Cloning And Functional Verification Of DXS And LIS Gene From Lavender

Lavender is the main characteristic economic crop in Xinjiang.The lavender essential oil extracted from its flower spikes is widely used because of its multiple effects.Lavender essential oil major components of monoterpene compounds in the highest proportion,including linalool,lavender acetate,linalyl acetate content of how much direct impact on the yield and quality of lavender essential oil.Directly regulating the content of terpenoids in lavender at the gene level,thereby improving the yield and quality of lavender essential oil,is of great significance to improving the current status of the domestic lavender industry.1-deoxy-D-Xylulose-5 phosphate synthase(DXS)is an important rate-limiting enzyme in the first reaction of the MEP pathway,which catalyzes the synthesis of 1-deoxy-D-xylulose-5 phosphate(DXP)from pyruvate and glyceraldehyde 3-phosphate.Linalool synthase(LIS)is the terminal enzyme that synthesizes monoterpene components in the MEP pathway,which can catalyze the formation of linalool from geranyl pyrophosphate(GPP).In this study,the DXS1,LIS1 and LIS2 genes of lavender were cloned from the highoil lavender variety’Zahua’.Through bioinformatics analysis,expression pattern analysis,subcellular localization analysis,prokaryotic expression analysis and enzyme activity detection analysis of three genes,we had initially grasped the expression patterns and molecular functions of the above genes,which laid the foundation for probing their regulatory mechanisms in the terpenoid metabolism pathway of lavender.1.Sequence analysis showed that lavender DXS1 had a open reading frame(ORF)of 2181 base pairs encoding a 726 amino acid protein,lavender LIS1 had a ORF of 1809 base pairs encoding a 602 amino acid protein,lavender LIS2 had a ORF of 1695 base pairs encoding a 726 amino acid protein.The DXS1 protein of lavender was highly conserved and closely related to the DXS protein of Lavandula angustifolia,Isodon rub escens and Plectranthus barbatus.LIS1 and LIS2 had functional groups in conserved domains DDXXD,RRX8W and DDXXTXXXE,and LIS1 was closely related to linalool synthase in broad-leaved Lavandula latifolia and Salvia splendens.LIS2 protein was closely related to linalool synthase in Lavandula angustifolia and Lavandula x intermedia.2.The expression level of DXS1 gene in the floral organs of the high-oil lavender variety’Zahua’ at different developmental stages was higher than its expression in the low-oil variety’French Blue’.DXS1 gene had the highest expression level in senescence stage of ’Zahua’ floral organ,and the highest expression level in the full bloom stage of ’French blue’ flower organ.DXS1 gene expression was highest in the calyx of’Zahua’ and highest expression in ’French blue’ stamens.DXS1 gene expression in five different tissues of’Zahua’ floral organs was higher than ’French blue’(except pistils and stamens).The expression level of LIS1 gene in the different developmental stages of ’Zahua’ flower organs was higher than that in ’French blue’.The expression of this gene was highest during the senescence stage of the two varieties.The expression of LIS1 gene was the highest in calyx and the lowest in stamen in different tissues of’Zahua’,while the expression of LIS1 gene was only a little in stamen in French blue,and almost no in other tissues.The expression level of LIS2 gene in different tissues and different developmental stages of ’Zahua’ flower organs was lower than that in ’French blue’.LIS2 gene had the highest expression in senescence stage and French blue half-opening stage,LIS2 gene was highly expressed in the calyx of the two varieties,and LIS2 gene expression was significantly higher than LIS1 gene in different developmental stages and different tissues of the two varieties.Combining with the content of terpenoids in floral organs,the analysis results showed that the total content of camphor and borneol in the floral organs of’French Blue’ was similar to the changes in the express trend of DXS1 gene at different developmental stages ofFrench Blue’.The total content of linalool and its derivatives in linalool was similar to that of LIS2 gene express trend in different tissues of floral organs in’Zahua’.3.After the construction pCAMBIA1304-DXS1,pCAMBIA1304-LIS1,pCAMBIA1304-LIS2 recombinant vectors were constructed,and recombinant proteins were expressed in arabidopsis protoplasts.The results of subcellular localization analysis showed that DXS1-GFP and LIS1-GFP localized in the chloroplast,The analysis results of LIS2-GFP are not available yet.4.The results of prokaryotic expression analysis showed that pET-28a(+)-DXS1 induced the maximum expression at 37℃ and IPTG concentration of 0.8 mM for 4 h;pET-28a(+)-LIS1 induced the maximum expression at 37℃ and IPTG concentration of 1.0 mM for 4 h;pET-28a(+)-LIS2 induced the maximum expression at 37℃ and IPTG concentration of 1.0 mM for 6 h;Solubility identification found that the recombinant proteins DXS1,LIS1,and LIS2 are all insoluble inclusion body proteins.Recombinant proteins DXS1,LIS1 and LIS2 were purified and recovered.The enzyme activity test results showed that LIS1 protein had monoterpene synthase activity and sesquiterpene synthase activity,and was involved in the synthesis of linalool and p-Menth-8(10)-ene-2,9-diol,and LIS2 protein had monoterpene synthase activity and was involved in the synthesis of linalool.