高寒藏药材麻花艽及其体细胞杂种齐墩果酸合成关键酶-β-amyrin合成酶基因克隆及其功能鉴定
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摘要
次生代谢产物关键酶基因的克隆及转化是植物次生代谢工程的研究热点。通过超量表达相关限速酶基因,来提高目标代谢物产量是植物代谢工程的主要策略之一。由于大多数植物次生代谢途径网络关系复杂;克隆基因及其功能鉴定的有效方法还较少,且非常耗时、效率低,结果所获得的次生代谢途径中的相关基因较少。目前,在基因水平上萜类合成途径研究较为清楚,三萜类代谢途径中的一些基因已被克隆,但有关中药材有效成分合成相关基因的发掘才刚刚开始。
高寒藏药材—麻花艽主治黄疸性肝炎、胆结石等疾病,其野生资源匮乏。狭叶柴胡是著名的抗病毒感冒中药材。利用不对称体细胞杂交可转移并提高药效成分含量,实现药用成分合成基因在不同科、属、种间的相互转移。本实验室已获得齐墩果酸含量高的狭叶柴胡与麻花艽体细胞杂种,克隆了杂种的齐墩果酸合成关键酶—β-amyrin合成酶基因片段(蔡云飞博士论文,2006),但是,有关β-amyrin合成酶全长基因的克隆及其在体细胞杂种中的存在形式尚未有系统深入的研究。
齐墩果酸是齐墩果烷型五环三萜化合物,是抗肝炎的有效成分之一。β-amyrin是齐墩果酸合成的前体物质,β-amyrin经氧化和羧化反应使28位C-上的烷基变为羧基,形成齐墩果酸,二者仅存在一个基团的差异。β-amyrin合成酶为氧化角鲨烯环化酶(OSCs)家族的一员,是合成β-amyrin及其下游产物齐墩果酸的关键酶。
本研究以麻花艽、狭叶柴胡及其体细胞杂种为材料,开展抗肝炎有效成分齐墩果酸合成关键酶—β-amyrin合成酶基因的克隆、功能鉴定及其在体细胞杂种中的存在形式的研究。首次分离了麻花艽、狭叶柴胡及其体细胞杂种的9个β-amyrin合成酶的全长新基因;完成了β-amyrin合成酶GsAS1和GsAS2在原核大肠杆菌、真核酵母中的功能验证;实现了GsAS1和GsAS2对麻花艽胚性愈伤组织的转化,获得了齐墩果酸含量高的转基因植株,为进一步研究转基因植株及后代齐墩果酸代谢的分子机制奠定了基础。
另一方面,本文系统地分析了β-amyrin合成酶基因在狭叶柴胡与麻花艽体细胞杂种中的存在方式、表达模式及其与齐墩果酸含量的关系,发现体细胞杂种的β-amyrin合成酶基因均来源于麻花艽,其表达水平与齐墩果酸含量关系密切,为进一步深入研究体细胞杂种中齐墩果酸代谢的调控机理提供了理论依据。分析了在诱导子茉莉酸甲酯(MeJA)处理下,齐墩果酸代谢途径中相关酶基因表达水平的差异,发现了MeJA通过诱导β-amyrin合成酶基因的过量表达来提高齐墩果酸的含量,从而证明了该基因为麻花艽及其体细胞杂种齐墩果酸合成的关键酶基因。利用改进的气相色谱-质谱(P&T-GC-MS)联用技术分离并鉴定了中药材水母雪兔子和盐地风毛菊的挥发性有机化合物成分,首次在盐地风毛菊中发现了2个未知成分,为中药材资源的进一步开发和利用奠定了基础。
主要研究过程及实验结果包括:
1.齐墩果酸合成关键酶—β-amyrin合成酶基因的克隆
采用RACE-PCR技术,从麻花艽、狭叶柴胡及其体细胞杂种2Q、3Q、3Q1'中分离了9个β-amyrin合成酶基因的全长cDNA,分别命名为:GsAS1(Genbank号为FJ790411)、GsAS2、Bs AS、GsAS1a、GsAS1b、GsAS1c、GsAS2a、GsAS2b、GsAS2c。序列分析表明,它们的ORF核苷酸长度分别为:2268bp、2277bp、2289bp、2268bp、2277bp、2268bp、2277bp、2268bp、2277bp。分别编码755、758、762、755、758、755、758、755、758个氨基酸,蛋白质分子量均为88kDa。它们的蛋白质序列中均含有氧化角鲨烯环化酶家族成员的特征序列:即4个拷贝的QW阅读框[(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW]和1个DCTAE阅读框。其核苷酸和氨基酸同源性分别为:GsAS1/GsAS2为81.73%和81.13%;BsAS/GsAS1为78.90%和79.79%;BsAS/GsAS2为83.49%和89.11%:GsAS1a/GsAS1b/GsAS1c为100%;GsAS2a/GsAS2b/GsAS2c为99.96%和100%。上述9个基因的核苷酸和蛋白质序列同源性分别为91.3%和90.4%。
系统树分析表明,上述基因均属于双子叶植物单功能的β-amyrin合成酶基因,氨基酸序列在进化上与人参、白桦、青蒿和紫菀等具有较高的同源性。用上述基因引物对柴胡、麻花艽及其杂种的基因组DNA进行PCR扩增,首次发现GsAS2、GsAS2a、GsAS2b、GsAS2c为没有内含子的β-amyrin合成酶基因。
核苷酸序列比对发现,来自杂种的GsAS1a、GsAS1b、GsAS1c与麻花艽GsAS1序列完全一致;GsAS2b与麻花艽GsAS2序列完全一致;GsAS2a和GsAS2c与麻花艽GsAS2序列分别有1个和2个碱基的突变,但其编码的氨基酸序列一致,表明杂种的上述基因来源于供体,这些基因渐渗到了柴胡基因组中,并在杂种中表达。
2.齐墩果酸代谢途径中相关基因的表达分析
1)β-amyrin合成酶基因在麻花艽中的表达分析
半定量RT-PCR和Real-time PCR分析表明,GsAS1和GsAS2在麻花艽叶片中高表达,在根和茎中的表达量甚微。GsAS1在叶中的表达量是根中的2.8倍,茎中的4.5倍;GsAS2在叶中的表达量是根中的5.1倍,茎中的4.2倍。
2)麻花艽体细胞杂种及其双亲齐墩果酸合成相关基因表达及其与齐墩果酸含量关系
选用β-amyrin合成酶、FPP合成酶、鲨烯合成酶、鲨烯环氧酶和环阿屯醇合成酶基因对杂种2Q、3Q、3Q1'及其双亲进行半定量RT-PCR分析,结果表明,β-amyrin合成酶和鲨烯合成酶基因的表达水平为:杂种3Q和3Q1'高于双亲,2Q低于双亲及3Q和3Q1'。环阿屯醇合成酶基因(分支途径基因)表达水平:2Q高于双亲及3Q和3Q1',3Q1'低于双亲及3Q和2Q,表明,3Q和3Q1'合成β-amyrin能力高于2Q。不同杂种中齐墩果酸合成相关基因表达水平存在明显差异,同一基因表达水平在不同杂种中亦存在明显差异。HPLC分析表明,麻花艽、狭叶柴胡及其杂种2Q、3Q、3Q1'齐墩果酸含量分别为0.195、0.129、0.108、0.473、0.350 mg/g,其中,2Q含量最低(低于双亲及3Q、3Q1'),3Q最高,3Q和3Q1'高于双亲。表明,3Q和3Q1'的β-amyrin合成酶基因表达水平高,其齐墩果酸含量高,2Q的β-amyrin合成酶基因表达水平低,其含量亦低。由此而知,杂种齐墩果酸合成相关基因表达与其含量呈正相关。
3)诱导子MeJA对麻花艽齐墩果酸合成相关基因的表达及齐墩果酸含量的影响
克隆齐墩果酸代谢途径相关的10个基因:HMG-CoA合酶、HMG-CoA还原酶、GPP合成酶、FPP合成酶、IPP异构酶、鲨烯合成酶、鲨烯环氧酶、环阿屯醇合成酶、羽扇豆醇合成酶和β-amyrin合成酶基因片段,分析MeJA处理下,上述基因的表达变化。
