CN115820687B - 柚子中绿原酸生物合成途径基因CgHCT和CgC3′H及其应用 - Google Patents
柚子中绿原酸生物合成途径基因CgHCT和CgC3′H及其应用 Download PDFInfo
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Abstract
本发明公开了一种柚子中绿原酸生物合成途径基因CgHCT和CgC3'H及其应用,属于植物基因工程技术领域。本发明公开了一种CgHCT和CgC3'H基因在促进柚子绿原酸合成中的应用,所述CgHCT基因的核苷酸序列如SEQ ID NO.1所示,所述CgC3'H基因的核苷酸序列如SEQ ID NO.3所示。本发明基于代谢物的全基因组关联分析方法,对CGA生物合成方面存在显著差异的柑橘品种进行研究,通过物质CGA定位到HCT酰基转移酶编码基因CgHCT,再通过同源检索和共表达分析获得香豆酸3'‑羟化酶编码基因CgC3'H,经实验证实,两个基因均参与CGA的生物合成途径,促进柚子中CGA含量积累。
Description
技术领域
本发明涉及植物基因工程技术领域,特别是涉及一种柚子中绿原酸生物合成途径基因CgHCT和CgC3'H及其应用。
背景技术
柑橘(Citrus reticuLata Blanco)为芸香科、柑橘属的植物,是橘、柑、橙、金柑,柚、枳等的总称,主要分布在北纬30°-南纬30°之间,属于热带及亚热带区域的常绿果树(除枳以外)。柑橘是全球最重要的经济作物之一,也是我国南方栽培面积最广、经济地位最重要的果树。柑橘果实具有十分丰富的营养价值,富含能参与人体新陈代谢、调节有关生理活动、对人体保健和疾病防治有重要作用的生物活性物质,其果肉、皮、核和络均可单独入药。柑橘品种繁多、资源丰富,富含酚类物质,约2/3的类黄酮和1/3的酚酸,酚类物质种类和含量也是评估柑橘质量非常重要的指标。酚类物质是植物中含量最丰富的次生代谢产物,具有抗氧化、抗炎、抗癌、抗菌等作用,对降低多种疾病的发生率,防御紫外线辐射等效果显著。柑橘果皮可作为植物酚酸的重要来源,酚酸作为抵抗癌症和心脏病的潜在保护因子,受到越来越多的关注。
绿原酸(CGA,5-O-caffeoylquinic acid)是柑橘中重要的酚酸之一,是植物体在有氧呼吸过程中经莽草酸途径产生的一种苯丙素类物质,作为植物体内最丰富的有益酚酸之一。CGA有“植物黄金”之美誉,具有抗氧化、抗炎、抗病原菌、抗老化、抑制肿瘤、保护心血管等免疫调节和防御作用等多种生物活性,以及具备天然提取的安全性,可作为营养保健品和食品添加剂。此外,研究发现CGA在遗传和健康代谢相关疾病中能调节脂质代谢和葡萄糖,因此其具有强大的生物学功能和良好的发展前景。现已发现CGA在高等双子叶植物和蕨类植物中广泛存在,其中在金银花、向日葵等忍冬科及菊科植物中存在较高的积累量,但在柑橘中含量较低。目前的研究表明,CGA存在三种可能的合成途径:途径一,咖啡酰辅酶A和奎宁酸在羟基肉桂辅酶A/奎宁酸肉桂酸羟基转移酶(HQT)的催化下合成CGA;途径二,HCT先催化合成乙酰转移酶得到对香豆酰奎宁酸,然后由香豆酸-3-羟化酶(C3'H)在NADPH存在的情况下将对香豆酰奎宁酸转化为CGA;途径三,羟基化肉桂酰D-葡萄糖:奎宁酸羟基化肉桂酰转移酶(HCGQT)催化咖啡酰D-葡萄糖与奎宁酸反应生成CGA。但是,柑橘中合成CGA的途径具体是哪种,以及其合成途径的关键基因有哪些,现有技术中尚未有深入研究。
发明内容
本发明的目的是提供一种柚子中绿原酸生物合成途径基因CgHCT和CgC3'H及其应用,以解决上述现有技术存在的问题,CgHCT基因和CgC3'H基因均参与CGA的生物合成途径,促进柚子中CGA含量积累,为促进柚子产生高含量的绿原酸提供了新思路。
