CN104894146A - 一种δ12-脂肪酸脱氢酶基因的新用途 - Google Patents
一种δ12-脂肪酸脱氢酶基因的新用途 Download PDFInfo
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Abstract
一种Δ12-脂肪酸脱氢酶基因的新用途。本发明公开了一种从圆红冬孢酵母YM25235中分离的编码Δ12-脂肪酸脱氢酶基因序列的新用途，即应用在生产多不饱和脂肪酸中，基因编码产物具有Δ15-脂肪酸脱氢酶的活性，能催化亚油酸转化成α-亚麻酸。
Description
技术领域
本发明属于生物技术领域和遗传工程领域，涉及一种从圆红冬孢酵母YM25235分离的Δ12-脂肪酸脱氢酶基因序列（GenBank登录号：KJ502671.1）在生产α-亚麻酸中的应用。
背景技术
多不饱和脂肪酸（Polyunsaturated fatty acids，PUFAs）是指含有两个或两个以上双键、碳原子数为18～22的直链脂肪酸。很多研究表明，诸如花生四烯酸AA（Arachidonic acid ARA，20:4n-6）、二十碳五烯酸EPA（Eicosapentaenoic acid， C20:5n-3）和二十二碳六烯酸DHA（Docosahexaenoic acid，22:6n-3）等长链多不饱和脂肪酸是细胞膜的重要组成成分，同时也是动物和人体中激素类（Hormone-like）生物活性物质的前体，在细胞膜的结构和功能调节、抗肿瘤抗炎、促进细胞生长、防止心脑血管疾病、有利于大脑视网膜形成、免疫调节和基因表达等很多重要的生理调节功能。
PUFAs广泛存在于各种生物中，其合成是从18C的饱和脂肪酸合成途径上扩展的，在真核生物中饱和的硬脂酸（Stearic acid）经过Δ9-脂肪酸脱氢酶的催化转化成油酸（Oleic acid，OA），然后经Δ12-脂肪酸脱氢酶的催化进一步转化成亚油酸（Linoleic acid，LA），后者进一步进入n-3和n-6途径在不同脂肪酸脱氢酶（Fatty acid desaturease）和延长酶（Elongase）交替催化下，合成各种不同的PUFAs，因此Δ12-脂肪酸脱氢酶催化合成LA是PUFAs合成关键步骤。
Δ12-脂肪酸脱氢酶(Δ12-fatty acid desaturases)又称油酸脱氢酶(oleate desaturases)属于一类膜整合酶，由于分离和鉴定膜结合蛋白的难度很大，因此很难得到包括Δ12-脂肪酸脱氢酶在内的各种膜结合蛋白性质脱氢酶高级机构，而只能通过对酶的核苷酸序列进行初步研究，或者在异源的受体内表达来研究酶的活性。序列对比的结果表明，编码Δ12-脂肪酸脱氢酶的核苷酸序列具有共同的机构特性：存在三个组氨酸保守区，形成四次跨膜的结构，这些都是维持脱氢酶所必须的。
自首次从耐冷的蓝细菌（Synechocystis sp.）PCC6803中克隆到Δ12-脂肪酸脱氢酶基因以来，已经从不同植物、动物和微生物中分离到很多类似的功能基因序列，功能分析结果表明Δ12-脂肪酸脱氢酶主要功能是在含有1个双键油酸（Δ9）的第12和13位碳原子之间插入一个双键，形成含有2个双键的亚油酸（Δ9, 12）。但是，近年来对一些真菌和原生动物来源的Δ12-脂肪酸脱氢酶体外功能验证结果表明，该酶具有多功能特点。比如线虫（Caenorhabditis elegans）Δ12-脂肪酸脱氢酶CeFAT-2具有双功能的Δ12/Δ15-脂肪酸脱氢酶活性，其可以催化油酸同时形成亚油酸和α-亚麻酸；高山被孢霉（Mortierella alpina）IS-4中的ω3-脂肪酸脱氢酶（MAW3）也被证明具有三功能的Δ12/Δ15/ω3-脂肪酸脱氢酶活性。这些表明，某些生物可以利用同一个脱氢酶就可以合成多种PUFAs，属于适应性进化。
目前由于PUFAs在人类健康中的重要作用，其市场需求也迅速增长。尽管有些PUFAs的生产已经产业化，还有许多也在加紧开发利用之中，但是除了LA外，现有来源在产量和质量上都不能满足日益增长的市场需求。因此，从不同生物中克隆PUFAs合成相关的脂肪酸脱氢酶基因，再与不同表达载体连接后转入不同的宿主细胞进行表达，利用宿主产生各种特定PUFAs具有极大应用前景。发掘PUFAs合成的高效、特异性新基因，也为新一代转基因脂肪酸产品的品质改良奠定基础。
发明内容
本发明的目的是提供一种Δ12-脂肪酸脱氢酶基因的新用途，即利用圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶基因生产α-亚麻酸的应用，该基因在GenBank登录号为KJ502671.1。
