CN113430215A - 乙酰CoA合成酶基因RKACS1及其应用 - Google Patents
乙酰CoA合成酶基因RKACS1及其应用 Download PDFInfo
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Abstract
本发明公开了一种乙酰CoA合成酶基因RKACS1，其核苷酸序列如SEQID NO:1所示，该基因编码的氨基酸序列如SEQ ID NO:2所示；该基因是红冬胞酵母（Rhodosporidium kratochvilovae）YM25235中分离获得的乙酰CoA合成酶基因，将该基因转化到红冬孢酵母YM25235中，实验结果显示，乙酰CoA合成酶基因RKACS1的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素和油脂含量的增加，本发明通过基因工程手段对微生物进行改造，来提高微生物体内类胡萝卜素和油脂的产量，为大规模商业化生产类胡萝卜素和油脂奠定基础。
Description
技术领域
本发明属于生物和遗传工程技术领域，涉及一种乙酰CoA合成酶基因RKACS1，具体涉及从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中克隆的乙酰CoA合成酶基因RKACS1以及将该基因与载体连接，转入酵母细胞中提高该基因的表达水平并促进类胡萝卜素和油脂的合成。
背景技术
类胡萝卜素（carotenoids）是自然界中广泛存在的一类天然色素，一般呈现黄色、红色或橙红色，目前在高等植物、动物、真菌等生物体中已发现的天然类胡萝卜素有800多种，常见的蔬菜水果中均存在丰富的类胡萝卜素，如柑橘、芒果、南瓜等。大部分类胡萝卜素是一类由8个首尾相连的类异戊二烯组成的C40类萜化合物及其衍生物，有些类胡萝卜素含C45或C50骨架，被称为高类胡萝卜素，也有少部分碳骨架少于C40的类胡萝卜素，被称为脱辅基类胡萝卜素；所有的类胡萝卜素均含有聚异戊二烯结构。
依据类胡萝卜素化学结构的组成可将类胡萝卜素分为胡萝卜素(carotene)和叶黄素(xanthophyll)两大类，其中，胡萝卜素是只含C、H两种元素的α-胡萝卜素、β-胡萝卜素、γ-胡萝卜素和番茄红素等；叶黄素是一类氧化后的胡萝卜素，含有C、H、O三大元素，可形成羟基、酮基、羧基、甲氧基等含氧官能团，如叶黄素、玉米黄质、虾青素等；含氧基团使类胡萝卜素分子结构发生复杂多样的变化，极性的改变使类胡萝卜素易于与机体脂肪酸、糖和蛋白等结合，形成多种功能不同的活性分子。类胡萝卜素具有较强的吸光性，通常在波长430~570nm之间有吸收峰，目前普遍采用反相高效液相色谱法，配合紫外可见光检测器、质谱、核磁共振或二极管阵列检测器对样品中的类胡萝卜素进行定性定量分析。
类胡萝卜素在人类的营养与健康领域起着十分重要的作用，α-胡萝卜素、β-胡萝卜素、γ-胡萝卜素和β-隐黄素等可以在双加氧酶的作用下形成维生素A，它们因此被称为维生素A原活性物质，是维生素A的主要来源。作为人体必需的微量营养素，类胡萝卜素具有多种功能，如抗氧化、抑制并消除体内的自由基、减慢衰老等。医学研究表明，除以上功能外，类胡萝卜素还具有抗癌、预防眼底黄斑病变和白内障、预防心血管疾病、预防非酒精性脂肪肝、加强机体免疫力等功能。此外，类胡萝卜素还能与动物蛋白相结合，使生物体呈现出体色，具有一定的保护作用。目前类胡萝卜素已被***粮食与农业组织（FAO）和世界卫生组织（WHO）等国际组织定为A类营养素，已在50多个国家获批为营养、色素双重功用的食品添加剂，被广泛应用于医药、食品、保健、美容等行业。迄今为止，已见报道的类胡萝卜素的生产工艺包括天然提取法、化学合成法和微生物发酵法，其中微生物发酵生产类胡萝卜素具有生物活性高、生产过程易于处理、产品安全和生产周期短等优点，近年来受到广泛关注。尽管已经有很多策略用来促进微生物中类胡萝卜素的合成，但是由于成本效益、色素产量和分离提取等方面的技术问题的限制，应用传统微生物发酵技术来生产各种不同类胡萝卜素在工业生产方面仍缺乏相应的策略。因此我们希望可以从生物工程角度着手，利用基因技术、物种发现、杂交培育等技术，得到高含量的原料，从而缓解目前天然类胡萝卜素的生产发展局限。近年来，结合经典遗传学和现代分子生物学方法来优化菌株原有的代谢途径和调节网络或者组装异源代谢途径使微生物高效益、低成本生产类胡萝卜素的代谢工程提供了一种潜在的替代途径。
