Cloning And Function Analysis Of A 3-ketoacyl-CoA Synthase Gene (MsKCS10) From Alfalfa (Medicago Sativa L.)

Alfalfa(Medicago sativa L.),an important perennial leguminous in the world,have high yield and good quality.However,the abiotic stress such as worldwide increasing drought and salt stress reducing alfalfa production.With the development of molecular breeding,the new technology provides a way out for alfalfa breeding in abiotic resistance to adapt the weak environment.The cuticular wax is a component of the cuticle that covers the surface of the plant.The accumulation of wax is related to the prevention of water dispersion and is contributes to plants' drought and salt tolerance.The plant cuticular wax is composed with Very Long Chain Fatty(VLCFAs)and other derivatives.The synthesis of very long chain fatty acids is mainly catalyzed by a complex enzyme.3-ketoacyl-CoA synthase(KCS)acts as the rate-limiting enzyme in the complex enzyme FAE and plays a key role in the synthesis of downstream products.Based on the Rapid-Amplification of cDNA Ends(RACE)technology,the gene MsKCS10 was cloned from alfalfa,and the function and characteristics of the gene were studied in this research.The research results are as follows:1.The MsKCS10 intermediate fragment sequence was cloned from the alfalfa leaves using PCR.And the complete cDNA sequence of MsKCS10 was amplified by RACE PCR.The full-length cDNA sequence of MsKCS10 was 1889?bp with an open-reading frame of 1614?bp encoding 537 amino acids.And the GenBank accession number was MK625177.After sequence alignment,the homology of the MsKCS10 gene to the Medicago truncatula KCS10 gene was 98.36%.The phylogenetic tree was constructed using the neighbor-joining method,and the analysis showed MsKCS10 gene had the highest homology with Medicago truncatula KCS10 gene.Real-time quantitative PCR(RT-qPCR)was used to analyze the expression in different tissue.The analysis showed MsKCS10 was mainly expressed in leaves and stems and hardly expressed in roots in alfalfa.And the expression of MsKCS10 gene in the aboveground parts of nine alfalfa cultivars was also analyzed by RT-qPCR.In addition,the transcriptional expression of this gene was also induced by abiotic stress.The analysis showed that under drought conditions,MsKCS10 was up-regulated faster,and under salt stress,it was slower.2.The transient expression vector pCAMBIA1300-MsKCS-GFP was constructed and injected into tobacco leaves by Agrobacterium-mediated transformation.And the protein MsKCS10 was located on the endoplasmic reticulum;The overexpression vector pCAMBIA1301-MsKCS was constructed then infected with Agrobacterium-mediated and leaf disc method.Transgenic tobacco was successfully obtained after tissue culture.Overexpression MsKCS10 in tobacco showed higher activity of antioxidant enzyme,accumulation of soluble matter and lower damage of cell membrane under the drought and salt stress.Furthermore,transgenic tobacco leaves revealed an increase in N-alkane content or fatty acid content.The increase of N-alkane content is mainly concentrated on the increase of C31 and C33 alkane;the increase of fatty acid content is concentrated in the content of C20 and C22 fatty acids,indicating that MsKCS10 is involved in tobacco wax biosynthesis and participates in the decarbonylation pathway.Scanning electron microscopy(SEM)results revealed that a special form of helical waxy crystal appeared on the leaves of transgenic tobacco.Chlorophyll leaching rate of transgenic tobacco was lower than that of the control group.At the same time,the water retention capacity of transgenic tobacco leaves was better than that of the control group.3.Based on the promoter region of fruit-specific promoter PG gene from GenBank,specific primers were designed.The tomato fruit-specific promoter PG was cloned from the Monkey-Maker tomato genome,and the fruit-specific promoter overexpression vector PBI121-PG promoter-MsKCS was constructed.Agrobacterium-mediated transformation was introduced into Micro-Tom tomato by plant tissue culture.It was found that the transgenic micro-Tom tomato was more durable than the control group.Furthermore,the peel of transgenic tomato revealed an increase in straight-chain alkanes,fatty acids and aldehydes.It was indicated that MsKCS10 participates in the decarbonylation pathway in the waxy synthesis of transgenic tomato fruit epidermis,the same as in tobacco leaves.In addition,after SEM,it was found that the transgenic tomato epidermis showed the same special waxy crystal as the epidermis of transgenic tobacco leaves.It is suspected that this may be due to the combination of N-alkane and fatty acid content.4.Overexpression vector pCABIAM3301-MsKCS was construct through In-fusion technology.And the interference vector pK7GWIWG2-MsKCS was constructed by Gateway method.Overexpression MsKCS10 alfalfa and RNAi-MsKCS10 interference alfalfa were obtained by Agrobacterium-mediated method.The SEM results of overexpression MsKCS10 in alfalfa and MsKCS10-RNAi interference alfalfa showed that the waxy crystals of overexpression alfalfa leaf epidermis had denser waxy crystals,and the waxy crystals of interference alfalfa were decreased.In addition,overexpressing MsKCS10 gene alfalfa had higher C30 alcohol content or N-alkane content,while RNAi-MsKCS10 transgenic alfalfa showed lower C30 alcohol content or N-alkane content,indicating that MsKCS10 was involved in both decarbonylation and acyl reduction pathways in wax synthesis of transgenic alfalfa leaves.In a nut shell,MsKCS10 mainly responds to plant drought stress,and participates in the decarbonylation pathway in wax synthesis of transgenic tobacco leaves and tomato fruit epidermis.In addition,spiral waxy crystals precipitated in the cuticle of both transgenic tobacco leaf and tomato peel.Combined with the waxy components analysis,this may be due to the combined effect of the increase of linear alkanes and fatty acids.In addition,MsKCS10 is involved in both decarbonylation pathway and acyl reduction pathway in wax synthesis of transgenic alfalfa leaves.