Isolation Of Cadinene Synthase Genes(Cdn1-C) In Gossyium Australe And G.arboreum And Functional Analysis Of Their Promoters

Cotton is an important economic crop and oil crop in the world.It provides human with high quality natural textile fiber and cottonseeds which is rich in oil,vitamins,protein,mineral elements and other natural nutrients.As a result,cotton exhibits a broad applicational prospect.Gossypol,which shows high toxicity to human beings and non ruminant animals,is widely existed in plant tissues and organs,including cottonseed.This directly limits the full use of cottonseed for human.Gossypol is an important natural phytoalexin,which can enhance the plant's resistance and adaptability to adverse environment.Previous studies have shown that?+?-?-cadinene synthase,which can catalyse farnesyl pyrophosphate to produce gossypol precursors?+?-?-cadinene,is the key enzyme for gossypol formation.Although cadinene synthase is important for gossypol biosynthesis as the rate limiting enzyme,there are few reports regarding the expression pattern and regulation mechanism of cadinene synthase gene.In this work,two?+?-6-cadinene synthases genes?designated as GauCdn and GarCdn?and their promoters?designated as GauCdn-P and GarCdn-P?were isolated from Gossypium australe and Gossypium carboretum,respectively.VIGS system and transient expression system were used to identify the function of Cdn genes.This study will lay the foundation for further investigation of cadinene synthase gene regulation mechanism and biosynthetic pathway of gossypol.The DNA sequence of GauCdn and GarCdn were 2863bp and 2846bp,while the cDNA sequence were 1776bp and 1814bp,respectively.Gene structure analysis showed that they all contained 7 exons and 6 introns.The full length of ORF for these two gene both were 1665bp,encoding 554 amino acids.Amino acid sequence analysis exhibited that they all had the typical DDxxD and RR?x?₈W conserved domains of terpene synthase.In addition,there was a highly conserved domain RxR at the region of 35 amino acid residues in the upstream of DDxxD domain.Phylogenetic analysis showed that these two Cdn genes all belong to the Cdnl-C subfamily.Transmembrane domain prediction showed that the two corresponding proteins did not contain transmembrane signal peptide,belonging to the secreted protein.Physiochemical property analysis showed that the molecular formula for GauCdn protein was C₂₈₆₅H₄₄₁₁N₇₅₉O₈₆₂S₂₄,with the molecular weight being 64.05KDa,isoelectric point being 5.22 and belonging to an acidic protein.The negatively charged residue?Asp +Glu?number in the protein of GauCdn was 85 and the positively charged residues?Arg +Lys?number was 63,resulting in the unstable factor being 45.34,belonging to unstable protein.The hydrophilic coefficient of GauCdn was-0.433 and hydrophobic coefficient was 82.04.So we predict that the GauCdn protein is a hydrophobic protein.The molecular formula for GarCdn protein was C₂₈₇₂H₄₄₂₄N₇₆₂O₈₅₆,with the molecular weight being 64.09KDa,isoelectric point being 5.41 and belonging to an acidic protein.The negatively charged residue?Asp + Glu?number in the protein of GauCdn was 83 and the positively charged residues?Arg + Lys?number was 65,resulting in the unstable factor being 46.57,belonging to unstable protein.The hydrophilic coefficient of GauCdn was -0.425 and hydrophobic coefficient was 82.40.And we also predict that the GarCdn protein is a hydrophobic protein.The function of Cdn genes was investigated by virus induced gene silencing technologies.The results showed that the expression level of Cdn gene and the gossypol content in silenced plants were both significantly lower than that in the control plants.A positive correlation between the content of gossypol and the expression level of Cdn gene was also revealed in cotton leaves.This implied that Cdn gene is one of the key genes controlling the synthesis of gossypol.In order to study the expression regulation mechanism for cadinene synthase gene,we cloned the promoters of these two genes,named as GauCdn-P and GarCdn-P,respectively.The promoter sequence length cloned was 2626bp and 2455bp,respectively.Further analysis showed that their A/T contents were 59.86%and 75.19%,respectively,which is in consistent with the characteristics of eukaryotic promoter sequence.Cis-acting elements analysis found that both of these promoters contained TATA-Box and CAAT-Box,which is indispensable for the typical eukaryotes basal promoter.A variety of phytohormone inducing elements were presented in the sequence of promoter GauCdn-P,such as,salicylic acid response element TCA-element,methyl jasmonate response element CGTCA-motif,abscisic acid response element ABRE,auxin-responsive element TGA-element,GA responsive element P-box and GARE-motif.In addition,the sequence of GauCdn-P also contains ARE,LTR,MBS,Circadian,CCAAT-box and other cis-regulatory elements.Multiple phytohormone responsive elements also existed in the sequence of promoter GarCdn-P,such as salicylic acid response element TCA-element,abscisic acid response element ABRE,ethylene-responsive element ERE,GA response element GARE-motif.In addition,ARE,MBS,Skn-1,RY-element,CAT-box,and a large number of light-responsive elements were presented in the sequence of promoter GarCdn-P.In order to study the effect of phytohormones on promoter activities and the location of hormone responsive elements in the promoter region,we constructed several promoter deletion expression vectors and carried out transient expression experiments in tobacco.GUS activity detection results showed that MeJA and SA can very significantly enhance the activity of GauCdn-P,and the response elements were respectively located in the promoter region of -500bp?-276bp and -276bp?-7bp;ABA can significantly improve the activity of GauCdn-P,and the response element is found in the promoter region of -1806bp?-1491 bp.Meanwhile,the study found that SA can very significantly improve the activity of GarCdn-P,and there is likely to be multiple salicylic acid responsive elements in the promoter region of -594bp?-12bp;ABA can significantly improve the activity of GarCdn-P,and the abscisic acid response elements located in the promoter region of -1475 bp?-1008bp.