本发明采用基于代谢物的全基因组关联分析(Genome-Wide Association Study,GWAS)的方法,关联到与绿原酸(Chlorogenic acid,CGA)物质含量相关的酰基转移酶(hydroxycinnamoyl-CoA transferase,HCT)CgHCT基因,通过反向遗传学手段对CgHCT基因进行克隆和功能解析。植物中绿原酸的合成有三种可能的途径,其中以羟基桂皮酰辅酶A羟基桂皮酰转移酶(hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase,HQT)合成路径报道最多,发明人以NtHQT和SlHQT蛋白序列作为诱饵在柚子基因组中进行同源检索,但是没有发现同源基因,表明柚子中通过HCT途径合成对香豆酸,然后通过对香豆酸3'-羟化酶(C3'H)进行羟基化可能是柚子中形成CGA的唯一途径。重组CgHCT蛋白的体外酶促实验表明,该蛋白以奎宁酸为硫酯激活底物合成了对香豆酰奎宁酸。使用分离自酿酒酵母的微粒体进行的体外酶促分析表明,CgC3'H能够在NADPH存在的情况下将对香豆酰奎宁酸转化为CGA。CgHCT基因与CgC3'H基因参与CGA的生物合成途径,促进CGA含量积累,证实了两个基因的功能和合成途径,为柚子中产生高含量的绿原酸提供了新思路,为柚子的遗传改良提供新的基因资源。
为实现上述目的,本发明提供了如下方案:
本发明提供一种CgHCT和CgC3'H基因在促进柚子绿原酸合成中的应用,所述CgHCT基因的核苷酸序列如SEQ ID NO.1所示,所述CgC3'H基因的核苷酸序列如SEQ ID NO.3所示。
进一步地,所述CgHCT基因编码的蛋白氨基酸序列如SEQ ID NO.2所示,所述CgC3'H基因编码的蛋白氨基酸序列如SEQ ID NO.4所示。
进一步地,通过上调CgHCT蛋白和CgC3'H蛋白的表达水平,以促进柚子绿原酸的合成。
进一步地,促进所述柚子绿原酸的合成途径为:所述CgHCT蛋白以奎宁酸为底物合成对香豆酰奎宁酸,所述CgC3'H蛋白将所述香豆酰奎宁酸转化为绿原酸。
本发明还提供一种提高植物中绿原酸含量的方法,包括使受体植物中CgHCT蛋白和CgC3'H蛋白的表达量提高的步骤,所述CgHCT蛋白和CgC3'H蛋白的氨基酸序列如SEQ IDNO.2和SEQ ID NO.3所示。
进一步地,向受体植物中导入能够表达CgHCT蛋白和CgC3'H蛋白的DNA,得到转基因植物,所述转基因植物与所述受体植物相比,果实中绿原酸含量更高。
进一步地,所述能够表达CgHCT蛋白和CgC3'H蛋白的DNA为CgHCT蛋白和CgC3'H蛋白的编码基因,为SEQ ID NO.1-2所示的DNA。
进一步地,所述植物为柚子。
本发明公开了以下技术效果:
本发明基于代谢物的全基因组关联分析方法,对CGA生物合成方面存在显著差异的柑橘品种进行研究,通过物质CGA定位到HCT酰基转移酶编码基因CgHCT,再通过同源检索和共表达分析获得香豆酸3'-羟化酶编码基因CgC3'H,推测出柚子中绿原酸的合成途径为:CgHCT蛋白以奎宁酸为硫酯激活底物合成对香豆酰奎宁酸,CgC3'H在NADPH存在的情况下再将对香豆酰奎宁酸转化为CGA。并且对CgHCT蛋白和CgC3'H蛋白的功能验证,进一步证实了其在CGA合成过程中的作用,为柚子中产生高含量的绿原酸提供了新思路,并为柚子的遗传改良提供新的基因资源。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明通过GWAS筛选定位到7个候选基因的CGA含量的全基因组关联分析结果;
图2为本发明通过GWAS筛选定位到的候选基因与其他物种中已报道HCT基因的进化树分析;
图3为柚子中绿原酸代谢合成的推测途径;
图4为pGEX-6p-1载体的质粒图谱;
图5为酵母载体PYES2(ADH1)-URA的质粒图谱;
图6为CgHCT基因体外酶活结果;上图为CgHCT基因体外酶活色谱图;下图为对香豆酰奎宁酸质谱图;
图7为CgC3'H基因体外酶活结果。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1 GWAS定位CgHCT和CgC3'H
