Isolation And Activity Analysis Of Promoter Of JsGDS Gene Associated With Aroma Synthesis From Jasminum Sambac

GDS which catalyzes the reaction from substrate?-farnesyl pyrophosphate?FPP?to product germacrene?sesquiterpene compounds?is one of the important enzymes in downstream of MVA pathway in process of syntheses of aroma substances in Jasminum sambac?L.?Ait.For exploring mechanism of expression and regulation of GDS gene,genomic DNA was extracted from young leaves of Jasminum sambac.5'terminal regulatory sequence of JsGDS gene has been isolated by genomic walking technology to find cis-acting elements of promoter and to predict environmental factors that affect expression and regulation of JsGDS gene;five plant expression vectors with different lengths of 5'flanking deletion of the resulted 5'terminal regulatory sequence were constructed by Gateway technology and then transformed into Arabidopsis thaliana for obtaining transgenic plants;the activity of the JsGDS promoter and its deletion fragments at different growth stages of transgenic seedlings were detected by GUS staining analysis to determine the minimum length and tissue specificity of expression of JsGDS,which will provide theoretical basis for further exploring the regulation mechanism of jasmine aroma,the main results are as follows:1.The 5'terminal regulatory sequence of JsGDS gene was cloned successfully from Jasminum sambac by genome walking technology and cis-acting elements of promoter of JsGDS gene were found:the pure genomic DNA from Jasminum sambac was extracted successfully using commercial Plant Genomic DNA Extraction Kit?CTAB method?,and used for template of subsequent genomic walking;the 1646 bp 5'terminal regulatory sequence of JsGDS gene was obtained by genomic walking;cis-acting elements of JsGDS promoter were found by promoter online analysis softwares:Plant CARE and PLACE,and the result showed that besides core element of plant promoter?TATA-box and CAAT-box?were found,the other important cis-acting elements such as light reaction elements including AE-box,AT₁-box,Box-4,I-box,TCT-motif,etc.,hormones responsive-related elements including abscisic acid relative element?ABRE?,Me JA related elements?CGTCA-motif and TGACG-motif?,gibberellin related element?GARE-motif?,and anaerobic induction element?ARE?,which indicated that expression and regulation of JsGDS gene is closely related to environmental factors such as light and exogenous hormones,etc.2.The five expression vectors with different lengths of 5'terminal flanking deletion were constructed by Gateway technology:according to the distribution of cis-acting elements in the 5'terminal regulatory sequence of JsGDS gene,the five fragments with different lengths of 5'terminal flanking regulatory sequence including JsGDS?-1646?-1?,JsGDSP1?-926?-1?,JsGDSP2?-666?-1?,JsGDSP3?-406?-1?and JsGDSP4?-206?-1?were amplified by PCR,And were used to fused into plant expression vector?p GWB633?with reporter gene GUS by Gateway technology.3.The activity of JsGDS gene promoter was detected by transformation with the resulted plant expression vectors containing different lengths of 5'terminal flanking deletion into Arabidopsis thaliana:the above resulted plant expression vectors containing different lengths of 5'terminal flanking deletion were transformed into Agrobacterium?GV3101?to infect petals of Arabidopsis thaliana for T₀transgenic plants;T₁positive transgenic plants were screened by Basta resistance and PCR;the resulted T₂transgenic plants were used for GUS staining to detect the activity of JsGDS gene promoter,the results showed that the all T₂transgenic plants had activities of GUS gene which were the strongest in seedlings at 7 days,and GUS gene only expressed on bud tips;the results of GUS staining in transgenic plants at blooming stage at 35days showed that all five promoter fragments had activity of GUS gene,and the JsGDS promoter activity was the strongest,and the JsGDSP1 and JsGDSP3 were the weakest,and all five deletions have tissue-specific expression,besides JsGDS and JsGDSP2 expressed in inflorescences,pods and axillary buds,JsGDS and JsGDSP2 also expressed in leaves and cauline leaves,respectively;JsGDSP1 and JsGDSP3 only expressed in axillary buds,and JsGDSP4 only expressed in inflorescences and axillary buds.Therefore,the minimum length of the JsGDS promoter is at least206 bp,and there may be negative regulatory elements in fragment between-926?-666 and-406?-206,and the key cis-acting elements related with expression of leaves and seed pods in fragment between-666to-406,and the key elements related with floral organ in the JsGDSP4.