CN103525842B - 一种δ12-脂肪酸脱氢酶基因及其重组表达载体 - Google Patents
一种δ12-脂肪酸脱氢酶基因及其重组表达载体 Download PDFInfo
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- CN103525842B CN103525842B CN201310494126.4A CN201310494126A CN103525842B CN 103525842 B CN103525842 B CN 103525842B CN 201310494126 A CN201310494126 A CN 201310494126A CN 103525842 B CN103525842 B CN 103525842B
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Abstract
本发明公开了一种从圆红冬孢酵母YM25235中分离的编码Δ12-脂肪酸脱氢酶的核苷酸序列，其核苷酸序列如SEQ ID NO：1所示，该基因编码的氨基酸序列如SEQ ID NO：2所示，通过构建重组载体并在酿酒酵母中表达，表达产物具有Δ12-脂肪酸脱氢酶功能，能催化油酸转化成亚油酸。
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Δ12-脂肪酸脱氢酶基因及其重组表达载体，具体涉及从酵母---圆红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中克隆的Δ12-脂肪酸脱氢酶基因及将该基因直接与不同载体连接，转入细菌、酵母、植物或动物中，利用其编码Δ12-脂肪酸脱氢酶产生不饱和脂肪酸。
背景技术
Δ12-脂肪酸脱氢酶(Δ12-fatty acid desaturases)又称油酸脱氢酶(oleate desaturases)。Δ12-脂肪酸脱氢酶属于一类膜整合酶，由于分离和鉴定膜结合蛋白的难度很大[MaKeon，1981，酶学方法，12141-12147；wang等，1988，植物生理学生物化学，26:777-792]，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底物特异性。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[Kyte et al，1982，J.Mol.Biol.157:105-132]，这些都是维持脱氢酶所必须的。
Δ12-脂肪酸脱氢酶的主要作用是在单不饱和脂肪酸油酸(oleic acid)的第12和13位碳原子之间插入一个双键，形成含有2个双键的多不饱和脂肪酸-亚油酸(linoleic acid)。Δ12-脂肪酸脱氢酶是油酸脱氢形成亚油酸的关键酶，是多不饱和脂肪酸(polyunsaturated fatty acid)代谢ω-3、ω-6途径的限速酶，控制着植物细胞中大多数不饱和脂肪的合成，只存在于植物和微生物中，人和哺乳动物细胞中缺乏在脂肪酸的第9位碳原子以上位置引入不饱和双键的脱氢酶，这些多不饱和脂肪酸必须从食物中获取。因此，亚油酸和α-亚油酸是人体必须脂肪酸，膳食性摄入亚油酸和α-亚油酸，经Δ6-脂肪酸脱氢酶催化转化成γ-亚油酸和十八碳四烯酸，它们又在其它相关酶的催化下可进一步转化成其它长链多不饱和脂肪酸[Horrobin DF，1992，Prog Lipid Res，31:163-194]。这些长链多不饱和脂肪酸是有机体组织生物膜组成成分，起到维持细胞正常功能和增加机体抗逆性的作用[Napier JA et al，1999，Curr.Opin.Plant Biol，2:123-127]，因此包括亚油酸在内的多不饱和脂肪酸在保健和医疗方面有很广阔的应用前景。研究不饱和脂肪酸的合成和它的生理作用成为当前的热点。
对于脂肪酸脱氢酶的生物学功能，许多实验室采用了从各种生物体中克隆相关的基因并转入各种受体中，在受体中观察受体的脂肪酸改变的方法。目前，已从动物、植物、微生物等不同来源中分离到许多Δ12-脂肪酸脱氢酶基因的同源序列，并且证明具有相应的生物学功能。尤其是近几年来随着双功能Δ12-脂肪酸脱氢酶的发现[Edgra B. Cahoon et al，2001，THE JOURNAL OF BIOLIGICAL CHEMISTRY，26：2637-2643.]和共轭油酸在医疗保健功能的发掘，人们越来越认识到不饱和脂肪酸的重要性，并且常规的生物化学方法很难分离纯化膜结合的Δ12-脂肪酸脱氢酶，研究其在自然环境中的生化特性比较困难，因此人们的注意力又开始转向对Δ12-脂肪酸脱氢酶的研究上。
