| DXS enzyme is the first key enzyme in the MEP pathway of terpene biosynthesis.Studies have shown that in the process of synthetic secondary metabolism,overexpression or inhibition of DXS can cause changes in the content of downstream synthetase-encoding genes and metabolites.Masson pine（Pinus massoniana Lamb.）is an important coniferous tree for turpentine,and it is necessary to carry out research on the synthesis of its terpenoids.This article focuses on the tissue expression pattern,subcellular localization,prokaryotic expression,eukaryotic expression,promoter cloning and transient expression of PmDXS,and explores its potential biological functions in order to subsequently reveal its terpenoid synthesis mechanism and horsetail in Masson pine.The resistance molecular breeding mechanism of pine provides some help and reference.The main findings are as follows:The DXS gene sequence in other plants was excavated by Genbank and compared with the Masson pine transcriptome database to obtain a partial sequence of the PmDXS.After PCR,c DNA end RACE,and software splicing technology,the full-length sequence of the PmDXS was cloned（Genbank ID:MK970590）2513 bp.ORF predicts that the open reading frame is 2223 bp.The protein has a molecular weight of 74 k D and is a hydrophilic protein with molecular formula C3536H5669N979O1035S27,isoelectric point PI 8.54;we found that it contains the core sequence of the TPP＿enzyme,TPP＿enzyme＿PYR super family and the PLN02582 multifunctional domain,and the unique ammonium disulfate binding site of the synthetase family and a transketolase domain of 1-deoxyxylulose-5-phosphate（DXS）family.Through real-time fluorescent quantitative PCR technology,the expression level of PmDXS gene in different tissues and different abiotic stresses（mechanical damage,15%penetrant,hydrogen peroxide,ethephon,methyl jasmonate and salicylic acid）was analyzed,and we found that the expression level of roots was higher.After 6 kinds of abiotic stress or hormone treatment,the expression level was up-regulated in a short time.It is speculated that this gene plays an important role in the synthesis of terpenoids in Masson pine and participates in the synthesis of terpenoid secondary products when resisting adversity stress.The 35S::PmDXS::GFP subcellular localization fusion expression vector was constructed,and the Nicotiana benthamiana was transiently expressed by injection.The results showed that the PmDXS protein subcellular localized in the chloroplast and belonged to the plastid protein;analysis of codon preference was determined It was expressed in Escherichia coli,and the prokaryotic expression vector Trans B（DE3）/p ET28a-PmDXS was constructed using DNA recombination technology,and the recombinant bacteria were observed under different abiotic stresses such as sodium chloride,mannitol,p H=5,and p H=9.Biological function,it was found that the number of colonies under different stresses was significantly better than that of the control group p ET28a,and it showed positive regulation under adversity stress.Tail-PCR technology was used to clone the upstream part of the promoter sequence of the PmDXS gene at 1024bp.After transient transformation,it was found that roots,stems,leaf tissues and leaf discs treated with different hormones（ABA,IAA,Me JA,SA）had GUS activity,indicating that the DXS promoter is an inducible promoter,and can drive the expression of the GUS reporter gene in N.benthamiana;the construction of the p CAMBIA1302-PmDXS overexpression fusion vector into the ecotype Columbia-0 Arabidopsis thaliana,measured in phenotype under different stress medium plates（sodium chloride,salicylic acid,methyl jasmonate,mannitol）,enzyme-linked immunoassay analysis of pigment and enzyme activity content of transgenic plants and wild-type plants,indicating that transgenic A.thaliana The chlorophyll a,chlorophyll b,carotenoid and DXS enzyme activity content of PmDXS overexpressing A.thaliana were significantly higher than those of wild-type A.thaliana during germination,growth and development under stress conditions.As the first key enzyme in the MEP pathway,PmDXS gene is involved in the response of Masson pine to stress,and plays an important role in the signal transduction process of hormone/abiotic stress. |