(1)不同浓度MeJA诱导的目标基因表达模式:选用0、20、50、100、200和500 uM的MeJA处理麻花艽愈伤组织,取处理24h的材料进行半定量RT-PCR分析,结果表明,在20和50 uM下基因表达量高,为适宜的处理浓度。MeJA处理可显著提高β-amyrin合成酶基因及其上游途径的GPP合成酶、FPP合成酶、IPP异构酶、鲨烯合成酶和鲨烯环氧酶基因表达,对分支途径的羽扇豆醇合成酶和环阿屯醇合成酶基因影响不大。
(2)不同时间MeJA诱导的目标基因表达模式:选用50 uM的MeJA处理麻花艽悬浮细胞系及其愈伤组织,分别取0h、6 h、12 h、24h、48h、3 d、5d的悬浮系及0h、6 h、12 h、24h、48h、4 d、7 d、10 d、14 d和21 d的愈伤组织进行半定量RT-PCR和Real-time PCR分析,结果表明,悬浮细胞系在MeJA处理6 h,β-amyrin合成酶、FPP合成酶和鲨烯合成酶基因表达上调,12 h达到最高,以后呈下降趋势。愈伤组织在MeJA处理6 h,β-amyrin合成酶基因表达明显上调,24 h达到最大,一直维持到10 d;鲨烯环氧酶基因表达亦略有上调,并维持到14 d;在MeJA处理24h,GPP合成酶、FPP合成酶、IPP异构酶基因表达明显上调,亦一直维持到14 d。环阿屯醇合成酶和羽扇豆醇合成酶基因的表达在6 h亦有略微上调,表明,MeJA对β-amyrin合成酶基因表达的影响最大,证实了该基因是齐墩果酸合成途径的重要关键酶基因。
(3)MeJA处理麻花艽愈伤组织对齐墩果酸含量的影响:麻花艽愈伤组织在0.1 mMMeJA处理不同时间下的HPLC分析表明,随着处理时间的延长,齐墩果酸含量持续增加,在24 h-48 h间其增长率最高,到21 d时达到最高,为0.964 mg/g,比对照(未处理)增加5倍多。表明,MeJA能够显著提高齐墩果酸含量。
(4)MeJA处理下β-amyrin合成酶基因表达与齐墩果酸含量的关系:MeJA处理12 h后,β-amyrin合成酶基因表达水平高,齐墩果酸含量亦增加,表明,β-amyrin合成酶基因表达水平与齐墩果酸含量呈正相关。
4)水杨酸对麻花艽齐墩果酸合成相关基因表达的影响
取50 uM水杨酸处理不同时间的麻花艽液体悬浮细胞系,对齐墩果酸代谢途径中主要的酶基因HMG-CoA合酶、IPP异构酶、FPP合成酶、鲨烯环氧酶、β-amyrin合成酶和环阿屯醇合成酶基因进行半定量RT-PCR分析。结果表明,仅有FPP合成酶基因表达在处理6 h时略有上调,其它基因表达没有变化,表明,水杨酸对齐墩果酸代谢途径相关基因表达没有影响。
3.β-amyrin合成酶基因的功能鉴定
1)原核细胞大肠杆菌的表达验证:构建了带有GsAS1与GsAS2的大肠杆菌表达载体(pET28(a)-GsAS1和pET28(a)-GsAS2),转化到表达型菌株BL21中,阳性克隆经IPTG诱导,提取蛋白进行SDS-PAGE分析,均获得了分子量大小为88.0 kDa的β-amyrin合成酶谱带。Western-blot分析表明,亦获得了一条β-amyrin合成酶谱带。
2)酵母真核细胞的表达验证:构建了酵母表达载体pPICZA-GsAS1和pPICZA-GsAS2,转入毕赤酵母X-33中,SDS-PAGE分析发现,转GsAS1和GsAS2的酵母菌与对照相比均含有β-amyrin合成酶谱带,分子量大小为88.0kDa,证明该基因为可靠的β-amyrin合成酶基因。气相-质谱法(GC-MS)测定转GsAS1和GsAS2酵母表达产物发现,酵母转化菌同β-amyrin标准品均在15.20 min处存在一个峰,该峰具有β-amyrin所具有的五环三萜结构:218(C环结构峰);203[m/z 218-CH3],证明GsAS1和GsAS2能够表达,其产物具有β-amyrin合成酶活性,可在酵母中直接催化底物2,3-氧化角鲨烯生成β-amyrin,而未转基因酵母未检测出β-amyrin。
3)麻花艽胚性愈伤组织的基因转化:采用gateway技术,构建了GsAS1和GsAS2的过表达载体(pK7WG2D-GsAS1和pK7WG2D-GsAS2)和RNAi载体(pK7GWIWG-GsAS1和pK7GWIWG-GsAS2)。利用基因枪法,将上述载体分别转入培养7d的麻花艽胚性愈伤组织(分化频率高,达90%以上),暗培养24h,在高浓度(50 mg/L)Kan下筛选出抗性愈伤组织。抗性愈伤组织在IB分化培养基上分化成苗后,用25 mg/L Kan进一步筛选再生植株,获得了111株抗性植株(GsAS1过表达系27株、GsAS2过表达系34株、GsAS1的RNAi系29株、GsAS2的RNAi系21株)。抗性植株总DNA用35S启动子引物进行PCR检测,GsAS1和GsAS2的过表达和RNAi植株转化频率分别为2.86%、3.13%、4.00%、2.94%。HPLC测定表明,GsAS1过表达植株齐墩果酸含量为对照植株的1.17-2.03倍,GsAS2过表达植株齐墩果酸含量为对照植株的1.67-2.46倍,GsAS2过表达植株齐墩果酸含量高于GsAS1过表达植株,而GsAS1和GsAS2的RNAi株系齐墩果酸含量比对照植株低很多。由此可见,GsAS1和GsAS2的过表达均促进了转基因植株齐墩果酸的积累,GsAS2的贡献较GsAS1大。
4.中药材干植株材料化学成分的代谢谱研究
水母雪兔子(雪莲)(Saussurea involucrate)和盐地风毛菊(Saussurea lacostei)的水溶性物质代谢图谱分析表明,水母雪兔子中共检出29种挥发性有机化合物(VOCs),盐地风毛菊中共检出24种VOCs,15种VOCs同时存在于两种材料中。首次在盐地风毛菊中发现2种新VOCs(a,a-二甲基-苯甲醇和1,8-亚乙基萘)。
Recently,the research hot spot of plant secondary metabolic engineering is turned into cloning and transfomation of genes coding key enzymes,of which the overexpression has been one of major strategies to increase the yield of target metabolin in this area.However,the related genes in the pathway were still known a little,according to the complexity of the relationship between metabolic pathways,the limited modus operandi to character functional gene.At present, the biosynthetic pathway of terpene was investigated at gene level and several genes were cloned. However,it is just started the research of gene discovery in Chinese crude drug.