目前，不饱和脂肪酸主要来源于动物、植物和低等生物等，与动植、物油生产PUFAs相比，微生物油脂的生产有很多优点，如生产周期短、微生物方法生产不受场地、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。目前分离到的脱氢酶和延伸酶效率较低且大多数对脂肪酸底物链长、双键数目及双键位置不具有严格的专一性，除目标产物外，还会生产一些非特异脂肪酸。由于这些脂肪酸的作用还不清楚，所以它们的出现不利于转基因植物的商业化应用，因此需要继续筛选具有高的底物特异性的脱氢酶和延伸酶。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油料植物，直接改善油脂品质，更加具有实际应用价值。本研究通过多种手段发掘多不饱和脂肪酸合成的高效、特异性新基因，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，这是从圆红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，该基因核苷酸序列如SEQ ID NO：1所示或该核苷酸序列的片段，或与SEQ ID NO：1互补的核苷酸序列，该基因序列长为1341bp（碱基），其中1-1341为编码Δ12-脂肪酸脱氢酶成熟多肽的开放阅读框；该基因编码的氨基酸序列如SEQ ID NO：2所示的多肽或其片段。
本发明另一目的是提供一种含有Δ12-脂肪酸脱氢酶基因的重组表达载体，是将SEQ ID NO：1所示基因直接与不同表达载体（质粒、病毒或运载体）连接所构建的重组载体。
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。
本发明另一目的是提供一种含有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。
用本发明所述的核苷酸序列或含有核苷酸序列的重组载体优化宿主细胞可用本领域的技术人员熟知的方法进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期手机菌体，用CaCl2、电穿孔等方法进行。当宿主是真核生物，可选用DNA转染法。显微注射、电穿孔、脂质体包装等方法。
本发明的核苷酸序列是DNA形式的，DNA形式包括cDNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的，编码成熟多肽的编码区序列可以与SEQ ID NO:2所示的编码区序列相同或是简并的变异体。
本发明还包括编码圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶的核苷酸序列的片段。如本发明所用，术语“片段”是指能编码基本上保持本发明天然的相同的生物学功能或活性的多肽的核苷酸序列。通过本文的阐述，这样的片段、被认为在本领域技术人员的知识范围之内。
本发明中的多肽和核苷酸序列优选以分离的形式提供，更佳地被纯化至均质。
本发明中特异核苷酸序列能用多种方法获得。例如，用本领域熟知的杂交技术分离核苷酸序列。这些技术包括但不局限于：（1）用探针与基因组或cDNA文库杂交以检出同源的核苷酸序列；和（2）表达文库的抗性筛选以检出具有共同结构特征的克隆的核苷酸序列片段。
本发明的DNA片段序列也能用下列方法获得：（1）从基因组DNA分离双链DNA序列；（2）化学合成DNA序列以获得所述多肽的双链DNA。
上述提到的方法中，分离基因组DNA最不常用。DNA序列的直接化学合成法是经常选用的方法。更经常选用的方法是DNA序列的分离。分离感兴趣的cDNA的标准方法是从高表达该基因的供体细胞分离mRNA并进行转录，形成质粒或噬菌体cDNA文库。提取mRNA的方法已有多种成熟的技术，用试剂盒也可从商业途径获得。而构建cDNA文库也是通常的方法。当结合聚合酶链式反应技术（PCR）时，即使极少的表达产物也能克隆。
本发明提供的核苷酸序列是一种高效的特异性的脂肪酸脱氢酶，可以将其与载体连接后转化至微生物细胞体内生产不饱和脂肪酸，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为本发明的圆红冬孢酵母YM25235 Δ12-脂肪酸脱氢酶和少根根霉Δ12-脂肪酸脱氢酶的氨基酸序列同源性比较图；
图2为本发明所构建的酿酒酵母表达载体pY3RKD12；
图3为本发明含pYES3/CT空载体的转基因酵母的气相色谱图；
图4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：从圆红冬孢酵母（Rhodosporidium toruloides）YM25235中分离Δ12-脂肪酸脱氢酶的核苷酸序列采用生工生物工程（上海）股份有限公司的Ezup柱式真菌基因组DNA抽提试剂盒（产品编号: SK8259）提圆红冬孢酵母YM25235的基因组DNA，取1ul为模板进行聚合酶链式反应，根据所发表的Δ12-脂肪酸脱氢酶同源序列的组氨酸保守区Ⅰ和Ⅲ区氨基酸序列设计简并引物（引物1和引物2），以上述提取的DNA模板在PCR仪（BIOER公司）上进行PCR扩增，反应所用引物、组份和扩增条件如下：
引物1：FD12CRF1: 5`-CA(T/C)GA(A/G)TG(T/C)GGICA(T/C)CA(A/G)(G/T)CITT-3`
引物2：FD12CRR3: 5`-(A/T)A(A/G)TG(A/G)TGI(A/G)(A/G/C)IAC(A/G)TGIGT-3`
