Research Article

Cloning and expression analysis of the Lonicera japonica Thunb. chlorogenic acid synthetase gene (LjCCoAOMT1) in rice

Published: March 26, 2014
Genet. Mol. Res. 13 (1) : 2166-2176 DOI: https://doi.org/10.4238/2014.March.26.5
Cite this Article:
(2014). Cloning and expression analysis of the Lonicera japonica Thunb. chlorogenic acid synthetase gene (LjCCoAOMT1) in rice. Genet. Mol. Res. 13(1): gmr2767. https://doi.org/10.4238/2014.March.26.5
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Abstract

Complete coding DNA sequences of a closely related chlorogenic acid synthetase gene (LjCCoAOMT1) were isolated from Lonicera japonica Thunb. by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). LjCCoAOMT1 was subsequently overexpressed in Escherichia coli and a 25-kD protein was detected by electrophoresis and western blot analysis. High-performance liquid chromatography (HPLC) analysis showed that recombinant LjCCoAOMT1 methylates the caffeic acid substrate to generate ferulic acid. Further analysis showed that the chlorogenic acid content was significantly correlated with the expression level of LjCCoAOMT1 in various tissues of L. japonica Thunb. at different developmental stages. A plant expression vector containing LjCCoAOMT1 was constructed and Agrobacterium-mediated transgenic rice was successfully obtained. Light treatment analysis showed that LjCCoAOMT1 transgenic rice was more sensitive than wild-type rice in responding to the changes in lighting conditions. Although gibberellic acid (GA3) could promote the growth of both wild-type and LjCCoAOMT1 transgenic rice, LjCCoAOMT1 transgenic rice appeared to be more sensitive to GA3. Furthermore, high concentrations of GA3 significantly facilitated the growth of LjCCoAOMT1 transgenic rice.

Complete coding DNA sequences of a closely related chlorogenic acid synthetase gene (LjCCoAOMT1) were isolated from Lonicera japonica Thunb. by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). LjCCoAOMT1 was subsequently overexpressed in Escherichia coli and a 25-kD protein was detected by electrophoresis and western blot analysis. High-performance liquid chromatography (HPLC) analysis showed that recombinant LjCCoAOMT1 methylates the caffeic acid substrate to generate ferulic acid. Further analysis showed that the chlorogenic acid content was significantly correlated with the expression level of LjCCoAOMT1 in various tissues of L. japonica Thunb. at different developmental stages. A plant expression vector containing LjCCoAOMT1 was constructed and Agrobacterium-mediated transgenic rice was successfully obtained. Light treatment analysis showed that LjCCoAOMT1 transgenic rice was more sensitive than wild-type rice in responding to the changes in lighting conditions. Although gibberellic acid (GA3) could promote the growth of both wild-type and LjCCoAOMT1 transgenic rice, LjCCoAOMT1 transgenic rice appeared to be more sensitive to GA3. Furthermore, high concentrations of GA3 significantly facilitated the growth of LjCCoAOMT1 transgenic rice.

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