Gentiana straminea Maxim.(Mahuaqinjiao) is an important medicinal plant in China,and has anti-hepatitis activity,while its natural resources were threatened by excessive exploitation. Bupleurum scoronerifolium is one of the famous Chinese medicinal materials with an anti-cold activity.Somatic hybridization has been proven to increase the contents of effective components in the somatic hybrids and realize the transfer of the genes related with the medicinal components in interspecific,intergeneric or even inter-familial plants.The somatic hybrids of B.scoronerifolium and G.straminea with higher content of oleanolic acid had already been obtained in our lab.Theβ-amyrin synthase gene,which plays an important role in the synthesis of oleanolic acid,has a different expression in the parents and the hybrid(B.scoronerifolium with one copy,G.straminea with two and hybrid with two).However,cloning,function and existing way ofβ-amyrin synthase gene in the parents and the hybrids had short of systemic and comparative analysis.
Oleanolic acid,having anti-hepatitis activity,belongs to oleanane type of pentacyclic triterpenoid.β-amyrin should be oxygenized and carboxylated at the C-28 position to form oleanolic acid.β-amyrin synthase,a member of oxidative squqlene family,is the key enzyme to synthesizeβ-amyrin and its downstream product oleanolic acid.
In this work,the clone and functional analysis ofβ-amyrin synthase gene in the biparents and hybrids between B.scoronerifolium and G.straminea was studied.We isolated and clonedβ-amyrin synthase genes from the biparents and hybrid clones for the first time;verified the functiona of GsAS1and GsAS2 by E.coli,yeast;realized the transformation of GsAS1and GsAS2 in embryogenic calli of G.straminea;obtained many transgenic plants with higher content of oleanolic acid.It was laid a theoretical foundation for the advanced research about the regulation mechanism of oleanolic acid metabolism.
On the other hand,we analyzed systematically the form existed and expression pattern ofβ-amyrin synthase gene in the somatic hybrids;investigated the relation between the gene expression level and the content of oleanolic acid;compared the difference of expression of the genes related with the oleanolic acid metabolic pathway under methyl jasmonic acid treatment; found the overexpression ofβ-amyrin synthase gene with the accumulation of oleanolic acid under methyl jasmonic acid treatment.In addition,we separated and identified the volatiles organic compounds in Chinese herb Saussurea involucrate and Saussurea lacostei.Two unknown components were detected by modified P&T-GC-MS which was laid a foundation for the exploitation of Chinese crude drug resources.
The main process and results of this research were as follows:
1.Cloning ofβ-amyrin synthase gene related with oleanollc acid metabolism
Nine full-length DNA ofβ-amyrin synthase genes were isolated by RACE-PCR from G. straminea,B.scoronerifolium and their somatic hybrids(2Q、3Q、3Q1'),named GsAS1(GI: FJ790411),GsAS2,Bs AS,GsAS1a,GsAS1b,GsAS1c,GsAS2a,GsAS2b and GsAS2c respectively. Their open reading frame consists of 2268bp、2277bp、2289bp、2268bp、2277bp、2268bp、2277bp、2268bp、2277bp respectively,and predicted to encode a 755,758,762,755,758,755, 758,755,758 residue protein with molecular mass 88.0 kD.Their protein sequences shared the characteristic sequence of oxidative squqlene family,which contains 4 copy of QW reading frame[(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW]and 1 copy of DCTAE reading frame. Their nucleotide and amino acids sequence homology were as following:GsAS1/GsAS2 were 81.73%and 81.13%,BsAS/GsAS1 were 78.9%and 79.79%,BsAS/GsAS2 were 83.49%and 89.11%,GsAS1a/GsAS1b/GsAS1c were 100%,GsAS2a/GsAS2b/GsAS2c were 99.6%and 100.0%.Compared with the nucleotide and amino acids sequence of the 9 genes above, nucleotide and amino acids homology were 91.3%and 90.4%respectively.
The phylogenetic analysis showed that all genes above are closely related to other plant OSCs,and particularly to the mono-functionalβ-amyrin synthases,while their amino acids sequence had higher homologous with Panax ginseng、Bupleurum Chinense、Artemisia annua and Aster sedifolius.GsAS2,GsAS2a,GsAS2b,GsAS2c didn't contain any intron by PCR,with their genomic DNA as the template using primers mentioned above.
The analysis of nucleotide sequence indicated that GsAS1a/GsAS1b/GsAS1c(from hybrids) were consistent with GsAS1,GsAS2b were consistent with GsAS2(from G.straminea) respectively,while GsAS2a and GsAS2c had small mutation in GsAS2 with 1 and 2 bases respectively.Therefore,the nucleotide sequence homology analysis among above genes mentioned indicated that they all have the function ofβ-amyrin synthase.
2.Expression profiling of the genes related with oleanolic acid synthesis pathway
1) Expression pattern of GsAS1 and GsAS2
The results of RT-PCR and Real-time PCR analyse showed that the expression of both GsAS1 and GsAS2 in the leaf was higher than in the roots and shoots.GsAS1 and GsAS2 are expressed in a tissue-specific manner,with its expression in the leaves being～2.8-fold and～5.1-fold than that in the roots,and～4.5-fold and～4.2-fold than that in the stems.
