Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L)

被引:98
作者
Yamaguchi, S
Saito, T
Abe, H
Yamane, H
Murofushi, N
Kamiya, Y
机构
[1] INST PHYS & CHEM RES,FRONTIER RES PROGRAM,WAKO,SAITAMA 35101,JAPAN
[2] UNIV TOKYO,BIOTECHNOL RES CTR,BUNKYO KU,TOKYO 113,JAPAN
[3] UNIV TOKYO,DEPT APPL BIOL CHEM,BUNKYO KU,TOKYO 113,JAPAN
关键词
D O I
10.1046/j.1365-313X.1996.10020203.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [H-3]CDP to [H-3]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.
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页码:203 / 213
页数:11
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