目的 探究灰氈毛忍冬Lonicera macranthoides中查耳酮合酶LmCHS1基因?qū)S酮類成分合成的作用。方法 以灰氈毛忍冬葉的cDNA為模板，利用RT-PCR技術(shù)克隆得到了LmCHS1基因開放閱讀框（open reading frame,ORF）序列，并對其基因序列進(jìn)行生物信息學(xué)分析。通過農(nóng)桿菌侵染的方式，構(gòu)建本氏煙草瞬時轉(zhuǎn)化體系，對LmCHS1編碼蛋白進(jìn)行亞細(xì)胞定位；紫外分光光度法檢測煙草葉片及灰氈毛忍冬樣品中總黃酮含量；構(gòu)建原核表達(dá)載體，誘導(dǎo)大腸桿菌得到重組蛋白；利用qRT-PCR檢測LmCHS1基因在灰氈毛忍冬花、莖和葉中的表達(dá)情況。結(jié)果 克隆得到的基因長度為1 182 bp，共編碼393個氨基酸，分子式為C₁₉₁₄H₃₀₇₀N₅₁₄O₅₆₃S₁₇，理論等電點(diǎn)為6.34，屬于親水蛋白。亞細(xì)胞定位結(jié)果表明LmCHS1蛋白主要定位在細(xì)胞質(zhì)，與大部分物種中具催化黃酮前體合成功能的CHS蛋白的亞細(xì)胞定位一致。原核表達(dá)得到的重組蛋白，大小為43 000?？傸S酮的含量測定結(jié)果顯示過表達(dá)LmCHS1煙草葉片總黃酮含量高于空載組，并具統(tǒng)計(jì)學(xué)差異。聯(lián)合表達(dá)量和總黃酮含量結(jié)果分析發(fā)現(xiàn)，LmCHS1表達(dá)量與總黃酮含量呈正相關(guān)。結(jié)論 LmCHS1蛋白定位在細(xì)胞質(zhì)中，正向調(diào)控黃酮類物質(zhì)的合成。

Objective To investigate the function of chalcone synthase of Lonicera macranthoides (LmCHS1) gene in the flavonoid biosynthesis. Methods The open reading frame (ORF) sequence of LmCHS1 was cloned from leaf cDNA using RT-PCR technology, followed by comprehensive bioinformatic analysis. The subcellular localization of the encoded protein was determined by constructing a transient transformation system in Nicotiana benthamiana via Agrobacterium infection. Total flavonoid content was quantified by ultraviolet spectrophotometry in both tobacco leaves and L. macranthoides samples. Prokaryotic expression vectors were constructed to obtain recombinant proteins through Escherichia coli induction. qRT-PCR was employed to detect the expression levels of LmCHS1 gene in flowers, stems, and leaves of L. macranthoides. Results The cloned gene was 1 182 bp in length, encoding 393 amino acids, with a molecular formula of C₁₉₁₄H₃₀₇₀N₅₁₄O₅₆₃S₁₇ and a theoretical isoelectric point of 6.34, which belongs to a hydrophilic protein.. Subcellular localization demonstrated cytoplasmic localization of LmCHS1 protein, consistent with that of most CHS proteins which catalyze the synthesis of flavonoid precursors in other species.. The recombinant protein obtained from prokaryotic expression was 43 000 in size. Flavonoid quantification showed significantly higher content in tobacco leaves overexpressing LmCHS1 compared to empty vector controls, with statistical significance. The integrated analysis of qRT-PCR data and total flavonoid content measurements revealed a positive correlation between the expression level of LmCHS1 and the total flavonoid content. Conclusion LmCHS1 protein s localized in the cytoplasm and positively regulates flavonoid biosynthesis.

R286.12

國家自然科學(xué)基金資助項(xiàng)目（82373992）；國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系（CARS-21）；湖南省中藥材產(chǎn)業(yè)技術(shù)體系專項(xiàng)（HARS-11）；2020年湖南省一流專業(yè)建設(shè)點(diǎn)：中藥資源與開發(fā)；湖南中醫(yī)藥大學(xué)重點(diǎn)學(xué)科中藥學(xué)科（校行發(fā)規(guī)字[2023]2號）