2) Expression of the genes related and the content of oleanolic acid in the hybrids and the biparents
The expression levels ofβ-amyrin synthase(AS),farnesyl pyrophosphate synthase(FPPS), squalene synthase(SQS),squalene epoxidase(SE) and cycloartenol synthase(CAS) in hybrids 3Q,3Q1',2Q and both parent lines were analyzed by RT-PCR.The results showed that the expression level of AS and SQS is 2Q< biparent lines<3Q=3Q1' and the expression level of CAS is 3Q1' 3) Expression of genes related and the content of oleanolic acid following elicitation by MeJA.
Ten genes(HMG-CoA synthase,HMG-CoA reductase,GPP synthase,FPP synthase,IPP isomerase,squalene synthase,squalene epoxidase,cycloartenol synthase,lupeol synthase andβ-amyrin synthase) related with the oleanolic acid synthesis was cloned and their expression levels under MeJA treatment were analyzed by RT-PCR.
(1) Gene expression pattern under different concentrations of MeJA treatment
The genes expression levels of G.Straminea callus was analyzed by RT-PCR under 0uM、20 uM、50 uM、100 uM、200 uM and 500uM MeJA.The results indicated that the expression ofβ-amyrin synthase and GPP synthase,FPP synthase,IPP isomerase,squalene synthase,squalene epoxidase in G.Straminea Callus were up-regulated,cycloartenol synthase and lupeol synthase expression were not changed significantly by treated with 20 and 50 uM MeJA.
(2) Genes expression pattern under different time of MeJA treatment
The genes expression levels of G.Straminea suspension cell after 0、6h、12h、24h、48h、3d、5d under 50 uM MeJA treatment and G.Straminea callus after 0、6h、12h、24h、48h、4d、7d、10d、14d and 21d under 50 um MeJA treatment were analyzed by RT-PCR.The expression ofβ-amyrin synthase,FPP synthase and squalene synthase in suspension cell was up-regulated after 6h MeJA treatment and reached the highest expression level at 12h before decline.After 6h MeJA treatment,the expression ofβ-amyrin synthase in callus was enhanced markedly and the expression of squalene epoxidase,cycloartenol synthase and lupeol synthase was slightly enhanced.β-amyrin synthase reached the highest expression level at 24h and held the line to 10d. The higher expression of squalene epoxidase maintained to 14d.After 1d MeJA treatment,GPP synthase,FPP synthase and IPP isomerase had a significant up-regulation and also held the levl towards 14d.The results above suggested that MeJA has a remarkable effect onβ-amyrin synthesis and proved theβ-amyrin synthase is a key gene in oleanolic acid metabolic pathway.
(3) The content of oleanolic acid content in G.straminea callus following elicitation by MeJA
The oleanolic acid content of G.straminea callus treated after different time under 0.1 mM MeJA was continuously increased by HPLC.It reached the highest increase rate during 24h and 48h.It had the highest content 0.964mg/g at 21d which was 5 times as the control line, demonstrated that the MeJA treatment can remarkably increase the oleanolic acid content.
(4) The relationship between the expression level ofβ-amyrin synthase and the content of oleanolic acid under the MeJA treatment
After 12h MeJA treatment,the expression ofβ-amyrin synthase elevated and the content of oleanolic acid also increased,demonstrated that the expression level ofβ-amyrin synthase was positive correlated with the content of oleanolic acid.
4) Expression of the genes related with oleanolic acid synthesis under Salicyli acid(SA) treatment
HMG-CoA synthase,FPP synthase,IPP isomerase,squalene epoxidase,cycloartenol synthase andβ-amyrin synthase are the key genes in oleanolic acid metabolic pathway.Their expression levels of G.straminea suspension cell after 0h,6h,12h,24h,48h,4d and 7d under 50 um SA treatment wer analyzed by RT-PCR.The result only shows a slightly up-regulation on the FPP synthase at 6h,suggesting that SA has no affection on the expression of the genes related with oleanolic acid synthesis.
3.Functional verification of GsAS1 and GsAS2
1) Functional verification in prokaryotic cells(E.coli)
The E.coli expression vectors pET28a-GsAS1 and pET28a-GsAS2 were constructed and transformed into E.coli BL21.The protein of the positive clone was extracted after induced by IPTG then analyzed by SDS-PAGE.A 88kDa band was detected which is identity withβ-amyrin synthase.Western-blot analysis determined that GsAS1 is a His-tag protein and its molecular weight is bigger(>90kDa) than expected due to some post-translational modification.
2) Functional verification in Pichia pastoris
The yeast expression vectors pPICZA-GsAS1 and pPICZA-GsAS2 were constructed and transformed into Pichia pastoris X-33.SDS-PAGE analyze detected an 88kDa band in transformed P.pastoris strains which identity toβ-amyrin synthase,proved that GsAS1 and GsAS2 are functionalβ-amyrin synthase gene.GC-MS analysis showed the positive transformed P.pastoris strains had an identical product at 15.20min withβ-amyrin synthase and shared the same construction withβ-amyrin synthase(m/z 426[M+];411[M+-CH3];393 [M+-H2O-CH3];218(C-ring fragment peak);203[m/z 218-CH3]),while the control strain did not have this product.The results proved GsAS1and GsAS2 can be expressed in P.pastoris and its product has the identity bioactivity withβ-amyrin which is synthesisβ-amyrin synthase which can catalyse epoxy squalane intoβ-amyrin.
3) Functional identification by expressed in G.Straminea
Plant overexpression vector(pK7WG2D) and RNAi vector(pK7GWIWG) were constructed using gateway technique and transferred into 7d grown callus of Gentiana Straminea by particle gun.After 24hours dark cultivation,the calli was selected in the culture medium with 50mg/L kanamycin.Surviving calli were then transferred to differentiation medium(IB).Rooted seedlings were continuely selected by kanamycin in the differentiation medium.A total of 111 resistant lines(GsAS1overexpression plants 27 lines,GsAS2 overexpression plants 34 lines, GsAS1 RNAi plants 29 lines and GsAS2 RNAi plants 21 lines) were gained in this way.GsAS1 and GsAS2 specific PCR primers were designed to conform by amplify a fragment of GsAS1 and GsAS2 from genome of resistant plants.The transformation rate was caculated (GsAS1overexpression 2.86%,GsAS2 overexpression 3.13%,GsASIRNAi 4.00%and GsAS2 RNAi 2.94%).HPLC analysis determined that the G.Straminea overexpressed GsAS1 contents 17%-113%more oleanolic acid than the control line and the G.Straminea overexpressed GsAS2 contents 67%-146%more oleanolic acid than the control line.As expected the RNAi lines had lower oleanolic acid content than the control line It proves that over expression the GsAS1 and GsAS2 increased the accumulation of oleanolic acid in transgenic plants and GsAS2 made much greater contribution than GsAS1.
