目的 以金銀花Lonicerae Japonicae Flos中糖基轉(zhuǎn)移酶為研究對(duì)象，進(jìn)行糖基轉(zhuǎn)移酶LjUGT73C1基因的生物信息學(xué)分析、基因合成與亞克隆、表達(dá)分析和純化。方法 利用前期鹽脅迫金銀花組學(xué)研究構(gòu)建的金銀花轉(zhuǎn)錄組數(shù)據(jù)庫(kù)注釋信息，篩選得到金銀花中關(guān)鍵糖基轉(zhuǎn)移酶LjUGT73C1，進(jìn)行基因合成和亞克隆；利用基因重組技術(shù)構(gòu)建了原核表達(dá)載體Pet-30a-LjUGT73C1，遺傳轉(zhuǎn)化大腸桿菌BL21（DE3）感受態(tài)；應(yīng)用SDS-PAGE凝膠電泳和Western blotting檢測(cè)蛋白的表達(dá)量、檢測(cè)不同誘導(dǎo)條件下基因的表達(dá)情況，最后經(jīng)親和色譜進(jìn)行純化。結(jié)果 通過(guò)基因合成與亞克隆得到糖基轉(zhuǎn)移酶LjUGT73C1的全長(zhǎng)序列，理論編碼氨基酸數(shù)目為493，等電點(diǎn)（PI）預(yù)測(cè)為6.27，理論相對(duì)分子質(zhì)量為55 530。構(gòu)建了LjUGT73C1基因的原核表達(dá)載體并且在大腸桿菌中誘導(dǎo)表達(dá)得到重組蛋白，應(yīng)用His親和色譜柱進(jìn)行蛋白純化并經(jīng)SDS-PAGE凝膠電泳檢測(cè)，確定為金銀花LjUGT73C1蛋白。結(jié)論 首次報(bào)道了鹽脅迫金銀花中糖基轉(zhuǎn)移酶基因LjUGT73C1，并進(jìn)行表達(dá)分析和純化，為進(jìn)一步研究金銀花中苯丙素的生物合成及表達(dá)調(diào)控研究奠定了基礎(chǔ)。

Objective The LjUGT73C1 gene, which was a glycosyltransferase gene from Jinyinhua (Lonicerae Japonicae Flos, LJF), was performed bioinformatics analysis, gene synthesis with subcloning, expression analysis and purification.Methods Based on the transcriptome database annotation information of LJF constructed in the previous salt stress omics study, the key glycosyltransferase LjUGT73C1 was screened, then gene synthesis and subcloning were performed. The prokaryotic expression vector Pet-30a-LjUGT73C1 was constructed by gene recombination technology, and transformed into competent Escherichia coli BL21 (DE3). SDS-PAGE gel electrophoresis and Western blot were used to detect the expression of proteins, and the expression of gene under different induction conditions was detected. Finally, the protein was purified by affinity chromatography. Results The full-length sequence of glycosyltransferase LjUGT73C1 was obtained by gene synthesis and subcloning. The theoretical number of amino acids encoded was 493, the predicted isoelectric point (PI) was 6.27, and the theoretical molecular weight was 55 530. The prokaryotic expression vector of gene LjUGT73C1 was constructed and the recombinant protein was induced and expressed in E. coli. The protein was purified by His affinity chromatography and identified as LjUGT73C1 by SDS-PAGE. Conclusion The LjUGT73C1 glucosyltransferase gene in LJF under salt stress was first reported, and its expression analysis and purification were carried out. This study laid a foundation for further study on the biosynthesis and expression regulation of phenylpropanoids in LJF.

R286.12

江蘇省基礎(chǔ)研究計(jì)劃自然科學(xué)基金資助項(xiàng)目（BK20220479）;江蘇省普通高校自然科學(xué)研究項(xiàng)目（22KJB360007）;國(guó)家中醫(yī)藥管理局高水平中醫(yī)藥重點(diǎn)學(xué)科建設(shè)項(xiàng)目（國(guó)中醫(yī)藥人教函［2023］85號(hào)）