关键词: 苦荞麦, 芦丁水解酶, 氨基酸序列, 昆虫表达系统, 槲皮素

Abstract: In order to obtain the gene encoding rutin-hydrolyzing enzyme in Fagopyrum tartaricum (FtRHE), the enzyme was obtained from the seeds of the tartary buckwheat cultivar ‘Yunqian 1’ and its peptide mass sequence was determined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) and then transcriptomic analysis and gene amplification were carried out. Results showed that molecular mass of RHE was about 62 kDa, and RHE was a β-glycosidase as evident from its peptide mass fingerprint. FSISWSR was a characteristic peptide fragment of RHE with mass-to-charge ratio of 914.428 6. Based on the DNA sequences obtained from the transcriptome of tartary buckwheat seeds, FtRHE gene was obtained by reverse transcription PCR amplification using specific primers. The open reading frame of FtRHE consisted of 1 539 bases, encoding 512 amino acids with amino acid residues 1–29 making upa signal peptide. Multiple sequence alignment showed that RHE shared a 54%–62% similarity with other β-glycosidases from various plants, indicating a low level of gene conservation. Bacmid-FtRHE was constructed by DNA recombination, and then transfected into insect cell Sf9. FtRHE was successfully expressed with higher rutin hydrolysis activity, indicating that the obtained FtRHE was the gene encoding rutin-hydrolyzing enzyme in tartary buckwheat seeds. This study may provide a theoretical basis for the breeding of buckwheat cultivars with high rutin content and without bitter or astringent taste and for the biotransformation of rutin into quercetin.

Key words: tartary buckwheat, rutin-hydrolyzing enzyme (RHE), amino acid sequence, insect expression system, quercetin