4.Study on metabolic profiling of S.involucrate and S.lacostei
The analysis of water-soluble substance in S.involucrate and S.lacostei showed that there were 29 volatile organic compounds(VOCs) in S.involucrate and 24 VOCs in S.lacostei.15 VOCs of them existed in both materials.What's more,a,a-dimethyl-benzenemethanol and acenaphthylene was first found in S.lacostei
高寒藏药材—麻花艽主治黄疸性肝炎、胆结石等疾病,其野生资源匮乏。狭叶柴胡是著名的抗病毒感冒中药材。利用不对称体细胞杂交可转移并提高药效成分含量,实现药用成分合成基因在不同科、属、种间的相互转移。本实验室已获得齐墩果酸含量高的狭叶柴胡与麻花艽体细胞杂种,克隆了杂种的齐墩果酸合成关键酶—β-amyrin合成酶基因片段(蔡云飞博士论文,2006),但是,有关β-amyrin合成酶全长基因的克隆及其在体细胞杂种中的存在形式尚未有系统深入的研究。
齐墩果酸是齐墩果烷型五环三萜化合物,是抗肝炎的有效成分之一。β-amyrin是齐墩果酸合成的前体物质,β-amyrin经氧化和羧化反应使28位C-上的烷基变为羧基,形成齐墩果酸,二者仅存在一个基团的差异。β-amyrin合成酶为氧化角鲨烯环化酶(OSCs)家族的一员,是合成β-amyrin及其下游产物齐墩果酸的关键酶。
本研究以麻花艽、狭叶柴胡及其体细胞杂种为材料,开展抗肝炎有效成分齐墩果酸合成关键酶—β-amyrin合成酶基因的克隆、功能鉴定及其在体细胞杂种中的存在形式的研究。首次分离了麻花艽、狭叶柴胡及其体细胞杂种的9个β-amyrin合成酶的全长新基因;完成了β-amyrin合成酶GsAS1和GsAS2在原核大肠杆菌、真核酵母中的功能验证;实现了GsAS1和GsAS2对麻花艽胚性愈伤组织的转化,获得了齐墩果酸含量高的转基因植株,为进一步研究转基因植株及后代齐墩果酸代谢的分子机制奠定了基础。
另一方面,本文系统地分析了β-amyrin合成酶基因在狭叶柴胡与麻花艽体细胞杂种中的存在方式、表达模式及其与齐墩果酸含量的关系,发现体细胞杂种的β-amyrin合成酶基因均来源于麻花艽,其表达水平与齐墩果酸含量关系密切,为进一步深入研究体细胞杂种中齐墩果酸代谢的调控机理提供了理论依据。分析了在诱导子茉莉酸甲酯(MeJA)处理下,齐墩果酸代谢途径中相关酶基因表达水平的差异,发现了MeJA通过诱导β-amyrin合成酶基因的过量表达来提高齐墩果酸的含量,从而证明了该基因为麻花艽及其体细胞杂种齐墩果酸合成的关键酶基因。利用改进的气相色谱-质谱(P&T-GC-MS)联用技术分离并鉴定了中药材水母雪兔子和盐地风毛菊的挥发性有机化合物成分,首次在盐地风毛菊中发现了2个未知成分,为中药材资源的进一步开发和利用奠定了基础。
主要研究过程及实验结果包括:
1.齐墩果酸合成关键酶—β-amyrin合成酶基因的克隆
采用RACE-PCR技术,从麻花艽、狭叶柴胡及其体细胞杂种2Q、3Q、3Q1'中分离了9个β-amyrin合成酶基因的全长cDNA,分别命名为:GsAS1(Genbank号为FJ790411)、GsAS2、Bs AS、GsAS1a、GsAS1b、GsAS1c、GsAS2a、GsAS2b、GsAS2c。序列分析表明,它们的ORF核苷酸长度分别为:2268bp、2277bp、2289bp、2268bp、2277bp、2268bp、2277bp、2268bp、2277bp。分别编码755、758、762、755、758、755、758、755、758个氨基酸,蛋白质分子量均为88kDa。它们的蛋白质序列中均含有氧化角鲨烯环化酶家族成员的特征序列:即4个拷贝的QW阅读框[(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW]和1个DCTAE阅读框。其核苷酸和氨基酸同源性分别为:GsAS1/GsAS2为81.73%和81.13%;BsAS/GsAS1为78.90%和79.79%;BsAS/GsAS2为83.49%和89.11%:GsAS1a/GsAS1b/GsAS1c为100%;GsAS2a/GsAS2b/GsAS2c为99.96%和100%。上述9个基因的核苷酸和蛋白质序列同源性分别为91.3%和90.4%。
系统树分析表明,上述基因均属于双子叶植物单功能的β-amyrin合成酶基因,氨基酸序列在进化上与人参、白桦、青蒿和紫菀等具有较高的同源性。用上述基因引物对柴胡、麻花艽及其杂种的基因组DNA进行PCR扩增,首次发现GsAS2、GsAS2a、GsAS2b、GsAS2c为没有内含子的β-amyrin合成酶基因。
核苷酸序列比对发现,来自杂种的GsAS1a、GsAS1b、GsAS1c与麻花艽GsAS1序列完全一致;GsAS2b与麻花艽GsAS2序列完全一致;GsAS2a和GsAS2c与麻花艽GsAS2序列分别有1个和2个碱基的突变,但其编码的氨基酸序列一致,表明杂种的上述基因来源于供体,这些基因渐渗到了柴胡基因组中,并在杂种中表达。
2.齐墩果酸代谢途径中相关基因的表达分析
1)β-amyrin合成酶基因在麻花艽中的表达分析
半定量RT-PCR和Real-time PCR分析表明,GsAS1和GsAS2在麻花艽叶片中高表达,在根和茎中的表达量甚微。GsAS1在叶中的表达量是根中的2.8倍,茎中的4.5倍;GsAS2在叶中的表达量是根中的5.1倍,茎中的4.2倍。
2)麻花艽体细胞杂种及其双亲齐墩果酸合成相关基因表达及其与齐墩果酸含量关系
选用β-amyrin合成酶、FPP合成酶、鲨烯合成酶、鲨烯环氧酶和环阿屯醇合成酶基因对杂种2Q、3Q、3Q1'及其双亲进行半定量RT-PCR分析,结果表明,β-amyrin合成酶和鲨烯合成酶基因的表达水平为:杂种3Q和3Q1'高于双亲,2Q低于双亲及3Q和3Q1'。环阿屯醇合成酶基因(分支途径基因)表达水平:2Q高于双亲及3Q和3Q1',3Q1'低于双亲及3Q和2Q,表明,3Q和3Q1'合成β-amyrin能力高于2Q。不同杂种中齐墩果酸合成相关基因表达水平存在明显差异,同一基因表达水平在不同杂种中亦存在明显差异。HPLC分析表明,麻花艽、狭叶柴胡及其杂种2Q、3Q、3Q1'齐墩果酸含量分别为0.195、0.129、0.108、0.473、0.350 mg/g,其中,2Q含量最低(低于双亲及3Q、3Q1'),3Q最高,3Q和3Q1'高于双亲。表明,3Q和3Q1'的β-amyrin合成酶基因表达水平高,其齐墩果酸含量高,2Q的β-amyrin合成酶基因表达水平低,其含量亦低。由此而知,杂种齐墩果酸合成相关基因表达与其含量呈正相关。
3)诱导子MeJA对麻花艽齐墩果酸合成相关基因的表达及齐墩果酸含量的影响
克隆齐墩果酸代谢途径相关的10个基因:HMG-CoA合酶、HMG-CoA还原酶、GPP合成酶、FPP合成酶、IPP异构酶、鲨烯合成酶、鲨烯环氧酶、环阿屯醇合成酶、羽扇豆醇合成酶和β-amyrin合成酶基因片段,分析MeJA处理下,上述基因的表达变化。
