Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba
Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba
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DOI：
10.1007/s00299-010-0943-4
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发表时间：
2011-01-01
影响因子：
6.2
通讯作者：
Yuan, Honghui
中科院分区：
文献类型：
--
作者：
Cheng, Hua;Li, Linling;Yuan, Honghui
查尔酮异构酶（CHI, EC 5.5.1.6）是类黄酮生物合成途径中催化查尔酮立体特异性异构化为相应的(2S)-黄酮的关键酶之一。本研究从银杏叶中分离到了查尔酮异构酶的cDNA序列和基因组序列（简称GbCHI）。该基因含有2个内含子和3个外显子，编码244个氨基酸的肽段，预测分子质量为26.29 kDa， pI为7.76。RQPCR结果显示，GbCHI在双叶中以组织特异性的方式表达。UV-B照射或5-氨基乙酰丙酸或三种植物生长调节剂（乙烯、脱落酸和氯灭菊酯）处理也能上调GbCHI的表达，这些作用与GbCHI启动子区域的分析一致。以pET-28a为载体，在大肠杆菌中成功表达了重组蛋白。体外酶活性测定表明，重组GbCHI蛋白可催化6′-羟基查尔酮生成柚皮素。RQPCR分析显示，CHI活性与CHI基因转录水平变化相关，GbCHI活性与银杏叶总黄酮水平也呈正相关，表明CHI是调控银杏叶总黄酮积累的关键基因。
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.