Study On Molecular Cloning And Functional Analysis Of DXS Gene From Lilium

Lilium spp.is a well-known bulb flower in the world,which has high ornamental value and economic value.In general,the Trumpet and Aurelian hybrids and Oriental hybrids emitted strong floral fragrance,but the floral scent of the Asiatic lilies were light,affecting its practical value.The main reason of the difference of floral aroma in lilies is the different release of terpenes.So this study focus on the key gene DXS involved in the synthesis pathway of terpenes.The results are as follows:(1)The cDNA sequence of the 1-deoxy-D-xylulose-5-phosphate synthase(DXS)gene from the ‘Entertainer' was cloned using RACE methods,named as Le DXS(Gen Bank accession number MF576067).The full length cDNA was 2471 bp,including a 2142 bp open reading frame that encodes 713 amino acids with a molecular weight of 76.3 k D.Phylogenetic analysis indicates that Le DXS belongs to the plant DXSII cluster.It is predicted that Le DXS is expressed in the chloroplasts by subcellular localization.(2)Le DXS gene was expressed in the petals of all cultivars based on qRT-PCR analysis.Le DXS had a higher expression level in strong-scented lilies compared to weak-scented and non-scented lilies.The correlation analysis between the expression level of Le DXS and the total amount of monoterpenes showed that there was no significant correlation between them.(3)When the Le DXS gene was introduced into tobacco by Agrobacterium-mediated method,GUS histochemical staining analysis showed that the efficiency of transient transformation was up to 70%.In this study,2 kanamycin resistant callus were obtained.(4)Sterile plantlets were obtained after Lilium formolongi ‘Julius' seeds were sowed in MS medium for 30 days.And the ger mination rate was 39.2%.With the leaves of seedling as explants,the optimal medium for callus induction by sifting was MS+1.5 mg/L 2,4-D+30 g/L sucrose+6 g/L agar,the rate of callus induction was 20.43%;the optimum medium for callus proliferation was MS+2.0 mg/L PIC+30 g/L sucrose+6 g/L agar,its regeneration rate was 0.0203 g/d;and the best medium for differentiation was MS basal medium,differentiation rate was 100%.These results will benefit our biologically functional understanding of DXS and establishment of the genetic transformation system of lily,and further research of floral fragrance breeding of lilies.