(1)不同浓度MeJA诱导的目标基因表达模式:选用0、20、50、100、200和500 uM的MeJA处理麻花艽愈伤组织,取处理24h的材料进行半定量RT-PCR分析,结果表明,在20和50 uM下基因表达量高,为适宜的处理浓度。MeJA处理可显著提高β-amyrin合成酶基因及其上游途径的GPP合成酶、FPP合成酶、IPP异构酶、鲨烯合成酶和鲨烯环氧酶基因表达,对分支途径的羽扇豆醇合成酶和环阿屯醇合成酶基因影响不大。
(2)不同时间MeJA诱导的目标基因表达模式:选用50 uM的MeJA处理麻花艽悬浮细胞系及其愈伤组织,分别取0h、6 h、12 h、24h、48h、3 d、5d的悬浮系及0h、6 h、12 h、24h、48h、4 d、7 d、10 d、14 d和21 d的愈伤组织进行半定量RT-PCR和Real-time PCR分析,结果表明,悬浮细胞系在MeJA处理6 h,β-amyrin合成酶、FPP合成酶和鲨烯合成酶基因表达上调,12 h达到最高,以后呈下降趋势。愈伤组织在MeJA处理6 h,β-amyrin合成酶基因表达明显上调,24 h达到最大,一直维持到10 d;鲨烯环氧酶基因表达亦略有上调,并维持到14 d;在MeJA处理24h,GPP合成酶、FPP合成酶、IPP异构酶基因表达明显上调,亦一直维持到14 d。环阿屯醇合成酶和羽扇豆醇合成酶基因的表达在6 h亦有略微上调,表明,MeJA对β-amyrin合成酶基因表达的影响最大,证实了该基因是齐墩果酸合成途径的重要关键酶基因。
(3)MeJA处理麻花艽愈伤组织对齐墩果酸含量的影响:麻花艽愈伤组织在0.1 mMMeJA处理不同时间下的HPLC分析表明,随着处理时间的延长,齐墩果酸含量持续增加,在24 h-48 h间其增长率最高,到21 d时达到最高,为0.964 mg/g,比对照(未处理)增加5倍多。表明,MeJA能够显著提高齐墩果酸含量。
(4)MeJA处理下β-amyrin合成酶基因表达与齐墩果酸含量的关系:MeJA处理12 h后,β-amyrin合成酶基因表达水平高,齐墩果酸含量亦增加,表明,β-amyrin合成酶基因表达水平与齐墩果酸含量呈正相关。
4)水杨酸对麻花艽齐墩果酸合成相关基因表达的影响
取50 uM水杨酸处理不同时间的麻花艽液体悬浮细胞系,对齐墩果酸代谢途径中主要的酶基因HMG-CoA合酶、IPP异构酶、FPP合成酶、鲨烯环氧酶、β-amyrin合成酶和环阿屯醇合成酶基因进行半定量RT-PCR分析。结果表明,仅有FPP合成酶基因表达在处理6 h时略有上调,其它基因表达没有变化,表明,水杨酸对齐墩果酸代谢途径相关基因表达没有影响。
3.β-amyrin合成酶基因的功能鉴定
1)原核细胞大肠杆菌的表达验证:构建了带有GsAS1与GsAS2的大肠杆菌表达载体(pET28(a)-GsAS1和pET28(a)-GsAS2),转化到表达型菌株BL21中,阳性克隆经IPTG诱导,提取蛋白进行SDS-PAGE分析,均获得了分子量大小为88.0 kDa的β-amyrin合成酶谱带。Western-blot分析表明,亦获得了一条β-amyrin合成酶谱带。
2)酵母真核细胞的表达验证:构建了酵母表达载体pPICZA-GsAS1和pPICZA-GsAS2,转入毕赤酵母X-33中,SDS-PAGE分析发现,转GsAS1和GsAS2的酵母菌与对照相比均含有β-amyrin合成酶谱带,分子量大小为88.0kDa,证明该基因为可靠的β-amyrin合成酶基因。气相-质谱法(GC-MS)测定转GsAS1和GsAS2酵母表达产物发现,酵母转化菌同β-amyrin标准品均在15.20 min处存在一个峰,该峰具有β-amyrin所具有的五环三萜结构:218(C环结构峰);203[m/z 218-CH3],证明GsAS1和GsAS2能够表达,其产物具有β-amyrin合成酶活性,可在酵母中直接催化底物2,3-氧化角鲨烯生成β-amyrin,而未转基因酵母未检测出β-amyrin。
3)麻花艽胚性愈伤组织的基因转化:采用gateway技术,构建了GsAS1和GsAS2的过表达载体(pK7WG2D-GsAS1和pK7WG2D-GsAS2)和RNAi载体(pK7GWIWG-GsAS1和pK7GWIWG-GsAS2)。利用基因枪法,将上述载体分别转入培养7d的麻花艽胚性愈伤组织(分化频率高,达90%以上),暗培养24h,在高浓度(50 mg/L)Kan下筛选出抗性愈伤组织。抗性愈伤组织在IB分化培养基上分化成苗后,用25 mg/L Kan进一步筛选再生植株,获得了111株抗性植株(GsAS1过表达系27株、GsAS2过表达系34株、GsAS1的RNAi系29株、GsAS2的RNAi系21株)。抗性植株总DNA用35S启动子引物进行PCR检测,GsAS1和GsAS2的过表达和RNAi植株转化频率分别为2.86%、3.13%、4.00%、2.94%。HPLC测定表明,GsAS1过表达植株齐墩果酸含量为对照植株的1.17-2.03倍,GsAS2过表达植株齐墩果酸含量为对照植株的1.67-2.46倍,GsAS2过表达植株齐墩果酸含量高于GsAS1过表达植株,而GsAS1和GsAS2的RNAi株系齐墩果酸含量比对照植株低很多。由此可见,GsAS1和GsAS2的过表达均促进了转基因植株齐墩果酸的积累,GsAS2的贡献较GsAS1大。
4.中药材干植株材料化学成分的代谢谱研究
水母雪兔子(雪莲)(Saussurea involucrate)和盐地风毛菊(Saussurea lacostei)的水溶性物质代谢图谱分析表明,水母雪兔子中共检出29种挥发性有机化合物(VOCs),盐地风毛菊中共检出24种VOCs,15种VOCs同时存在于两种材料中。首次在盐地风毛菊中发现2种新VOCs(a,a-二甲基-苯甲醇和1,8-亚乙基萘)。
Recently,the research hot spot of plant secondary metabolic engineering is turned into cloning and transfomation of genes coding key enzymes,of which the overexpression has been one of major strategies to increase the yield of target metabolin in this area.However,the related genes in the pathway were still known a little,according to the complexity of the relationship between metabolic pathways,the limited modus operandi to character functional gene.At present, the biosynthetic pathway of terpene was investigated at gene level and several genes were cloned. However,it is just started the research of gene discovery in Chinese crude drug.
