植物学报
植物學報
식물학보
ACTA BOTANICA SINICA
2002年
2期
181-187
,共7页
王伟%石青%朱平%欧阳涛%李秾%程克棣%WANG Wei%SHI Qing%ZHU Ping%OUTANG Tao%LI Nong%CHENG Ke-di
王偉%石青%硃平%歐暘濤%李秾%程剋棣%WANG Wei%SHI Qing%ZHU Ping%OUTANG Tao%LI Nong%CHENG Ke-di
왕위%석청%주평%구양도%리농%정극체%WANG Wei%SHI Qing%ZHU Ping%OUTANG Tao%LI Nong%CHENG Ke-di
中国红豆杉%紫杉醇生物合成%紫杉烯合酶
中國紅豆杉%紫杉醇生物閤成%紫杉烯閤酶
중국홍두삼%자삼순생물합성%자삼희합매
Taxus chinensis%Taxol biosynthesis%taxadiene synthase
紫杉烯合酶是一种二萜环化酶,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯.利用PCR扩增同源探针筛选cDNA文库,克隆了一个编码中国红豆杉(Taxus chinensis (Pilg.) Rehd.)紫杉烯合酶3′端的2 151 bp的cDNA片段,也通过PCR扩增得到了该基因5′端的611 bp的cDNA片段,将这两个cDNA片段拼接在一起,得到长2 712 bp的cDNA片段,具有一个2 586个碱基的开放阅读框架(ORF),编码包括质体转移肽在内的共862个氨基酸残基;该酶与太平洋红豆杉紫杉烯合酶有97%的同源性(identity),与其他植物萜类环化酶也有较高的同源性.利用融合表达载体pET-32a在大肠杆菌BL21trxB中表达,所表达的融合蛋白以包含体形式存在.包含体经过变性、复性和再折叠,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶.用毛细管气相色谱-质谱联用对酶促反应产物进行分析,结果表明,融合的紫杉烯合酶能催化产生4(5),11(12)-紫杉烯.
紫杉烯閤酶是一種二萜環化酶,催化牛兒基牛兒基焦燐痠形成紫杉醇生物閤成過程中的中間體紫杉烯.利用PCR擴增同源探針篩選cDNA文庫,剋隆瞭一箇編碼中國紅豆杉(Taxus chinensis (Pilg.) Rehd.)紫杉烯閤酶3′耑的2 151 bp的cDNA片段,也通過PCR擴增得到瞭該基因5′耑的611 bp的cDNA片段,將這兩箇cDNA片段拼接在一起,得到長2 712 bp的cDNA片段,具有一箇2 586箇堿基的開放閱讀框架(ORF),編碼包括質體轉移肽在內的共862箇氨基痠殘基;該酶與太平洋紅豆杉紫杉烯閤酶有97%的同源性(identity),與其他植物萜類環化酶也有較高的同源性.利用融閤錶達載體pET-32a在大腸桿菌BL21trxB中錶達,所錶達的融閤蛋白以包含體形式存在.包含體經過變性、複性和再摺疊,利用His殘基親和凝膠柱層析得到融閤的紫杉烯閤酶.用毛細管氣相色譜-質譜聯用對酶促反應產物進行分析,結果錶明,融閤的紫杉烯閤酶能催化產生4(5),11(12)-紫杉烯.
자삼희합매시일충이첩배화매,최화우인기우인기초린산형성자삼순생물합성과정중적중간체자삼희.이용PCR확증동원탐침사선cDNA문고,극륭료일개편마중국홍두삼(Taxus chinensis (Pilg.) Rehd.)자삼희합매3′단적2 151 bp적cDNA편단,야통과PCR확증득도료해기인5′단적611 bp적cDNA편단,장저량개cDNA편단병접재일기,득도장2 712 bp적cDNA편단,구유일개2 586개감기적개방열독광가(ORF),편마포괄질체전이태재내적공862개안기산잔기;해매여태평양홍두삼자삼희합매유97%적동원성(identity),여기타식물첩류배화매야유교고적동원성.이용융합표체재체pET-32a재대장간균BL21trxB중표체,소표체적융합단백이포함체형식존재.포함체경과변성、복성화재절첩,이용His잔기친화응효주층석득도융합적자삼희합매.용모세관기상색보-질보련용대매촉반응산물진행분석,결과표명,융합적자삼희합매능최화산생4(5),11(12)-자삼희.
Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.