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 1 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、52℃ 1min、72℃ 2min进行30个循环，最后72℃ 10min，琼脂糖凝胶电泳检测结果显示，扩增得到大小约750bp的片段，用多功能DNA纯化回收试剂盒(离心柱型)（北京百泰克生物技术有限公司）回收，把回收片段亚克隆到pMD-18T（TaKaRa公司产品）中，连接产物转化到用CaCl2法处理的大肠杆菌DH5α，在含氨苄青霉素（100ug/ml）的LB固体平板上过夜培养，挑取平板上生长的白色菌落，通过菌落PCR验证阳性克隆。将验证阳性的克隆子接入LB液体培养基（含100ug/ml氨苄青霉素）中过夜培养，用高纯质粒小量制备试剂盒（离心柱型）（北京百泰克生物技术有限公司）提取质粒，经测序（北京三博志远北京三博远志生物技术有限责任公司），测序结果显示所扩增得到的片段大小为704bp，通过其编码的氨基酸序列在Genebank数据库中用Blast程序进行同源检索，检索结果表明与该片段最相似的同源片段为Δ12-脂肪酸脱氢酶，但并不完全相同，证明所扩增的片段为新的潜在的脱氢酶基因的片段。
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3和引物4），以cDNA为模板进行PCR反应，引物序列如下：
引物3：RKD12F:5`-ATAAGCTTATGGCCGCTACCCTCCGCCAGC-3`（SEQ ID NO:3）
引物4：RKD12R:5`-ATGAATTCCTAGAGTCCCTCGACGCCCGAGT-3`（SEQ ID NO:4）
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SEQ ID NO:1所示的核苷酸序列。
实施例2：所分离核苷酸序列的同源性搜索
将推测的Δ12-脂肪酸脱氢酶的氨基酸序列在Genbank上进行同源性搜索，所得同源序列大部分为编码Δ12-脂肪酸脱氢酶的序列，少数为假定蛋白的序列，选择已做功能验证的Δ12-脂肪酸脱氢酶做同源性比较，其中与少根根霉的Δ12-脂肪酸脱氢酶同源性最高，同源性为42.83%，见图1，图1显示本发明的圆红冬孢酵母YM25235中分离Δ12-脂肪酸脱氢酶和少根根霉Δ12-脂肪酸脱氢酶（AAV52631.1）的氨基酸序列同源性比较图，RKD12为本发明的圆红冬孢酵母YM25235Δ12-脂肪酸脱氢酶，RAD12为少根根霉Δ12-脂肪酸脱氢酶，Consensus为两序列之间相同的氨基酸序列，这说明本发明提供的核苷酸序列所编码的酶具有潜在的Δ12-脂肪酸脱氢酶的功能。
实施例3： 酿酒酵母重组表达载体的构建
根据SEQ ID NO:1所示编码区序列，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HindⅢ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SEQ ID NO:1所示1-1341bp的序列一致；然后取25ul 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，质粒构建结果见图2，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4：酿酒酵母重组表达载体和空载体转化酿酒酵母细胞
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80ul分装于1.5ml预冷的离心管中，取1ug线性化的重组表达载体pY3RKD12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25uF，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5： 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0.4，30℃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200ul的正己烷回溶样品，最后用0.45um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1ul；分析软件：Anstar。
色谱分析结果见图3、4，图3显示含pYES3/CT空载体的转基因酵母，图4显示含重组质粒pY3RKD12的转基因酵母；与图3相比，图4中保留时间为13.8min对应的峰为亚油酸甲酯，合成亚油酸甲酯的亚油酸即为黏红酵母YM25079Δ12-脂肪酸脱氢酶催化油酸产生的亚油酸，说明本发明的Δ12-脂肪酸脱氢酶具有催化油酸生产亚油酸的能力，为一种有活性的Δ12-脂肪酸脱氢酶，可以将其应用于生产上。
SEQUENCE LISTING
<110> 昆明理工大学
<120> 一种Δ12-脂肪酸脱氢酶基因及其重组表达载体
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cgctcgagac gtcgtatccg cccttcgtcg ttccgaacct caccatcaag 150