Gentiana straminea Maxim.(Mahuaqinjiao) is an important medicinal plant in China,and has anti-hepatitis activity,while its natural resources were threatened by excessive exploitation. Bupleurum scoronerifolium is one of the famous Chinese medicinal materials with an anti-cold activity.Somatic hybridization has been proven to increase the contents of effective components in the somatic hybrids and realize the transfer of the genes related with the medicinal components in interspecific,intergeneric or even inter-familial plants.The somatic hybrids of B.scoronerifolium and G.straminea with higher content of oleanolic acid had already been obtained in our lab.Theβ-amyrin synthase gene,which plays an important role in the synthesis of oleanolic acid,has a different expression in the parents and the hybrid(B.scoronerifolium with one copy,G.straminea with two and hybrid with two).However,cloning,function and existing way ofβ-amyrin synthase gene in the parents and the hybrids had short of systemic and comparative analysis.
Oleanolic acid,having anti-hepatitis activity,belongs to oleanane type of pentacyclic triterpenoid.β-amyrin should be oxygenized and carboxylated at the C-28 position to form oleanolic acid.β-amyrin synthase,a member of oxidative squqlene family,is the key enzyme to synthesizeβ-amyrin and its downstream product oleanolic acid.
In this work,the clone and functional analysis ofβ-amyrin synthase gene in the biparents and hybrids between B.scoronerifolium and G.straminea was studied.We isolated and clonedβ-amyrin synthase genes from the biparents and hybrid clones for the first time;verified the functiona of GsAS1and GsAS2 by E.coli,yeast;realized the transformation of GsAS1and GsAS2 in embryogenic calli of G.straminea;obtained many transgenic plants with higher content of oleanolic acid.It was laid a theoretical foundation for the advanced research about the regulation mechanism of oleanolic acid metabolism.
On the other hand,we analyzed systematically the form existed and expression pattern ofβ-amyrin synthase gene in the somatic hybrids;investigated the relation between the gene expression level and the content of oleanolic acid;compared the difference of expression of the genes related with the oleanolic acid metabolic pathway under methyl jasmonic acid treatment; found the overexpression ofβ-amyrin synthase gene with the accumulation of oleanolic acid under methyl jasmonic acid treatment.In addition,we separated and identified the volatiles organic compounds in Chinese herb Saussurea involucrate and Saussurea lacostei.Two unknown components were detected by modified P&T-GC-MS which was laid a foundation for the exploitation of Chinese crude drug resources.
The main process and results of this research were as follows:
1.Cloning ofβ-amyrin synthase gene related with oleanollc acid metabolism
Nine full-length DNA ofβ-amyrin synthase genes were isolated by RACE-PCR from G. straminea,B.scoronerifolium and their somatic hybrids(2Q、3Q、3Q1'),named GsAS1(GI: FJ790411),GsAS2,Bs AS,GsAS1a,GsAS1b,GsAS1c,GsAS2a,GsAS2b and GsAS2c respectively. Their open reading frame consists of 2268bp、2277bp、2289bp、2268bp、2277bp、2268bp、2277bp、2268bp、2277bp respectively,and predicted to encode a 755,758,762,755,758,755, 758,755,758 residue protein with molecular mass 88.0 kD.Their protein sequences shared the characteristic sequence of oxidative squqlene family,which contains 4 copy of QW reading frame[(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW]and 1 copy of DCTAE reading frame. Their nucleotide and amino acids sequence homology were as following:GsAS1/GsAS2 were 81.73%and 81.13%,BsAS/GsAS1 were 78.9%and 79.79%,BsAS/GsAS2 were 83.49%and 89.11%,GsAS1a/GsAS1b/GsAS1c were 100%,GsAS2a/GsAS2b/GsAS2c were 99.6%and 100.0%.Compared with the nucleotide and amino acids sequence of the 9 genes above, nucleotide and amino acids homology were 91.3%and 90.4%respectively.
The phylogenetic analysis showed that all genes above are closely related to other plant OSCs,and particularly to the mono-functionalβ-amyrin synthases,while their amino acids sequence had higher homologous with Panax ginseng、Bupleurum Chinense、Artemisia annua and Aster sedifolius.GsAS2,GsAS2a,GsAS2b,GsAS2c didn't contain any intron by PCR,with their genomic DNA as the template using primers mentioned above.
The analysis of nucleotide sequence indicated that GsAS1a/GsAS1b/GsAS1c(from hybrids) were consistent with GsAS1,GsAS2b were consistent with GsAS2(from G.straminea) respectively,while GsAS2a and GsAS2c had small mutation in GsAS2 with 1 and 2 bases respectively.Therefore,the nucleotide sequence homology analysis among above genes mentioned indicated that they all have the function ofβ-amyrin synthase.
2.Expression profiling of the genes related with oleanolic acid synthesis pathway
1) Expression pattern of GsAS1 and GsAS2
The results of RT-PCR and Real-time PCR analyse showed that the expression of both GsAS1 and GsAS2 in the leaf was higher than in the roots and shoots.GsAS1 and GsAS2 are expressed in a tissue-specific manner,with its expression in the leaves being～2.8-fold and～5.1-fold than that in the roots,and～4.5-fold and～4.2-fold than that in the stems.