发明人对来自世界各地154份柑橘材料的1895975个SNPs,在绿原酸生物合成方面进行全基因组关联分析,利用亲缘关系(K)矩阵将种群结构建模为LMM中的随机效应,结果在1号、2号、3号和4号染色体上分别定位到了7个相关基因,分别为Chr1:Cg1g025190;Chr2:Cg2g025640;Chr3:Cg3g004610,Cg3g004640;Chr4:Cg4g024050,Cg4g024060,Cg4g024070(见图1),这几个基因序列获取途径为柑橘在线网站http://citrus.hzau.edu.cn/。为了探究候选基因的功能,将已报道的其他物种中的同源基因(“Versatility inacyltransferase activity completes chicoric acid biosynthesis in purpleconeflower”Fu et al 2021)和晚白柚基因组数据中这7个候选基因的氨基酸序列作为参考,使用MEGA5软件对7个候选糖基转移酶进行neighbor-joining系统进化分析。分析结果显示,只有Cg2g025640与先前报道的羟基肉桂酰辅酶A转移酶(HCT)蛋白聚在一起,其他基因形成独立的分支(见图2),说明候选基因Cg2g025640与这些物种中编码羟基肉桂酰辅酶A转移酶的基因功能相似,也可能催化绿原酸生物合成途径中的酰基化修饰步骤,本研究中将Cg2g025640命名为CgHCT,并从柚子基因组中对其进行基因克隆以进行功能验证。此外,值得注意的是,CGA的合成有三种可能的途径,其中HQT使用的途径已经被广泛报道,为了进一步探究HQT是否也存在于柚子基因组中,我们用NtHQT和SlHQT作为诱饵进行同源检索,但是在柚子基因组中没有发现其同源基因,这表明通过HCT合成对香豆酸,然后通过对香豆酸3'-羟化酶(C3'H)进行羟基化形成CGA可能是柚子的唯一途径。对绿原酸的化学结构分析显示,对香豆酰奎宁酸与绿原酸相比缺少一个羟基,结合文献得知这步催化反应可以由香豆酸-3-羟基化酶(Coumaric acid 3-hydroxylase,C3'H)催化完成。为了筛选绿原酸合成途径中最后一步的关键候选基因,我们使用了同源比对寻找候选基因的方法。芸香科植物芸香中合成绿原酸最后一步的关键酶RgC3'H已经报道,以其氨基酸序列(NCBI编号JF799117)为靶标,对柚子基因组进行同源基因搜索(identy values>40%,E values<1.0×10-60),最终找到与芸香中的C3'H序列一致性较高的同源基因Cg6g017470,将其命名为CgC3'H(identity values为90.35%)。图3为柚子中绿原酸代谢合成的推测途径。考虑到CgC3'H编码的蛋白是一种膜蛋白,因此将其从晚白柚基因组中克隆出来后,将选用酵母表达体系进行蛋白表达,进一步进行功能验证。
实施例2 分离克隆CgHCT和CgC3'H基因
发明人采用TRIZOL试剂(购自Invitrogen公司)从已经测序的柑橘叶片提取总RNA(提取方法根据上述TRIZOL试剂说明书),利用反转录试剂盒Supermix(购自北京全式金公司)将其反转录合成cDNA,反应条件:42℃ 30min,80℃ 5sec。以该cDNA为模板,以引物CgHCT-F(SEQ ID NO.5)和CgHCT-R(SEQ ID NO.6)扩增出CgHCT,其核苷酸序列如SEQ IDNO.1所示。
SEQ ID NO.5:5’-ttccaggggcccctgggatccATGATAATCAACCTGAAGGAATCGA-3’;
SEQ ID NO.6:5’-cagtcagtcacgatgcggccgcGATGAGTTTCAGTGGTGGTTTTCTAAA-3’;
SEQ ID NO.1:
>CgHCT-Cg2g025640.1-CDS(1bp-1293bp,direct)1293bp
ATGATAATCAACCTGAAGGAATCGACAATGGTGCGGCCGGCGGCGGAGACGCCGAGGGTGGCGCTGTGGAACGCGAACGTCGACCTGGTGGTGCCGCGGTTCCACACGCCCAGCGTCTACTTCTACCGGCCGACCGGCGCCGCCAACTTCTTCGACGCCGGGGTTCTCAAGGACGCACTGAGCAAGGCGCTGGTGCCGTTCTATCCGATGGCCGGCCGGCTGAAGCGGGACGACGACGGCCGTATCGAGATTGACTGCAACGCCGAGGGCGTCTTGCTCGTCGAGGCCGAAACGACGTCGTTGATCGATGATTTCGGAGACTTTGCTCCCACTCTGGAGCTGAAGCAGCTCATCCCAACGATCGACTACTCTGGTGGAATATCAACTTATCCCCTCTTAGTTTTGCAGATCACTCACTTCAAATGCGGTGGGGTCTCACTTGGTGTGGGTATGCAGCATCATGTGGCCGATGGATTTTCTGGTCTCCACTTTGTGAACACATGGTCAGATTTTGCTCGTGGTCTTGATCTTACCATCCCACCATTCATTGATCGAACTTTGCTTCGCTCCCGAGATCCACCTCAACCTGCATTTGAACATATTGAATACCAGCCTCCCCCTCCATTGAAACATCCTCTGAAATCAAGTTCTGAGACTACAGCTGTCTCAATTTTCAAATTAACCAGGGACCAACTCAACATCCTCAAAGCCAAGGCCAAAGAAGATGGTAACACTATCAATTATAGCTCATATGAGATGTTGGCGGGCCATGTGTGGAGGTCTGCATGCATCGCACGGGGGCTTACTGCTGATCAAGAGACCAAATTGTACATTGCAACAGATGGACGTGCTAGATTGCGGCCTCCTCTTCCACCTGGTTACTTTGGGAACGTGATCTTTACAGCTACACCAATGGCAGTCACGGGTGATCTCCAATCAAAGCCAATATGGTACGCTGCAAGTCGGATCCACGATGCATTGGTTCGGATGGACAACGACTATCTAAGGTCAGCTCTTGATTACTTAGAGCTTCAGCCTGATTTATCAGCTTTGGTTCGTGGTGCCCATACATTTAGGTGTCCAAATCTCGGGATTACTAGTTGGGTTAGGTTGCCAATACATGACGCTGATTTCGGGTGGGGTCGTCCCATTTTTATGGGACCTGGTGGGATTGCATATGAGGGCTTATCATTTATAATCCCAAGCCCAACCAATGATGGAAGCTTATCAGTTGCCATCTCTCTGCAAACTGAACACATGAAACTATTTGAGAAGTTAATATATGACATTTAG。
其编码的氨基酸序列如SEQ ID NO.2所示:
MIINLKESTMVRPAAETPRVALWNANVDLVVPRFHTPSVYFYRPTGAANFFDAGVLKDALSKALVPFYPMAGRLKRDDDGRIEIDCNAEGVLLVEAETTSLIDDFGDFAPTLELKQLIPTIDYSGGISTYPLLVLQITHFKCGGVSLGVGMQHHVADGFSGLHFVNTWSDFARGLDLTIPPFIDRTLLRSRDPPQPAFEHIEYQPPPPLKHPLKSSSETTAVSIFKLTRDQLNILKAKAKEDGNTINYSSYEMLAGHVWRSACIARGLTADQETKLYIATDGRARLRPPLPPGYFGNVIFTATPMAVTGDLQSKPIWYAASRIHDALVRMDNDYLRSALDYLELQPDLSALVRGAHTFRCPNLGITSWVRLPIHDADFGWGRPIFMGPGGIAYEGLSFIIPSPTNDGSLSVAISLQTEHMKLFEKLIYDI*。
以引物CgC3'H-F(SEQ ID NO.7)和CgC3'H-R(SEQ ID NO.8)扩增出CgC3'H,其核苷酸序列如SEQ ID NO.3所示。
SEQ ID NO.7:5’-ATACAATCAACTATCTCATATACAATGACTCTCCCACTCATCCCATTATCA-3’;
SEQ ID NO.8:5’-TACATGATGCGGCCCCTATCACATATCAGCGGCCACACGTTTA-3’;
SEQ ID NO.3:
>CgC3'H-Cg6g017470.1(1bp-1527bp,direct)1527bp
