Cloning And Functional Analysis Of 3-ketoacyl-Coenzyme Synthase(KCS)of Cuticular Waxes In Wheat(Triticum Aestivum L.)

Cuticular wax that covers the surface of the torrential plants serves as a barrier against environment,the main components of it are the Very Long Chain Fatty Acids(VLCFAs)and their derivates,involving in many plant physiological activities.KCS gene encodes ?-ketoacyl CoA synthase,a rate-limiting enzyme in the synthesis of VLCFAs,catalyzing the condensation reaction of VLCFAs elongation process and playing a key role in biosynthesis of precusors of culticular wax.In order to understand the function of KCS genes in biosynthesis of culticular wax,in this project,Gas Chromatography(GC)was used to detect the component and content of culticular wax in 3 different tissues from two common wheat materials(varieties).Transcriptone sequencing and related bioformatics methods were employed to acquire potential TaKCS genes.We cloned TaKCS1,TaKCS2,TaKCS3 and TaKCS4 from cDNA sample of Chinese Spring(CS),and primarily identified their roles by analyzing them in yeast.The expression features assays and heterologous expression in tomato cv.“Micro Tom” demonstrates the effect of TaKCS4 on cuticular wax.The primary results as follow.1.Based on the wax analysis of leaves in seedling stage,flag leaves in booting stage and glumes in early flowering stage,we found that,compared to CS,wax load in W87 is higher.At the seedling stage,the wax load on leaves is almost the same between two materials.The primary alcohols were dominant and the diketone could not be detected.With the growth extension,the relative content of primary alcohols was decreased and the amount of n-alkanes accumulated mostly.In the early stage of flowering,the content of diketone in wheat glume was dominant,followed by alkanes,then primary alcohols.Among them,the content of diketone in glumes of W87 was significantly higher than that in CS,but,for the content of nalkanes and primary alcohols,it is opposite.The dominant carbonchain length on leaves was changed from C28 to C28 and C30,with the development of plants.For glumes,C30 and C32 was dominant.2.In this study,several potential diketone biosynthesis genes and 93 KCSs were screened.Those KCS genes have specific expression patterns in different tissues and materials,consistent with the changes in the dominant carbonchain length in various tissues.Four TaKCS genes were cloned by transcriptome sequencing.Then,these genes were subjected to chromosome localization,amino acid primary sequence analysis,transmembrane domain analysis,functional domain analysis.The amino-acid sequences of four TaKCS were used to constructed the phylogenetic tree and compared with that of AtKCS genes and various KCS genes from some main crops.The relative content of saturated long chain fatty acids C16: 0 and C18: 0 was decreased,the relative content of C26: 0 sharply increased and the presence of C28: 0 was detected in the following heterologous expression in yeast.This has been shown that these four genes have the function of encoding ?-ketoacyl-CoA synthetase.3.The expression profile of the target genes was constructed by qPCR,including the relative expression level in different tissues,hormone treatments and abiotic stresses.It was found that,in the total RNA of 8 tissues in wheat at different stages and tissues,the expression level of TaKCS1 was higher in seedling leaves and roots than others and TaKCS2 expressed relatively higher in roots and pistils.Compared to other tissues,the expression level of TaKCS3 was relatively highest in roots,however,that of TaKCS4 was higher in leaves and genital organs and was lowest in roots.The relative expression level of TaKCS4 rapidly increased while application of ABA,drought,PEG and low temperature and it was gradually restored to normal level after a long period.The expression of TaKCS4 decreased continuously in dark treatment,indicating that its promoter region may own the light-regulated cis-elements.4.Subcellular localization indicated that TaKCS4 protein was located on the endoplasmic reticulum of tobacco epidermal cells.The heterologous overexpression of TaKCS4 in tomato showed that the surface wax load of transgenic T0 progeny increased significantly,among which the content of alkanes sharply increased.Compared with the empty vector,the leaf wax content of the transgenic plants increased by 2-4 times,and the contents of n-alkanes,primary alcohols,fatty acids and branched alkanes increased simultaneously.Among them,the increase of n-alkanes is obvious,and the content of C31 alkane and C30: 0-OH increases significantly.