultivate new colored cotton materials with rich colors and stable properties.With previously obtained transgenic materials,the color stability of mature fibers and transcriptomic changes in fibers of Fbl2A::GhPSY2D transgenic cotton was analyzed.Meanwhile,bioinformatic and expressional analysis of cotton CCO gene family were carried out.Virus-induced gene silencing(VIGS)was used to silence CCO gene dominantly expressed in upland cotton tissues.Furthermore,RNAi vector of CCO gene was construct and transformed into cotton.The main results are as follows:1.Color analysis of mature fiber of Fbl2A::GhPSY2D cotton plantThe phenotype of mature fibers of Fbl2A:: GhPSY2 D transgenic cotton was observed.The results showed that the mature fibers of the transgenic cotton were brown and yellow compared with the control.Observations for three successive generations showed that the color of mature fibers could be inherited steadily.2.Quality analysis of Fbl2A::GhPSY2D mature fiberThe lint content of Fbl2A::GhPSY2D transgenic cotton mature fibers was determined.The results showed that the lint content of the transgenic materials decreased significantly.At the same time,the micronaire value of Fbl2A::GhPSY2D mature fibers decreased significantly,which were 3.8 ± 0.35 and 3.85 ± 0.25,respectively.There was no significant change in other fiber quality indexes.3.Transcriptome analysis of 20 DPA(days post anthesis)fibers from transformant 212 of GhPSY2 D transgenic cottonThe transcriptomic analysis of the fibers of Fbl2A::GhPSY2D transgenic cotton 20 DPA showed that there were 8870 differentially expression genes(DEGs),including 5336 up-regulated genes and 3534 down-regulated genes.Further,KEGG enrichment analysis of DEGs was carried out.The up-regulated DEGs were mainly enriched in alpha-linolenic acid metabolism,plant-pathogen interaction,plant hormone signal transduction,phenylalanine metabolism,phenylpropanoid biosynthesis,cysteine and methionine metabolism,amino sugar and nucleotide sugar metabolism pathway.The down-regulated genes are mainly enriched in carbon fixation in photosynthetic organisms,photosynthesis-antenna proteins,glyoxylate and dicarboxylate metabolism,photosynthesis,carbon metabolism,circadian rhythm-plant,brassinosteroid biosynthesis and pentose phosphate pathway.4.Identification and evolution analysis of cotton CCO gene family membersUsing the CCO protein sequence of Arabidopsis thaliana as reference,the CCO protein family members were identified in the G.raimondii,G.arboreum and G.hirsutum protein databases,and 15,16,and 30 genes encoding CCO were identified,respectively.Cluster analysis was conducted between Arabidopsis CCO gene family members and cotton CCO gene family members.The results showed that CCO gene family in cotton could be divided into six clusters: CCD1,CCD4,CCD7,CCD8,CCD-like and NCEDs.5.Expression pattern of CCO genes in G.hirsutumAccording to the transcriptome data of upland cotton,heatmap analysis was carried out on the expression patterns of all members of CCO gene family in upland cotton tissues.The results showed that GhCCD1A/1D was predominantly expressed in most tissues compared with other gene expression levels.In addition,GhCCD4-2A was predominantly expressed in fibers of 20 days post anthesis(DPA).Meanwhile,five tissues(roots,stems,leaves,petals and 20-DPA fibers)of upland cotton were selected to detect the GhCCOs genes by qRT-PCR,which was consistent with heatmap results.6.Silencing CCO genes enhances carotenoid level in cotton leaves and fibersVIGS technique was used to silence the expression of GhCCD1 D and GhCCD4-2A genes in white cotton plants.Cotyledons of cotton showed obvious yellow color and carotenoid content increased significantly.In addition,the expression of GhCCD1 D and GhCCD4-2A genes was down-regulated by VIGS using Fbl2A::GhPSY2D as the receptor,and the color of 28 DPA fibers was deepened in silenced plants.7.Construction of RNA interference vector of CCO gene and cotton genetic transformationIn order to further regulate the expression of GhCCO gene,the RNAi vector(pLGN-pFbl2A-GhCCDs-RNAi)was constructed and transformed into cotton.At present,three transgenic seedlings have been obtained.Taken together,our results showed that the expression level of GhPSY2 D gene was up-regulated during the secondary wall synthesis stage of cotton fibers,which could make cotton fibers brown and yellow;and down-regulated expression of GhCCD1 D and GhCCD4-2A genes could further deepen the color of cotton fibers.The cotton fiber color could be improved by regulating the expression level of carotenoid metabolic genes.