ATGACTCTCCCACTCATCCCATTATCAATCATTTTCATCATCCTTGCATACAAGCTCTACCAACGGCTGAGATTCAAGCTCCCGCCCGGCCCTCGTCCCTTGCCAATCGTCGGAAACCTCTACGACATAAAGCCGGTGAGGTTCCGGTGCTTTGCGGAATGGGCTCAACAATACGGACCAATCATTTCGGTTTGGTTCGGCTCGACTTTGAACGTGATTGTGTCCAACACAGAATTGGCTAGAGAAGTGCTTAAAGAGCATGATCAACAATTGGCCGACAGGCATCGAAGTAGATCAGCTGCAAAGTTCAGCAGAGATGGCAAGGACCTCATTTGGGCCGATTATGGGCCTCACTACGTCAAGGTTCGTAAAGTCTGCACGCTCGAGCTTTTTACGCCAAAGAGATTACAGGCTCTGAGACCAATTAGAGAGGATGAAGTCACAGCCATGGTTGAATCCATTTTCAAAGACTGCACCGATCCTCAAAATTATGGGAAGAGTGTACTAGTGAAGAAGTATTTGGGAGCAGTGGCATTCAACAACATTACAAGGCTAGCGTTTGGGAAGAGATTCGTGAATTCAGAAGATGTGATGGACGAGCAAGGGAAAGAATTCAAGGCAATAGTAGCTAATGGGCTGAAGCTTGGGGCATCGCTAGCTATGGCTGAGCACATTCCATGGCTTCGTTGGATGTTCCCATTAGAGGAAGGGGCATTTGCCAAGCACGGCGAACGCCGAGACCGTCTTACTCGAGCTATCATGGAAGAGCACACACTTGCTCGCCAGAAGAGCGGTGGTACCAAGCAACACTTTGTTGATGCTTTGCTTACACTGCAAGAAAAATATGACCTAAGTGAAGACACTATCATTGGACTCCTCTGGGACATGATCACAGCGGGCATGGACACAACTGCAATCTCAACAGAATGGGGAATGGCCGAGCTCATTAGGAACCCTAGAGTGCAACAAAAGGCTCAAGAGGAACTAGACCGTGTGATAGGATTTGAACGTGTGATGATGGAAACCGATTTCTCAAACCTTCCTTACTTACAAGCTGTAGCCAAGGAGGCTCTAAGGTTGCACCCACCAACTCCCCTGATGCTTCCTCACCGTGCCAATGCCAATGTTAAGATAGGTGGCTACGATGTTCCTAAGGGATCAAACATTCACGTCAACGTATGGGCAGTAGCTCGGGATCCTGCAGTCTGGAAGGACCCGTTAGAGTTCCGGCCTGAGCGGTTCTTCGAGGAGGATGTGGACATGAAAGGCCATGATTTTAGGCTACTACCATTTGGTGCTGGTAGGAGAGTGTGCCCAGGTGCACAACTTGGTATAAATTTGGTCACGTCAATGTTGGGGCATCTATTACACCATTTTGCTTGGGCGCCGCCAGAGGGAGTGAAGCCAGAGGAAATTGACATGTCAGAAAATCCTGGATTGGTTACATATATGAAGACACCAGTACAGGCTGTGCCAACTCCTAGGCTGCCTTCGCACTTGTATAAACGTGTGGCCGCTGATATGTGA。
其编码的氨基酸序列如SEQ ID NO.4所示:
MTLPLIPLSIIFIILAYKLYQRLRFKLPPGPRPLPIVGNLYDIKPVRFRCFAEWAQQYGPIISVWFGSTLNVIVSNTELAREVLKEHDQQLADRHRSRSAAKFSRDGKDLIWADYGPHYVKVRKVCTLELFTPKRLQALRPIREDEVTAMVESIFKDCTDPQNYGKSVLVKKYLGAVAFNNITRLAFGKRFVNSEDVMDEQGKEFKAIVANGLKLGASLAMAEHIPWLRWMFPLEEGAFAKHGERRDRLTRAIMEEHTLARQKSGGTKQHFVDALLTLQEKYDLSEDTIIGLLWDMITAGMDTTAISTEWGMAELIRNPRVQQKAQEELDRVIGFERVMMETDFSNLPYLQAVAKEALRLHPPTPLMLPHRANANVKIGGYDVPKGSNIHVNVWAVARDPAVWKDPLEFRPERFFEEDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMLGHLLHHFAWAPPEGVKPEEIDMSENPGLVTYMKTPVQAVPTPRLPSHLYKRVAADM*。
