绞股蓝(Gynostemma pentaphyllun (Thunb.) Makino) 属于葫芦科绞股蓝属多年生草质藤本植物，其主要有效成分为绞股蓝皂苷，在叶片中含量最高，目前分离鉴定的绞股蓝皂苷已达160余种。现代药理学研究表明绞股蓝皂苷具有抗肿瘤、抗焦虑、抗动脉粥样硬化、调节免疫力、降血糖、降血脂等多种药理活性。绞股蓝皂苷属于三萜皂苷，因与人参皂苷具有相同或相似的化学结构而备受关注。氧化鲨烯环化酶(OSCs)是绞股蓝皂苷三萜骨架结构合成的关键酶，本研究对绞股蓝OSCs进行了克隆和鉴定，为进一步研究绞股蓝皂苷的合成与调控奠定了基础。

植物信号分子茉莉酸甲酯(MeJA)可以诱导绞股蓝皂苷上游合成途径中的法尼基焦磷酸合酶(FPS)和鲨烯合酶(SS)基因上调表达，因此，我们推测参与绞股蓝皂苷合成的OSCs基因也可能受MeJA调控。对MeJA处理0h, 6h, 12h, 24h的绞股蓝叶的转录组进行了Illumina测序，共获取202,535,156条raw reads，采用de novo组装共获得82,073条unigenes。通过基因注释和分类，发现在GO分析中，有22,662个unigenes得到注释，其中有45个基因参与次生代谢，有2,015个基因与代谢调节有关。在KEGG分析中，总共有291个基因与萜类代谢相关，其中有82个基因与萜类骨架合成相关，有42个基因与倍半萜和三萜的生物合成相关。

根据序列比对和注释结果，筛选到5个绞股蓝OSCs基因，分别为GpOSC1-5。序列分析发现在5个OSCs中能找到OSCs的保守基序DCTAE，MWCHCR和QW。利用real-time PCR技术对OSCs进行组织特异性和MeJA诱导表达分析，发现GpOSC1在绞股蓝叶中高表达，其余4个基因在茎中表达量最高。MeJA对GpOSC1，4的表达具有抑制作用，GpOSC2，3，5的表达在MeJA处理的初期受到抑制，随后表达量逐渐升高，在处理24小时后表达量达到最高。进化树分析发现GpOSC1属于β-香树脂合酶类；GpOSC2属于环阿屯醇合酶类，与已鉴定的西葫芦(Cucurbita pepo)环阿屯醇合酶具有87.8%的相似性；GpOSC3属于羊毛甾醇合酶类，与丝瓜(Luffa aegyptiaca)的羊毛甾醇合酶具有77.1%的相似性；GpOSC4属于葫芦二烯醇合酶类，与已鉴定的葫芦二烯醇合酶的相似性均大于80%。GpOSC5属于isomultiflorenol合酶类，与丝瓜(Luffa aegyptiaca)中isomultiflorenol合酶的相似性达到82.3%。

将基因GpOSC1-5分别克隆到酵母表达载体pYES2中，并转化酿酒酵母麦角甾醇缺陷型菌株GIL77。对外源基因诱导48小时后，离心收集菌体，碱法裂解细胞后用正己烷提取酶的催化产物，通过GC-MS分析，发现GpOSC1能够合成两个特异性产物（产物1和2），GpOSC4能够合成一个特异性产物（产物3）。通过比较保留时间和质谱图，发现这三个产物与标准品达玛烯二醇 II、羽扇豆醇、葫芦二烯醇、桦木醇和β-香树脂等具有显著差异。通过核磁共振对3个产物的结构解析工作正在进行中。

本研究通过转录组测序和基因的异源表达相结合，成功对绞股蓝OSCs进行了功能鉴定，不但为绞股蓝皂苷的代谢工程与合成生物学研究提供了有用的元器件，也为通过分子育种提高绞股蓝皂苷含量提供了有力的理论支撑。

Gynostemma (Cucurbitaceae) is a genus of perennial creeping herbs. Gypenosides (triterpenoid saponins), the main effective components of G. pentaphyllum, are mainly distributed in leaves. More than 160 glycosides were found in G. pentaphyllum. According to modern pharmacology, gypenosides have many pharmacological activities including anticancer function, antianxiety function, antidiabetics, anti-atherosclerosis, blood fat-reducing and immunoprotection. Gypenosides draw a lot of public attentions as their structural similarities to glycosides found in Panax ginseng. OSCs play an important role in the synthesis of triterpenoids backbone. In this study, we have cloned and identified OSCs from G. pentaphyllum and lay a solid foundation for elucidating gypenosides biosynthesis pathway and its regulations.

The plant signaling compound MeJA can induce the expression of FPS and SS, which belonging to the gypenosides upstream biosynthesis pathway. Therefore, we speculated that the OSCs also induced by MeJA in G. pentaphyllum. Based on this speculation, we analyzed the transcriptional levels of the G. pentaphyllum handled with MeJA by Illumina HiSeq^TM2000. After sequencing, we obtained 202 million raw reads and assembled into 82,073 unigenes. In GO analysis, 45 genes possibly involved in secondary metabolic process and 2,015 genes possibly involved in metabolic regulation. In KEGG analysis, 291 genes possibly involved in terpenoid metabolism, in which 82 genes are related to the terpene backbone biosynthesis and 42 genes are related to the synthesis of sesquiterpenes and triterpenoids.

Based on annotation and homology alignment, five OSCs named GpOSC1-5 were screened and several highly conserved motifs, including DCTAE, MWCHCR and QW, were found in these candidate OSCs. According to real-time PCR, GpOSC1 shows high expression in leaves and GpOSC2-5 show high expression in stems. GpOSC1 and GpOSC4 were downregulated by MeJA. GpOSC2, GpOSC3 and GpOSC5 were also suppressed in preliminary stage, but gradually increase until 24h. According to phylogenetic analysis, GpOSC1 clustered with β-Amyrin; GpOSC2 clustered with cycloartenol synthase and shared 87.8% similarity with identified CAS in Cucurbita pepo; GpOSC3 clustered with lanosterol synthase; GpOSC4 clustered with cucurbitadienol synthase and shared more than 80% similarity with identified CbQ; GpOSC5 clustered with isomultiflorenol synthase and shared 82.3% similarity with identified IMS in Luffa aegyptiaca.

For identifying the function of candidate OSCs, GpOSC1-5 were cloned into the yeast expression vector pYES2 and heterologously expressed in Saccharomyces cerevisiae strain GIL77 (gal2 hem3-6 erg7 ura3-167) lacking lanosterol synthase activity. After induction with galactose, the cells were collected, refluxed with 20% KOH/50% ethanol and extracted with the same volume of n-hexane. Reaction products were analyzed by GC-MS and the results showed that GpOSC1 can cyclize 2,3-oxidosqualene into two compounds (product 1 and 2) and GpOSC4 can cyclized 2,3-oxidosqualene into one compound (product 3). According to the analysis results, these three compounds’ retention time show distinct difference with authentic dammarenediol II, lupeol, β-amyrin, cucurbitadienol and betulin. The structures of these three products will be further studied by NMR.
    In this study, we identified the function of candidate OSCs based on the combination of RNA-seq and heterologous expression.This work not only provided useful components for gypenosides metabolic engineering and synthetic biology, but also provided theoretical support for molecular breeding of G.pentaphyllum.