Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli.
Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.
Wang
Qian
Q
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai, PR China.
Pi
Yan
Y
Hou
Rong
R
Jiang
Keji
K
Huang
Zhuoshi
Z
Hsieh
Ming-Shiun
MS
Sun
Xiaofen
X
Tang
Kexuan
K
D000085:Acetates / Q000494:pharmacology; D015139:Blotting, Southern; D029769:Camptotheca / Q000201:enzymology*; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D035264:Flowers; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D005816:Genetic Complementation Test; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D054883:Oxylipins / Q000494:pharmacology; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020156:Salicylic Acid / Q000494:pharmacology
D000085:Acetates; D003517:Cyclopentanes; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D054883:Oxylipins; D012333:RNA, Messenger; C072239:methyl jasmonate; D010088:Oxidoreductases; D020156:Salicylic Acid