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Expression patterns of an isoflavone reductase-like gene and its possible roles in secondary metabolism in Ginkgo biloba

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Abstract

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Our results showed that GbIRL1 belongs to the PCBER protein family. Besides, IRL1 gene was a novel gene regulating lignin change and also effecting the accumulation of flavonoids in Ginkgo.

Abstract

A cDNA encoding the IFR-like protein was isolated from the leaves of Ginkgo biloba L., designated as GbIRL1 (Accession no. KC244282). The cDNA of GbIRL1 was 1,203 bp containing a 921 bp open reading frame encoding a polypeptide of 306 amino acids. Comparative and bioinformatic analyses revealed that GbIRL1 showed extensive homology with IFLs from other gymnosperm species. Phylogenetic tree analysis revealed that GbIRL1 shared the same ancestor in evolution with other PCBERs protein and had a further relationship with other gymnosperm species. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The vitro enzyme activity assay by HPLC indicated that recombinant GbIRL1 protein could catalyze the formation the TDDC, IDDDC from DDDC, DDC. Tissue expression pattern analysis showed that GbIRL1 was constitutively expressed in stem and roots, especially in the parts of the pest and fungal infection, with the lower expression being found in 1- or 2-year old stem. The increased expression of GbIRL1 was detected when the seedlings were treated with Ultraviole-B, ALA, wounding and ethephon, abscisic acid, salicylic acid. Correlation analysis between GbIRL1 activity and flavonoid accumulation during Ginkgo leaf growth indicated that GbIRL1 might be the rate-limiting enzyme in the biosynthesis pathway of flavonoids in Ginkgo leaves. Results of RT-PCR analysis showed that the transcription level of change in GbIRL1 power correlated with flavonoid contents, suggesting IRL1 gene as a novel gene regulating lignin change and also effecting the accumulation of flavonoids in Ginkgo.

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Abbreviations

bp:

Base pair

HPLC:

High performance liquid chromatography

IPTG:

Isopropyl b-d-thiogalactoside, C9H18O5S

IFR:

Isoflavone reductase

IRL:

Isoflavone reductase-like protein

IRLP:

IRLgene promoter

ORF:

Open reading frame

PCBER:

Phenylcoumaran benzylic ether reductases

PCR:

Polymerase chain reaction

RACE:

Rapid amplification of cDNA ends

QRT-PCR:

Real-time quantitative PCR

SD:

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 and 31270717), Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratories (20011BLKF238 and 2011BH0030), and University-industry Cooperation Fund of Hubei Educational Office (CXY2009B009).

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Authors and Affiliations

  1. Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratories, Hubei Huanggang, 438000, China

    Cheng Hua, Li Linling, Xu Feng, Wang Yan, Yuan Honghui, Wu Conghua, Wang Shaobing, Liao Zhiqin, Hua Juan, Wang Yuping, Cheng Shuiyuan & Cao Fuliang

  2. College of Chemistry and Life Science, Huanggang Normal University, Huanggang, 438000, China

    Cheng Hua, Li Linling, Xu Feng, Wang Yan, Yuan Honghui, Wu Conghua, Wang Shaobing, Liao Zhiqin, Hua Juan & Wang Yuping

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

    Cheng Shuiyuan & Cao Fuliang

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  1. Cheng Hua
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  2. Li Linling
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  3. Xu Feng
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  4. Wang Yan
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  5. Yuan Honghui
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  6. Wu Conghua
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  7. Wang Shaobing
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  8. Liao Zhiqin
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  9. Hua Juan
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  10. Wang Yuping
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  11. Cheng Shuiyuan
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  12. Cao Fuliang
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Correspondence to Cheng Shuiyuan or Cao Fuliang.

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Communicated by H. Judelson.

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Hua, C., Linling, L., Feng, X. et al. Expression patterns of an isoflavone reductase-like gene and its possible roles in secondary metabolism in Ginkgo biloba . Plant Cell Rep 32, 637–650 (2013). https://doi.org/10.1007/s00299-013-1397-2

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