Cloning And Functional Analysis Of Capsaicin Synthase Gene Promoter In Capsicun. Chinense Jacq

Capsaicin is a unique alkaloid plant secondary metabolites in hot pepper(Capsicum annuum L.),it plays an important role in food, health, medicine, agricultureand military fields. Currently,the production of natural, non-polluting capsaicin is farbelow market demand.Improving capsaicin content in fine pepper,can meet the marketdemand, but also improve value-added of pepper, in favor of the adjustment innational agriculture industrial structure.To study the expression and regulation of keygene in capsaicin biosynthesis deeply, it can provide technical support to improve theamount of capsaicin, and provide important reference for orientation breeding peppers.Capsaicin synthase enzyme is the last enzyme in the synthesis of capsaicin, alsothe key rate-limiting enzyme. It is mainly located in the epidermal cells of the fruitplacenta vacuole membrane,, the capsaicin synthase catalytic first branched chain fattyacids and coenzyme A (COA) form isoC10-COA complex in the synthesis of capsaicin,then under the action of ATP and Mg2+cofactor, isoC10-COA-ylamine with syntheticvanilla capsaicin. Therefore, the study of capsaicin synthase gene promoter is importantfor parsing the regulation of gene expression; provide an important experiments basisfor the separation of transcription factor involved in the capsaicin biosynthesis andscreening theinteracting protein.Accordingly,it is important for interpretating syntheticcapsaicin transcriptional regulatory mechanisms.1. Based on the specific primer,CSPRO DNA sequence were isolated frompepper,it has1351bp. The promoter functional elements prediction shows CSPROsequences in addition to the core promoter sequence, as well as several cis-actingelements or binding sites with light, water stress, hormonal response and so on. Therewere ABRE cis-acting elements associated with the ABA，MYB binding sites,W-boxwhich induced with stressand damage signal transduction and GA,cis-acting elementsunit associated with MeJA, pollen-specific expression elements and theGTGA-motify responses light reaction and so on.2. The experiment constructed three5’deletion plant expression vector, and thefull-length sequence were for genetic transformation, get13T1transgenic Arabidopsisplants,41T2transgenic positive plants.The GUS staining resules show CSPRO drivenGUS express in flower only, in other tissues or organs were not blue GUS stainingdetected. GUS staining results are consistent with T2generation. Based on the aboveresults suggest CSPRO is anther-specific promoter.3. In order to verify the start activity in cis-acting elements，using the method ofpoint mutations to mutant the sequence of MeJA and MYB,and then construct plantexpression vector for arabidopsis genetic transformation. Get three MeJA mutanted T1transgenic positive plants,two MeJA together with MYB mutanted T1transgenicpositive plants,forty-three MeJA T2transgenic positive plants.4. In order to verify the cis-acting element related to MeJA induced by methyljasmonate or not, the Arabidopsis of total length of MeJA mutanted wewe treated withdifferent concentrations of methyl jasmonate. Carried out on the plant leaf flower GUSstaining, GUS staining results showed that, MeJA mutant Arabidopsis flowers withGUS staining was no significant difference, MeJA cis-acting function needs to befurther verified.