可用本领域的技术人员熟知的方法来构建含编码圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶的核苷酸序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等[Sambroook,et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989]。所述的编码圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶的核苷酸序列可有效连接到表达载体的恰当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体的PL启动子；真核启动子包括CMV早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。增强子是DNA表达的顺式作用因子，通常大约有10-300bp，作用于启动子以增强基因的转录。如腺病毒增强子。
本发明提供一种含有Δ12-脂肪酸脱氢酶基因或上述重组表达载体的宿主细胞，该细胞是细菌细胞、真菌细胞、植物细胞或动物细胞，或这些宿主细胞的后代，宿主细胞的代表性例子有：大肠杆菌；真菌细胞或酵母；植物细胞如油菜、烟草、大豆；昆虫细胞如果蝇S2或Sf9；动物细胞如CHO、COS或Bowes黑素瘤细胞等。
本发明所述应用中还包括将圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶基因与载体连接后转化至微生物细胞体内生产α-亚麻酸和其他多不饱和脂肪酸，具有产物特异性高、生产周期短、生产不受场地、气候、季节的影响及利用不同的菌种和培养基适合开发功能油脂等优点，可以用于商业化生产，直接改善油脂品质，更加具有实际应用价值。本发明应用基因工程技术构建特异性生产多不饱和脂肪酸的转基因酵母细胞生产不饱和脂肪酸，具有操作简单、成本低、可行性高等优点，为新一代转基因脂肪酸产品的品质改良奠定基础。
附图说明
图1为本发明所构建的酿酒酵母表达载体pY3RKD12；
图2为本发明含pYES3/CT空载体的转基因酵母的气相色谱图；
图3为本发明含重组质粒pY3RKD12的转基因酵母的气相色谱图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中使用的试剂和方法，如无特殊说明，均采用常规试剂和使用常规方法。
实施例1：参考酵母菌一些种已知和潜在Δ12-脂肪酸脱氢酶基因序列，设计特异性引物（引物1和引物2），以cDNA为模板进行PCR反应，引物序列如下：
引物1：RKD12F:5`-ATAAGCTTATGGCCGCTACCCTCCGCCAGC-3`（SEQ ID NO:1）
引物2：RKD12R:5`-ATGAATTCCTAGAGTCCCTCGACGCCCGAGT-3`（SEQ ID NO:2）
先用真核细胞总RNA提取试剂盒（Promega公司产品）试剂盒提取细胞的总RNA，利用反转录试剂盒（fermentas公司产品）反转录合成第一条cDNA，然后以其为模板进行PCR反应，PCR扩增体系（25 μL）组成如下：
10×Ex Taq Buffer 2.5 μL
dNTP（2.5 μmol/L） 2 μL
模板 3 μL
P1（10 μmol/L） 2 μL
R1（10 μmol/L） 2 μL
Ex Taq DNA polymerase（5U/μL） 2 μL
无菌ddH2O 补足至25 μL
扩增条件：94℃变性4min，再用94℃ 1min、55℃ 1min、72℃ 2min进行30个循环，最后72℃ 10min；PCR扩增产物采用上述相同的方法鉴定、测序，用DNAMAN软件进行序列分析，结果显示得到了一段1341bp长的序列，如GenBank登录号为KJ502671.1中所示的核苷酸序列。
实施例2：酿酒酵母重组表达载体的构建
根据实施例1所得序列的编码区序列，设计出一对基因特异性扩增引物（引物3和引物4）分离其潜在的开放阅读框序列：
引物3：RKD12F:5`-ATAAGCTTATGGCCGCTACCCTCCGCCAGC-3`
引物4：RKD12R:5`-ATGAATTCCTAGAGTCCCTCGACGCCCGAGT-3`