PCR反应体系:10ul(Template 0.5ul,Buffer 5ul,dNTP 2ul,DMSO 0.2ul,Enzyme0.2ul,Primer Pairs 0.6ul,ddH2O补足至10ul);PCR反应条件:95℃预变性2min;94℃10sec,60℃ 30sec,72℃ 2min,35个循环;72℃延伸5min。
实施例3 CgHCT和CgC3'H基因载体的构建与蛋白表达
大肠杆菌表达载体的构建方法如下:首先将实施例2中扩增获得的目的基因的cDNA序列通过同源重组克隆技术连入pGEX-6p-1载体(见图4),筛选阳性克隆并测序确认,发明人将此克隆命名为pGEX-6p-1-CgHCT。阳性克隆pGEX-6p-1-CgHCT质粒测序正确后用于大肠杆菌原核蛋白表达并进行纯化。
酵母载体构建方法如下:首先将实施例2中扩增获得的PCR产物通过同源重组克隆技术连入酵母表达载体PYES2(ADH1)-URA(见图5),发明人将此克隆命名为PYES2(ADH1)-URA-CgC3'H。阳性克隆PYES2(ADH1)-URA-CgC3’H质粒测序正确后用于蛋白表达,即通过酵母菌株表达CgC3'H基因的蛋白。具体步骤如下:1)先在含2%葡萄糖的泛基诺SD/-Ura(备注:SD/-Ura=Sc-Ura)培养基(货号YGM003A-3)中30℃,280rpm/min震荡培养至OD600大约1.2-1.4;2)1000转离心5min,弃上清;3)用200-300mL泛基诺Sc-Ura液体培养基悬浮细胞,充分混匀,离心弃上清;4)加500mL Sc-Ura培养基,继续震荡培养3h,使得细胞内的葡萄糖耗竭;5)离心弃上清,换成新的500mL Sc-Ura培养基继续震荡培养8-12h;6)1L酵母Cell离心后用TEK buffer{50mM Tris-HCl(pH=7.5),1mM EDTA-2Na,0.1M KCl}洗一次(30mL-50mL TEK buffer/样品);7)磷酸Buffer A{0.1M磷酸钾a potassium phosphate(pH=7.4),1M Sorbitol,14mMβ-巯基乙醇(按照1mL/L加,如20uL加20mL中)}洗2-3次(20mLBuffer A/样品),用6000rpm,4℃,离心5min,将细胞悬于Buffer B(Buffer A+1mM PMSF)+1体积玻璃珠,30Hz(振2min,冰上放置2min,重复10次);(40个1.5mL管/500uL buffer B=20mL);8)取上清至新的离心管中,玻璃珠用Buffer B洗3次,合并上清;9)离心,12000g10min,4℃;10)将离心后的上清于140,000g,90min,4℃,去掉上清,沉淀用Buffer C{0.1MPotassium phosphate(Ph=7.4),0.4M sucrose,0.5mM glutathione}漂洗后用Buffer D(Buffer C+10%甘油)保存于-70℃。
酵母蛋白表达中所用试剂的配制:
(1)主要培养基配方:
泛基诺SD/-Ura培养基:YNB 6.7g,Ura缺素剂0.62g,950mL的ddH2O,50mL的2%葡萄糖,加入1M KOH,调节pH为5.8。
(2)主要试剂母液配方:
1)Tris-Hcl(20X)母液
1M Tris-Hcl称60.57g Tris溶于300mL灭菌ddH2O水中后用浓盐酸调pH=7.5后,用灭菌ddH2O定容到500mL即可。
2)2X磷酸钾母液
每L KH2PO4母液中含有27.2g KH2PO4,即浓度为0.2mol/L的母液;
每L K2HPO4母液中含有45.644g K2HPO4-3H2O,即浓度为0.2mol/L的母液。
(3)主要试剂工作液配方:
1)TEK工作液:
取25mL的Tris-HCl(20X)母液+3.728g KCl+0.18612g EDTA-2Na盐后加水定容至500mL即可。(稀释后的工作液pH值也需要进行微调)。
2)3L磷酸钾工作液:分别用量筒取405mL K2HPO4母液和95mL KH2PO4母液,加灭菌ddH2O至1L定容即可(配3瓶)。
3)Buffer A:1000mL磷酸钾工作液+182.17g Sorbitol+1mL巯基乙醇(使用前单独加)。
