Identification And Characterization Of GhNCED Genes And Functional Analysis Of Them In Response To Drought Stress In Cotton(Gossypium Hirsutum)

9-cis-epoxy carotenoid dioxygenase(NCED)is a key rate-limiting enzyme in ABA biosynthesis which plays a crucial role in plants’ responses to abiotic stresses such as drought,high salt,high temperature and cold.In present study,31 NCED genes were identified from upland cotton(Gossypium hirsuta L.)using network biological information resources and phylogenetic analysis was conducted.The gene sequences of GhNCED family and the protein sequences encoded in cotton are highly conserved.There are many cis-acting elements related to abiotic stress and ABA signal transduction in their promoter regions.Analysis of the expression patterns of GhNCED family genes showed that the expression of most GhNCEDs genes was induced by drought stress,and the expression levels were significantly up-regulated.In order to reveal the important role of NCED family genes in cotton under drought stress,three GhNCED genes(GhNCED1-6/GhNCED2-1/GhNCED4-3)whose expression levels were significantly increased under drought stress were selected in this paper for further study.1.Identification and phylogenetic analysis of GhNCED gene family31 GhNCED genes were identified from cotton genome by using nine NCED gene sequences of Arabidopsis thaliana as "Query" sequences.Then these genes were named as GhNCED1-1 to GhCCD8-4 respectively,according to their sequence similarity with AtNCEDs.We analyzed the physical and chemical properties of GhNCED family including chromosomal location,number of amino acids,isoelectric point,and molecular weight of coding proteins.The subcellular localization of these proteins was also predicted.In addition,we selected the NCED protein sequences of 10 representative species from low to high evolutionarily to construct the rootless evolutionary tree.The results showed that these NCED could be divided into six branches,among which CladeⅠ appeared relatively late and only existed in monocotyledonous and dicotyledonous plants.On the other hand,Clade Ⅱ-Ⅵ were relatively ancient,which contain NCED homologous proteins even in lower plants such as Chlamydomonas,microalgae,mosses,and ferns,indicating that the NCED family plays an important role in plants.2.Conserved motif and gene structure Analysis of GhNCED familyA rootless evolutionary tree was constructed using 31 protein sequences of GhNCEDs to analyze the connection between the evolutionary relationship of GhNCED family with the conserved motif as well as gene structure.The results showed that in the same evolutionary branch,the gene structure and protein sequence were highly conserved,and the order of conserved motifs,the number and position of introns were highly consistent.3.Chromosomal collinearity and promoter element analysis of GhNCED genesTo further reveal the amplification mechanism of GhNCEDs in the cotton genome,we investigated the direct homologous genes of GhNCEDs from ancestral Asian cotton(G.rboretum and G.raimondii).The results showed that the NCED gene family in upland cotton was replicated for amplification.In addition,in order to predict the potential function of GhNCED gene,we also analyzed the cis regulatory elements in their promoters by using the PlantCare database.The results showed that there were many cis-acting elements related to plants’ responses to abiotic stress.4.Expression pattern analysis of GhNCED gene in cottonNine GhNCED genes which show significant expression differences with control in the drought transcriptome data obtained previously were selected out.And their drought-induced expression patterns were confirmed by qRT-PCR.The results showed that except GhNCED2-4,the other 8 genes were induced by drought treatment,with increasing expression levels in varying degrees.It is worth noting that the expression of GhNCED1-6 and GhNCED2-1 genes were significantly upregulated in cotton leaves after 4 to 8 days of drought treatment,the transcripts were increased by 3 to 6 times.The expression of GhNCED3-1 was also significantly increased in plants treated with drought for 4 and 8 days,however,there was no significant difference with control in the samples treated with drought for 6 days.The expression of GhNCED4-3 in cotton leaves treated with drought for 4 days was increased by four times,and then down-regulated,with no significant difference compared with the control.5.Functional study of GhNCED1-6/GhNCED2-1/GhNCED4-3 genes in drought responseAccording to the above results,three genes of GhNCED1-6/2-1/4-3 were selected for further study,and the three genes’ overexpression vectors were constructed to transform Arabidopsis thaliana and cotton,respectively.Comparing with WT,transgenic Arabidopsis thaliana plants overexpressing GhNCED1-6/4-3 showed stronger tolerance to drought and significantly increased survival rate.Besides,we also constructed vectors to inhibit the expression of NCED genes in cotton,and obtained GhNCED1-6/2-1/4-3 silenced transgenic cotton plants using the VIGS technique.Comparing with the control,the stomatal closure of GhNCEDs silenced plants was limited after drought treatment,and the content of ABA decreased,the content of protective enzymes(peroxidase and proline)decreased significantly.While the content of malondialdehyde and hydrogen peroxide were increased significantly,the water loss rate was accelerated and the tolerance to drought decreased significantly.The expression of ABA response genes also changed significantly,suggesting that GhNCED gene is involved in drought stress response of cotton through ABA signaling pathway.6.Subcellular localization and promoter activity analysis of GhNCED proteinWe constructed the fusion expression vector of three genes of GhNCED1-6/2-1/4-3 and the tag protein GFP,respectively,to transform cotton.Green fluorescence only existed in the cytoplasm,indicating that the fused proteins 35S::GhNCED1-6::GFP was localized in the cytoplasm of the cotton cell.Additionally,we constructed a GUS gene expression vector driven by GhNCED1-6 promoter to transform Arabidopsis thaliana,which found that the GhNCED1-6 promoter was expressed in root,stem,leaf and flower tissues.Moreover,after ABA and mannitol treatment,the expression of GUS gene increased,suggesting that GhNCED1-6 gene may be involved in the response to stress.