此两个引物的5‵端下划线部分分别含有EcoRⅠ和Hind Ⅲ酶切位点，所用的扩增条件和反应组分和以cDNA为模板的PCR反应相同，扩增产物的测序结果显示和GenBank登录号为KJ502671.1中所示1-1341bp的序列一致；然后取25μl PCR产物和10ul pYES3/CT（Invitrogen公司）分别进行双酶切，电泳回收酶切大片段，并用T4连接酶在16℃连接4h，连接产物转化大肠杆菌HD5α，通过质粒提取和PCR筛选阳性克隆，并进行测序鉴定，所构建的含有Δ12-脂肪酸脱氢酶基因的酵母表达命名为pY3RKD12。
实施例3：酿酒酵母重组表达载体pY3RKD12转化酿酒酵母细胞
挑取酿酒酵母菌株INVSC1单菌落于5ml YEPD液体培养基中，30℃摇床过夜培养，按1%的接种量转接于100ml的YEPD液体培养基中，30℃摇床摇至OD600于1.3-1.5之间；4℃、4000g离心5min收集沉淀细胞，用100ml预冷的无菌ddH2O洗涤细胞，4℃、4000g离心5min收集沉淀细胞，再用50ml预冷的无菌ddH2O洗涤细胞，4℃、4000g离心5min收集沉淀细胞，用20ml预冷的1M山梨醇洗涤细胞，4℃、4000g离心5min收集沉淀细胞，细胞用0.5ml的预冷的1M山梨醇悬浮，每80μl分装于1.5ml预冷的离心管中，取2μg重组表达载体pY3RKD12（附图1）和空载体pYES3/CT分别加入到80ul的感受态细胞中，混匀后转至0.2cm的电转杯中，冰上孵育5min，转入电转仪中电击（电击条件：电压1.5kV，电容25μF，电阻200Ω，电击时间为4-10ms），电击完毕后立即加入1ml 1M预冷的山梨醇并吹匀，30℃孵育2h后分别涂布于SC-Trp（无色氨酸）选择培养基上，置30℃培养3-4天。
实施例4： 酵母工程菌的诱导表达与脂肪酸甲酯的气相色谱分析
挑取分别转化了重组表达载体pY3RKD12和空载体pYES3/CT并在SC-Trp选择培养基（色氨酸合成缺陷型培养基）平板上出现的阳性转化子，分别接种于15ml 液体SC-Trp选择培养基（含2%葡萄糖），30℃摇菌培养24h，转接于100ml的SC-Trp选择培养基（含2%的半乳糖和1%的棉子糖）中，使其初始OD600为0.4，同时添加0.5mM外源性的油酸作为底物，30℃继续培养72h，4000g离心收集菌体，用去离子水洗涤三次，菌体于50℃烘干，研碎，取100mg干菌体粉末加入5ml 5%的KOH-CH3OH溶液中，70℃反应3h，反应结束后冷却至室温，用6M的盐酸调节溶液的pH值到2.0，加入4ml 14%BF3-CH3OH，70℃反应1.5h，合成脂肪酸甲酯；再加入10ml饱和的NaCl溶液，剧烈震荡混匀，转移至分液漏斗中，用8ml 1:4的氯仿：环己烷抽提两次，合并两次的提取液，把含有脂肪酸甲酯的溶剂用氮气吹干，用200μl的正己烷回溶样品，最后用0.45μm微孔滤膜过滤除去大颗粒杂质。
采用气相色谱分析仪器分析转基因酵母工程菌的诱导表达情况，气相色谱分析仪器为岛津GC-7A，柱子：弹性石英毛细管柱，0.32×30m，固相支持物为聚二乙二醇丁二酸酯镀膜物：聚酰亚胺。载气：N2，线速10cm/s。分流比：100:1，气化室温度：250℃，柱温：180℃，尾吹：50ml/min，检测器：氢火焰离子化检测器，把上述制备的脂肪酸甲酯化样品进行GC分析，上样量为1μl；分析软件：Anstar。
色谱分析结果见图2和图3，图2显示含pYES3/CT空载体的转基因酵母，图3显示含重组质粒pY3RKD12的转基因酵母；与图2相比，图3中出现保留时间为16.65min对应的新峰为α-亚麻酸甲酯，与α-亚麻酸甲酯标准品一致，说明本发明的Δ12-脂肪酸脱氢酶也具有催化亚油酸生产α-亚麻酸的能力，具有Δ15-脂肪酸脱氢酶活性，可以将其应用于生产α-亚麻酸等多不饱和脂肪酸上。
序列表
<110> 昆明理工大学
<120> 一种Δ12-脂肪酸脱氢酶基因的新用途
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> 人工序列
<400> 1
ataagcttat ggccgctacc ctccgccagc 30
<210> 2
<211> 31
<212> DNA
<213> 人工序列
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atgaattcct agagtccctc gacgcccgag t 31
Claims (1)
1.Δ12-脂肪酸脱氢酶基因在生产α-亚麻酸中的应用，该基因的GenBank登录号为KJ502671.1。
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