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acgccgcgta ccatatcgac cccgcattct cgtacgaggg cggcaagtac 300
ctcagcggct gggccggctt cgctgccaag tgggcgtgct ggtcgctgta 350
ctggacattg cagggatggg tcggcacagg gatttggatt ctaggccacg 400
agtgcggtca ccaggcgttc tcgacgtcca agacggtcaa caacacgatg 450
ggcctcttcc tccactcgtt cgtccttgtg ccgtaccact cgtggcgcat 500
ctcgcacgcc aagcaccacg ccgcgaccgg ccacctcacc cgcgacgagg 550
tcttcgtccc ccgcaccaag tcgttccgca agccggcgcc gacgggcaag 600
aagctcgagg tcgcgcacaa cattaagatc gacgagctcc ttgaagacgc 650
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acgaaccact tcgaccccaa gtcgctcgtc ttcgatgcgc gccaccgcca 800
ccaggtcctc ctctcggacg ccttcctcgt cggcatgctc agcctcatcg 850
ccctctttgg ccactacatg ggcggcttct cggcgaccgt caagtactac 900
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Pro Ile Lys Ala Thr Glu Thr Ser Tyr Pro Pro Phe Val Val Pro
40
Asn Thr Thr Ile Lys Glu Val Thr Gly Ala Ile Pro Ala His Cys
50 60
Phe Glu Arg Ser Ala Thr Arg Ser Ser Thr Tyr Val Val Ala Asp
70
Phe Ile MET Val Ala Ala Thr Gly Tyr Ala Ala Tyr His Ile Asp
80 90
Pro Ala Phe Ser Tyr Glu Gly Gly Lys Tyr Thr Ser Gly Trp Ala
100
Gly Phe Ala Ala Lys Trp Ala Cys Trp Ser Thr Tyr Trp Thr Leu
110 120
Gln Gly Trp Val Gly Thr Gly Ile Trp Ile Thr Gly His Glu Cys
130
Gly His Gln Ala Phe Ser Thr Ser Lys Thr Val Asn Asn Thr MET
140 150
Gly Thr Phe Thr His Ser Phe Val Thr Val Pro Tyr His Ser Trp
160
Arg Ile Ser His Ala Lys His His Ala Ala Thr Gly His Thr Thr
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Arg Asp Glu Val Phe Val Pro Arg Thr Lys Ser Phe Arg Lys Pro
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Ala Pro Thr Gly Lys Lys Thr Glu Val Ala His Asn Ile Lys Ile
200 210
Asp Glu Thr Thr Glu Asp Ala Pro Ile Tyr Arg Thr Gly Trp Thr
220
Thr Val Gln Gln Thr Phe Gly Trp Pro Ala Tyr Thr Phe Ser Asn
230 240
Ala Ser Gly Gln Thr Trp Tyr Pro Lys Trp Thr Asn His Phe Asp
250
Pro Lys Ser Thr Val Phe Asp Ala Arg His Arg His Gln Val Thr
260 270
Thr Ser Asp Ala Phe Thr Val Gly MET Thr Ser Thr Ile Ala Thr
280
Phe Gly His Tyr MET Gly Gly Phe Ser Ala Thr Val Lys Tyr Tyr
290 300
Phe Ile Pro Tyr Thr Thr Val Asn His Trp Thr Val MET Ile Thr
310