4)Buffer B:1L磷酸钾工作液+182.17g Sorbitol+0.17419g PMSF+1mL巯基乙醇(使用前单独加)。
5)Buffer C:500mL磷酸钾工作液+68.46g Sucrose+0.07683g glutathione。
6)Buffer D:取80mL buffer C+20mL甘油。
实施例4 CgHCT和CgC3'H的体外酶活验证
1.CgHCT蛋白的体外酶活测定
为了验证候选基因CgHCT的功能,发明人使用上述表达的CgHCT蛋白进行体外酶活测定。CgHCT蛋白体外酶活反应体系是20μL,100mM Na-Pi缓冲液pH7.0,1mM EDTA,p-coumaryl-CoA(购自YuanYe,http://www.shyuanye.com/index.htmL)50μL,奎宁酸1mM,纯化酶500ng。加入酶启动反应,37℃孵育30min,加入20μL冰冷甲醇终止反应。用空PYES2(ADH1)-URA重组酵母转化的微粒体制剂进行阴性对照反应。用NanoDrop ND-1000分光光度计测定280nm紫外吸光度测定总蛋白含量。所有反应混合物通过0.2μm过滤器(Millipore)过滤后用于液相色谱-质谱(LC-MS)分析。检测结果见图6,图6中上图为CgHCT基因体外酶活色谱图,上图中从上到下依次为奎宁酸标准品色谱图,实验组色谱图,对照组色谱图;下图为对香豆酰奎宁酸质谱图。结果表明该蛋白以奎宁酸为硫酯激活底物,并与预测结果一致合成了对香豆酰奎宁酸,产物经保留时间、裂解模式和紫外光谱证实CgHCT催化合成了对香豆酰奎宁酸。
2.CgC3'H蛋白的体外酶活测定
为了验证候选基因CgC3'H的功能,发明人使用上述表达的CgC3'H蛋白进行体外酶活测定。加入500μL 100mM Tris-HCl,含1mg总微粒体蛋白,500mM NADPH和200μM底物,在摇床中(120rpm,30℃)反应4h。加入500μL甲醇和涡旋可使反应停止。用空PYES2(ADH1)-URA重组酵母转化的微粒体制剂进行阴性对照反应。用NanoDrop ND-1000分光光度计测定280nm紫外吸光度测定总蛋白含量。所有反应混合物通过0.2μm过滤器(Millipore)过滤后用于LC-MS分析。检测结果见图7,图中从上到下依次为对香豆酰奎宁酸标准品色谱图,实验组色谱图,对照组色谱图,CGA标准品色谱图;结果表明反应产物出现明显的色谱峰并且其保留时间和裂解模式与化合物CGA标准品一致,说明CgC3'H能够在NADPH存在的情况下将对香豆酰奎宁酸转化为CGA。
上述1-2的LC-MS分析参数均如下:
(1)负离子模式:
柱子:C18,M/Z range 100-1200;
流动相A:去离子水,含有0.04%醋酸;
流动相B:乙腈,含有0.04%醋酸;
(2)质谱碎片离子对信息:
香豆酰奎宁酸:母离子(Q1):337,子离子(Q3):191、144、273;
绿原酸:母离子(Q1):353,子离子(Q3):191。
综合上述可知,本研究发现柚子中通过HCT途径合成对香豆酸,然后通过对香豆酸3'-羟化酶(C3'H)进行羟基化可能是柚子中形成CGA的唯一途径,并经体外酶促实验证实,CgHCT蛋白以奎宁酸为硫酯激活底物合成了对香豆酰奎宁酸,CgC3'H在NADPH存在的情况下再将对香豆酰奎宁酸转化为CGA,表明CgHCT基因与CgC3'H基因参与CGA的生物合成途径,可促进柚子中CGA含量积累,为柚子的遗传改良提供新的基因资源。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (1)
1.CgHCT和CgC3'H基因在促进柚子绿原酸合成中的应用,其特征在于,所述CgHCT基因的核苷酸序列如SEQ ID NO.1所示,所述CgC3'H基因的核苷酸序列如SEQ ID NO.3所示;
所述CgHCT基因编码的蛋白氨基酸序列如SEQ ID NO.2所示,所述CgC3'H基因编码的蛋白氨基酸序列如SEQ ID NO.4所示;
促进所述柚子绿原酸的合成途径为:CgHCT蛋白以奎宁酸为底物合成对香豆酰奎宁酸,CgC3'H蛋白在NADPH存在的情况下将所述香豆酰奎宁酸转化为绿原酸。
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