Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba.
Chalcone isomerase (CHI, EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthesis pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. In this investigation, both the cDNA sequence and the genomic sequence encoding the chalcone isomerase from Ginkgo biloba L. (designated as GbCHI) were isolated from the leaves. The GbCHI gene contained two introns and three extrons and encoded a peptide of 244 amino acids with a predicted molecular mass of 26.29 kDa and a pI of 7.76. RQPCR showed that GbCHI was expressed in a tissue-specific manner in G. biloba. Expression of GbCHI was also up-regulated by UV-B irradiation or treatment with 5-aminolevulinic acid or three plant growth regulator-ethylene, abscisic acid, and chlormequat-and these effects were consistent with analysis of the GbCHI promoter region. The recombinant protein was successfully expressed in an E.coli strain with the pET-28a vector. In vitro enzyme activity, assayed by HPLC, indicated that recombinant GbCHI protein could catalyze the formation of naringenin from 6'-hydroxychalcone. RQPCR analysis showed that CHI activity correlated with changes in transcription level of the CHI gene, GbCHI activity was also positively correlated with total flavonoid levels in ginkgo leaves, suggesting CHI as a key gene regulating flavonoid accumulation in ginkgo leaves.
College of Life Science and Engineering, Huanggang Normal University, Huanggang 438000, China.
Hua
H
Cheng
Linling
L
Li
Shuiyuan
S
Cheng
Fuliang
F
Cao
Yan
Y
Wang
Honghui
H
Yuan
D000040:Abscisic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000494:pharmacology; D001483:Base Sequence; D002716:Chlormequat / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods*; D005030:Ethylenes / Q000494:pharmacology; D005419:Flavonoids / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000528:radiation effects; D017343:Genes, Plant / Q000235:genetics*; D005875:Gibberellins / Q000494:pharmacology; D020441:Ginkgo biloba / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000528:radiation effects; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D018744:DNA, Plant; D005030:Ethylenes; D005419:Flavonoids; D005875:Gibberellins; D010940:Plant Proteins; D000040:Abscisic Acid; D000622:Aminolevulinic Acid; D002716:Chlormequat; C036216:ethylene; C007842:gibberellic acid; D019753:Intramolecular Lyases; C019865:chalcone isomerase