目的 克隆粗莖秦艽Gentiana crassicaulis的裂環(huán)馬錢(qián)苷合酶（secologanin synthase，SLS）基因GcSLS，分析其理化性質(zhì)、系統(tǒng)進(jìn)化關(guān)系、組織表達(dá)特性，為進(jìn)一步研究植物萜類化合物生物合成通路中的關(guān)鍵酶及其作用機(jī)制提供基礎(chǔ)資料。方法 依據(jù)粗莖秦艽轉(zhuǎn)錄組數(shù)據(jù)，注釋、篩選并克隆GcSLS基因；使用生物信息學(xué)工具分析其編碼的氨基酸序列、結(jié)構(gòu)域等，預(yù)測(cè)其編碼蛋白的理化性質(zhì)；使用MEGA11軟件分析進(jìn)化關(guān)系，并利用熒光定量PCR檢測(cè)GcSLS基因在不同組織中的相對(duì)表達(dá)水平。結(jié)果 從粗莖秦艽中成功克隆獲得GcSLS基因，其開(kāi)放閱讀框（open reading frame，ORF）序列全長(zhǎng)共計(jì)1 566 bp，編碼521個(gè)氨基酸，蛋白質(zhì)相對(duì)分子質(zhì)量為59 590，為堿性親水穩(wěn)定蛋白，具有細(xì)胞色素P450的保守域并含有1處跨膜結(jié)構(gòu)。熒光定量PCR結(jié)果顯示，GcSLS在粗莖秦艽根和花中的表達(dá)量顯著高于在葉和莖中的表達(dá)量；且該基因受茉莉酸甲酯（methyl jasmonate，MeJA）誘導(dǎo)后表達(dá)水平顯著上調(diào)。結(jié)論 獲得粗莖秦艽GcSLS序列基礎(chǔ)信息，分析序列特征及表達(dá)模式，可為深入探究GcSLS在萜類化合物合成通路中的功能提供科學(xué)資料。

Objective To clone secologanin synthase (SLS) gene from Gentiana crassicaulis (GcSLS) and carry out physical and chemical properties, system evolutionary relationships, organization expression characteristic analysis. Methods Based on the annotation results of transcriptome data, we cloned the GcSLS from G. crassicaulis, analyzed its amino acid coding sequences and predicted the protein domains, physical and chemical properties and structure features. MEGA 11 software was used for evolutionary analysis. Quantitative PCR was employed to detect the expression levels of GcSLS in different tissues. Results We successfully cloned the GcSLS, and the ORF of GcSLS was 1 566 bp in length, encoding a 521 aa protein with a molecular weight of 59 590. It was a hydrophilic protein with a transmembrane domain, P450 conserved domains and no signal peptides. The results of quantitative PCR showed that the expression of GcSLS in roots and flowers was significantly higher than that in leaves and stems, and the expression of GcSLS was significantly induced by methyl jasmonate (MeJA). Conclusion The basic information, sequence characteristics and expression pattern of GcSLS may provide scientific data for further investigation of the function of GcSLS in the synthesis pathway of terpenoids.

R286.12

國(guó)家自然科學(xué)基金面上項(xiàng)目（82073959）；西藏自治區(qū)藏醫(yī)藥管理局科研課題（JJKT2020017）