Study On Expression Analysis And Promoter Cloning Of CsCOMT Gene In Tea Plant

(-)-Epigallocatechin-3-O(3-O-methyl)gallate(EGCG3"Me)and(-)-epigallocatechin-3-O(4-O-methyl)gallate(EGCG4"Me)were O-methylated derivatives of EGCG EGCG3"Me and EGCG4"Me were receiving considerable interest because of their great potential for improving human health.They were found have better affection in anti-allergic,anti-toxins,inhibit lipid oxidation enzymes,especially anti-pollen hypersensitivity than catechins.In this study,the promoter sequence of EGCG-O-methyltransferase(CsCOMT)gene was cloned by genome walking from tea cultivars rich in EGCG3"Me&the transcription start site,core promoter and potential cis-acting elements were predicted.The content of EGCG3"Me and the relative expressed level of CsCOMT gene in different tea samples was also studied.Finally,thin-layer chromatography(TLC)method for the separation of catechins and methylated EGCG(EGCG3"Me and EGCG4"Me)was developed.The main results were as follows:1.The promoter sequence of CsCOMT gene was successfully cloned by Genome Walker,and the full-length sequence contained 1647 bp.The transcription start site,core promoter and potential cis-acting elements were predicted by line analysis software Promoter Prediction and PlantCARE.The results showed that the promoter contained some basic elements such as TATA box,CAAT box,as well as many other important cis-acting elements such as light-responsive elements,stress response element.2.The relative expression levels of CsCOMT gene in different tea samples was analyzed by real-time fluorescence quantitative PCR.The relative expression levels of CsCOMT was in the order of Summer tea>Autumn tea>Spring tea in the same tea cultivar,and they increased with the maturity of tea leaf.However,the relative expression level of CsCOMT in the second leaf was lowest among all the tea leaves.3.The content of EGCG3"Me was analyzed by high performance liquid chromatography(HPLC).The content of EGCG3 "Me was in the order of summer tea>autumn tea>spring tea in the same tea cultivar.The content of EGCG3"Me increased with the maturity of tea leaf and the fourth or fifth tealeaves had the highest content of EGCG3"Me.4.Thin-layer chromatography(TLC)method for the separation of seven related compounds:(+)-catechin(C),(-)-epicatechin(EC),(-)-epigallocatechin(EGC),(-)-epicatechin gallate(ECG),(-)-epigallocatechin gallate(EGCG),EGCG3" Me and EGCG4" Me was developed.The above-mentioned seven compounds have been resolved using polyamide TLC plates using a n-butanol-acetone-acetic acid(8:5:3,v/v).