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Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba

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Abstract

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.

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Abbreviations

bp:

Base pair

HPLC:

High performance liquid chromatography

IPTG:

Isopropyl β-d-thiogalactoside, C9H18O5S

PCR:

Polymerase chain reaction

RACE:

Rapid amplification of cDNA ends

RQPCR:

Real-time quantitative PCR

RT-PCR:

Reverse transcriptase polymerase chain reaction

S.D.:

Standard deviation

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

UV:

Ultraviolet

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Acknowledgments

This work was supported by the Natural Science Foundation of China (30971974), the Program for New Century Excellent Talents in University (NCET-04-0746), and University–industry Cooperation Fund of Hubei Educational Office (CXY2009B009).

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Authors and Affiliations

  1. College of Life Science and Engineering, Huanggang Normal University, Huanggang, 438000, China

    Hua Cheng, Linling Li, Shuiyuan Cheng, Yan Wang & Honghui Yuan

  2. College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, 210037, China

    Hua Cheng, Shuiyuan Cheng & Fuliang Cao

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  1. Hua Cheng
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  2. Linling Li
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  3. Shuiyuan Cheng
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  4. Fuliang Cao
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  5. Yan Wang
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  6. Honghui Yuan
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Correspondence to Shuiyuan Cheng.

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Communicated by C. Quiros.

First two authors contributed equally to this work.

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Cheng, H., Li, L., Cheng, S. et al. Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba . Plant Cell Rep 30, 49–62 (2011). https://doi.org/10.1007/s00299-010-0943-4

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