Tyr Thr Gln His Thr Asp Pro Gln Thr Pro His Tyr Ser Ala Asp
320 330
MET Trp Asn Phe Gln Arg Gly Ala Thr Cys Thr Ile Asp Arg Asn
340
MET Thr Gly Pro Val Gly Pro Tyr Thr MET His Gly Ile Thr Glu
350 360
Thr His Val Ala His His Ile Ser Ser Lys Ile Pro His Tyr His
370
Ala Trp Glu Ala Thr Glu Ala Thr Lys Asn Phe Thr Gly Glu His
380 390
Tyr His Ala Thr Asp Glu Asn MET Phe Val Ser Thr Trp Lys Ser
400
Tyr Lys Gln Cys Arg Tyr Val Glu Asp Glu Gly Gln Val Thr Phe
410 420
Tyr Lys Asp Ala Tyr Gly Arg Ala Arg Arg Thr Ala Val Ile Pro
430
Asp Val Pro Ser Asp Ser Gly Val Glu Gly Thr ***
440 447
<210> 3
<211> 30
<212> DNA
<213> 人工序列
<400> 3
ataagcttat ggccgctacc ctccgccagc 30
<210> 4
<211> 31
<212> DNA
<213> 人工序列
<400> 4
atgaattcct agagtccctc gacgcccgag t 31
Claims (2)
1.一种Δ12-脂肪酸脱氢酶基因，其核苷酸序列如SEQ ID NO：1所示，该基因编码的氨基酸序列如SEQ ID NO：2所示。
2.一种含有权利要求1所述Δ12-脂肪酸脱氢酶基因的重组表达载体。
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| CN103834672B (zh) * | 2014-03-17 | 2015-12-09 | 昆明理工大学 | 一种δ12-脂肪酸脱氢酶基因及其应用 |
| CN104212819B (zh) * | 2014-07-28 | 2016-05-25 | 昆明理工大学 | 一种δ12-脂肪酸脱氢酶基因及其应用 |
| CN108624600B (zh) * | 2018-05-22 | 2021-06-18 | 昆明理工大学 | 锌指转录因子基因RkMsn4的用途 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570116A (zh) * | 2004-05-13 | 2005-01-26 | 南开大学 | 少根根霉δ12－脂肪酸脱氢酶的核苷酸序列及其应用 |
| CN1644698A (zh) * | 2004-12-22 | 2005-07-27 | 南开大学 | 毕赤酵母△12－脂肪酸脱氢酶的核苷酸序列及其应用 |
| CN101696415A (zh) * | 2009-10-28 | 2010-04-21 | 南京农业大学 | 一种油酸脱氢酶基因及其所编码的蛋白质 |
| CN102533804A (zh) * | 2010-09-10 | 2012-07-04 | 兰州大学 | 白沙蒿Δ12脂肪酸脱氢酶(As FAD2)基因及用途 |
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2013
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570116A (zh) * | 2004-05-13 | 2005-01-26 | 南开大学 | 少根根霉δ12－脂肪酸脱氢酶的核苷酸序列及其应用 |
| CN1644698A (zh) * | 2004-12-22 | 2005-07-27 | 南开大学 | 毕赤酵母△12－脂肪酸脱氢酶的核苷酸序列及其应用 |
| CN101696415A (zh) * | 2009-10-28 | 2010-04-21 | 南京农业大学 | 一种油酸脱氢酶基因及其所编码的蛋白质 |
| CN102533804A (zh) * | 2010-09-10 | 2012-07-04 | 兰州大学 | 白沙蒿Δ12脂肪酸脱氢酶(As FAD2)基因及用途 |
Non-Patent Citations (2)
| Title |
|---|
| A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides;Zhiwei Zhu等;《nature communications》;20121009;1-11 * |
| Δ12-脂肪酸脱氢酶基因在大肠杆菌中的表达;李明春等;《微生物学通报》;20041231;第31卷(第4期);43-48 * |
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