2) Expression of the genes related and the content of oleanolic acid in the hybrids and the biparents
The expression levels ofβ-amyrin synthase(AS),farnesyl pyrophosphate synthase(FPPS), squalene synthase(SQS),squalene epoxidase(SE) and cycloartenol synthase(CAS) in hybrids 3Q,3Q1',2Q and both parent lines were analyzed by RT-PCR.The results showed that the expression level of AS and SQS is 2Q< biparent lines<3Q=3Q1' and the expression level of CAS is 3Q1'
Ten genes(HMG-CoA synthase,HMG-CoA reductase,GPP synthase,FPP synthase,IPP isomerase,squalene synthase,squalene epoxidase,cycloartenol synthase,lupeol synthase andβ-amyrin synthase) related with the oleanolic acid synthesis was cloned and their expression levels under MeJA treatment were analyzed by RT-PCR.
(1) Gene expression pattern under different concentrations of MeJA treatment
The genes expression levels of G.Straminea callus was analyzed by RT-PCR under 0uM、20 uM、50 uM、100 uM、200 uM and 500uM MeJA.The results indicated that the expression ofβ-amyrin synthase and GPP synthase,FPP synthase,IPP isomerase,squalene synthase,squalene epoxidase in G.Straminea Callus were up-regulated,cycloartenol synthase and lupeol synthase expression were not changed significantly by treated with 20 and 50 uM MeJA.
(2) Genes expression pattern under different time of MeJA treatment
The genes expression levels of G.Straminea suspension cell after 0、6h、12h、24h、48h、3d、5d under 50 uM MeJA treatment and G.Straminea callus after 0、6h、12h、24h、48h、4d、7d、10d、14d and 21d under 50 um MeJA treatment were analyzed by RT-PCR.The expression ofβ-amyrin synthase,FPP synthase and squalene synthase in suspension cell was up-regulated after 6h MeJA treatment and reached the highest expression level at 12h before decline.After 6h MeJA treatment,the expression ofβ-amyrin synthase in callus was enhanced markedly and the expression of squalene epoxidase,cycloartenol synthase and lupeol synthase was slightly enhanced.β-amyrin synthase reached the highest expression level at 24h and held the line to 10d. The higher expression of squalene epoxidase maintained to 14d.After 1d MeJA treatment,GPP synthase,FPP synthase and IPP isomerase had a significant up-regulation and also held the levl towards 14d.The results above suggested that MeJA has a remarkable effect onβ-amyrin synthesis and proved theβ-amyrin synthase is a key gene in oleanolic acid metabolic pathway.
(3) The content of oleanolic acid content in G.straminea callus following elicitation by MeJA
The oleanolic acid content of G.straminea callus treated after different time under 0.1 mM MeJA was continuously increased by HPLC.It reached the highest increase rate during 24h and 48h.It had the highest content 0.964mg/g at 21d which was 5 times as the control line, demonstrated that the MeJA treatment can remarkably increase the oleanolic acid content.
(4) The relationship between the expression level ofβ-amyrin synthase and the content of oleanolic acid under the MeJA treatment
After 12h MeJA treatment,the expression ofβ-amyrin synthase elevated and the content of oleanolic acid also increased,demonstrated that the expression level ofβ-amyrin synthase was positive correlated with the content of oleanolic acid.
4) Expression of the genes related with oleanolic acid synthesis under Salicyli acid(SA) treatment
HMG-CoA synthase,FPP synthase,IPP isomerase,squalene epoxidase,cycloartenol synthase andβ-amyrin synthase are the key genes in oleanolic acid metabolic pathway.Their expression levels of G.straminea suspension cell after 0h,6h,12h,24h,48h,4d and 7d under 50 um SA treatment wer analyzed by RT-PCR.The result only shows a slightly up-regulation on the FPP synthase at 6h,suggesting that SA has no affection on the expression of the genes related with oleanolic acid synthesis.
3.Functional verification of GsAS1 and GsAS2
1) Functional verification in prokaryotic cells(E.coli)
The E.coli expression vectors pET28a-GsAS1 and pET28a-GsAS2 were constructed and transformed into E.coli BL21.The protein of the positive clone was extracted after induced by IPTG then analyzed by SDS-PAGE.A 88kDa band was detected which is identity withβ-amyrin synthase.Western-blot analysis determined that GsAS1 is a His-tag protein and its molecular weight is bigger(>90kDa) than expected due to some post-translational modification.
2) Functional verification in Pichia pastoris
The yeast expression vectors pPICZA-GsAS1 and pPICZA-GsAS2 were constructed and transformed into Pichia pastoris X-33.SDS-PAGE analyze detected an 88kDa band in transformed P.pastoris strains which identity toβ-amyrin synthase,proved that GsAS1 and GsAS2 are functionalβ-amyrin synthase gene.GC-MS analysis showed the positive transformed P.pastoris strains had an identical product at 15.20min withβ-amyrin synthase and shared the same construction withβ-amyrin synthase(m/z 426[M+];411[M+-CH3];393 [M+-H2O-CH3];218(C-ring fragment peak);203[m/z 218-CH3]),while the control strain did not have this product.The results proved GsAS1and GsAS2 can be expressed in P.pastoris and its product has the identity bioactivity withβ-amyrin which is synthesisβ-amyrin synthase which can catalyse epoxy squalane intoβ-amyrin.
3) Functional identification by expressed in G.Straminea
Plant overexpression vector(pK7WG2D) and RNAi vector(pK7GWIWG) were constructed using gateway technique and transferred into 7d grown callus of Gentiana Straminea by particle gun.After 24hours dark cultivation,the calli was selected in the culture medium with 50mg/L kanamycin.Surviving calli were then transferred to differentiation medium(IB).Rooted seedlings were continuely selected by kanamycin in the differentiation medium.A total of 111 resistant lines(GsAS1overexpression plants 27 lines,GsAS2 overexpression plants 34 lines, GsAS1 RNAi plants 29 lines and GsAS2 RNAi plants 21 lines) were gained in this way.GsAS1 and GsAS2 specific PCR primers were designed to conform by amplify a fragment of GsAS1 and GsAS2 from genome of resistant plants.The transformation rate was caculated (GsAS1overexpression 2.86%,GsAS2 overexpression 3.13%,GsASIRNAi 4.00%and GsAS2 RNAi 2.94%).HPLC analysis determined that the G.Straminea overexpressed GsAS1 contents 17%-113%more oleanolic acid than the control line and the G.Straminea overexpressed GsAS2 contents 67%-146%more oleanolic acid than the control line.As expected the RNAi lines had lower oleanolic acid content than the control line It proves that over expression the GsAS1 and GsAS2 increased the accumulation of oleanolic acid in transgenic plants and GsAS2 made much greater contribution than GsAS1.
4.Study on metabolic profiling of S.involucrate and S.lacostei
The analysis of water-soluble substance in S.involucrate and S.lacostei showed that there were 29 volatile organic compounds(VOCs) in S.involucrate and 24 VOCs in S.lacostei.15 VOCs of them existed in both materials.What's more,a,a-dimethyl-benzenemethanol and acenaphthylene was first found in S.lacostei
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