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克隆并鉴定何首乌中二苯乙烯合成酶基因FmSTS2,并研究其在何首乌各组织的表达与二苯乙烯苷表达的关系,为研究二苯乙烯合成酶基因在二苯乙烯苷合成及其调控中的功能奠定基础。采用互补cDNA末端快速扩增技术克隆目的片段,得到一种二苯乙烯合成酶基因FmSTS2(GeneBank登录号:JX914503),其互补cDNA全长1 644 bp。将FmSTS2连接pET-28a原核表达载体并转化BL21大肠杆菌构建原核表达系统,Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,HPLC鉴定反应产物为含有二苯乙烯母核的白藜芦醇,证明FmSTS2是一种二苯乙烯合成酶基因。HPLC测定何首乌各组织二苯乙烯苷含量;RT-PCR检测何首乌的块根,老藤,茎,叶中表达FmSTS2的程度依次降低,与二苯乙烯苷在各个组织的含量高低一致。
Cloning and identification of Polygonum multiflorum stilbene synthase gene FmSTS2 and its expression in Polygonum multiflorum tissue expression and stilbene glycoside expression, in order to study the stilbene synthase gene in stilbene glycoside synthesis and its regulation Function laid the foundation. The target fragment was cloned by rapid amplification of cDNA ends, and a stilbene synthase gene FmSTS2 (GeneBank accession number: JX914503) was obtained. The complementary cDNA was 1 644 bp in length. FmSTS2 was ligated with pET-28a prokaryotic expression vector and transformed into E.coli BL21 to construct prokaryotic expression system. Ni-NTA affinity resin purified the soluble protein and then catalyzed the reaction with malonyl-COA and coumaroyl-COA as substrate. The reaction was identified by HPLC The product is resveratrol containing the stilbene nucleus, demonstrating that FmSTS2 is a stilbene synthase gene. The contents of stilbene glycoside in Polygonum multiflorum Thunb. Were determined by HPLC. The expression of FmstS2 in tuberous roots, old rattan, stems and leaves of Polygonum multiflorum was detected by RT-PCR.