Identification Of StDXS And StDXR Gene Of Senna Tora And The Functions In Tobacco Seedlings

Terpenoids are widely used in the field of medicine,especially in the field of traditional Chinese medicine.Therefore,the study of the key genes controlling terpenoid metabolism will lay a molecular foundation for the medicinal research of Senna tora.In previous laboratory studies,CDS sequences of 1-deoxy-D-xylulose-5-phosphate synthase(St DXS)and 1-deoxyD-xylulose-5-phosphate reductoisomerase(St DXR)were cloned from Senna tora and expressed in Escherichia coli to detect ?-carotene content.The transgenic tobacco overexpressed plants were also obtained.In this study,the St DXS and St DXR genomic fulllength sequences from transcriptome data were identified,and then the genes were analyzed for homology,physical and chemical properties,tertiary structure prediction and other bioinformatics,and the family genes were identified.Tobacco is a model plant for plant molecular biological engineering.In this study,St DXS and St DXR were overexpressed in tobacco,then the contents of photosynthetic pigments ?-carotene and Chlorophyl and the contents of endogenous hormones gibberellin(GA)and abscisic acid(ABA)were determined in transgenic tobacco plants,respectively.The role of the two genes in controlling terpenoid metabolism was verified by detecting the content of terpenoid metabolites downstream of MEP pathway.Phenotypic analysis was carried out under drought,salt and high temperature stress,respectively to further explore gene function.The main results were as follows:1.Firstly,the accuracy of CDS sequence information of St DXS and St DXR was verified,and the full length of the genome fragment was determined,and the exon and intron information of the gene was obtained.The St DXS gene structure contains 9 introns and 10 exons,and St DXR contains 11 introns and 12 exons.St DXS was closely related to Citrus clementina and Helianthus annuus,and St DXR was closely related to Glycine max.13 St DXS and 2 St DXR gene family members were identified in Senna tora genomes by hmmer 3.1b2 software.The results of promoter homeopathic element analysis and screening showed that the two types of genes may be involved in stress response and responses to various plant growth regulators.2.Use GFP as reporter gene to transform tobacco instantaneously.The fluorescence signal observed by laser confocal microscope confirmed that the expression product was located in chlorophyll,which was consistent with the results predicted by the subcellular localization prediction tools.This study clarified the localization of St DXS and St DXR gene expression products in cells,which will provide the basis for the study of protein function.3.The contents of ?-carotene,chlorophyll,GA and ABA in St DXS /St DXR gene positive transgenic tobacco were different from those in the wild type.The concentration of several terpenoids in St DXR transgenic tobacco was significantly higher than that in wild type tobacco.The average content of ?-carotene,chlorophyll,carotenoid,GA and ABA were 2.27,1.46,2.5,3.53 and 3.77 times of those of wild type,respectively.However,in St DXS transgenic tobacco,the content of GA was significantly lower than that of wild-type tobacco,while the contents of other substances were increased.The average content of ?-carotene,chlorophyll,carotenoid and ABA were 1.1 times,1.34 times,1.86 times and 4.32 times respectively of those of wild type.The results showed that St DXS and St DXR had different effects on the synthesis of these pigments and hormones in tobacco.4.Under drought,salt and high temperature stress conditions,plants showed certain growth inhibition phenotypes compared with non-stress treatments.However,the platelet phenotype of transgenic plants was significantly less inhibited than that of wild-type plants.Under 200 mmol/L mannitol simulated drought stress,the stress resistance of St DXS transgenic plants was significantly reflected in the increase of lateral root number and root hair,and the stress resistance of St DXR transgenic plants was significantly reflected in the increase of plant height,root elongation and root hair.Under 100 m M Na Cl stress,stress resistance of St DXS transgenic plants was significantly reflected in plant height,and stress resistance of St DXR transgenic plants was significantly reflected in plant height,lateral roots and root elongation.The advantages of transgenic plants under high temperature stress were shown in biomass and number of leaves.These phenotypic data will provide reference for later gene transcriptional analyses and gene function studies.This study will provide a basis for verifying the regulatory functions of St DXS and St DXR in terpenoid metabolism and exploring the functions in stress response.