油脂（lipids）是生物体进行多项生命活动的关键物质，常见的油脂包括亚油酸（LA）、α-亚麻酸（ALA）、γ-亚麻酸（GLA）、花生四烯酸（ARA）、二十碳五烯酸（EPA）以及二十二碳六烯酸（DHA）等功能性油脂。研究表明，ALA、DHA、EPA等ω-3脂肪酸作为人体重要的生命活性物质能有效促进人体生长发育，具有预防糖尿病、心脑血管疾病、抗癌、抗炎、降血脂、增强机体免疫力等生理功能，长期缺乏ω-3脂肪酸与ω-6脂肪酸易导致遗传性肥胖、机体代谢紊乱，进而影响人体健康；此外，LA、ARA、DHA等可作为药物添加剂，起到降低血糖血压、阻止妊娠、缓解呼吸道疫病、预防动脉硬化及消化***溃疡的作用。功能性油脂大多是人体必需而又无法自身合成的，ALA可从部分植物油中获得，DHA和EPA主要来源于鱼油和微生物油脂。但鱼油成本高、易受污染、品质不稳定、产量难以满足全球需求。
产油微生物的油脂含量高，其脂肪酸组成与植物油脂相似。与植物油脂相比，产油脂微生物具有不与粮争地、不受气候和环境影响、生长周期短、容易实现大规模生产等优势，因此利用微生物发酵生产油脂越来越受到关注。目前，已有30多种酵母被鉴定为产油酵母，这些产油酵母大多能通过在特定条件下培养或基因改造来提高细胞内油脂的积累量。因此我们希望可以从生物工程角度着手，利用基因技术、合成生物学等技术，有目的的改造菌株，提高现有菌株的油脂转化率，从而缓解目前油脂资源紧张的压力。近年来，结合经典遗传学和现代分子生物学方法来优化菌株原有的代谢途径和调节网络或者组装异源代谢途径为微生物高效益、低成本生产油脂的代谢工程提供了一种潜在的替代途径。
发明内容
本发明目的是提供一种乙酰CoA合成酶基因RKACS1，该基因从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，其核苷酸序列如SEQ ID NO:1所示或为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1977bp（碱基），编码的氨基酸序列如SEQ ID NO:2所示的多肽或片段。
本发明另一目的是提供乙酰CoA合成酶基因RKACS1的重组表达载体，是将SEQ IDNO: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。可用本领域技术人员熟知的方法来构建乙酰CoA合成酶基因RKACS1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；所述乙酰CoA合成酶基因RKACS1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***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含有乙酰CoA合成酶基因RKACS1或上述重组表达载体的宿主细胞。
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。
本发明另一目的是将上述乙酰CoA合成酶基因RKACS1应用在产类胡萝卜素中。
本发明另一目的是将上述乙酰CoA合成酶基因RKACS1应用在产油脂中。
本发明从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235的总RNA中分离得到乙酰CoA合成酶基因RKACS1，该基因全长1977bp；红冬孢酵母YM25235中RKACS1基因的过表达会引起细胞内这个基因的转录水平在一定程度上有一定的提高，说明外源基因在菌体内发生转录，继而翻译成相应蛋白，引起细胞内与类胡萝卜素或油脂合成相关的酶的表达量的提高，本研究结果有助于阐明红冬孢酵母YM25235中产类胡萝卜素及油脂机制，为揭示微生物提高类胡萝卜素及油脂产量机制提供参考，将有助于通过基因工程手段对其进行改造来提高类胡萝卜素及油脂含量，对类胡萝卜素及油脂的工业化生产提供良好的应用前景和经济效益，为大规模商业化生产类胡萝卜素及油脂奠定基础。
附图说明
图1为本发明的红冬孢酵母YM25235的RKACS1基因的PCR扩增图；1.DNA分子量标记DL2000；2.阴性对照；3.RKACS1的cDNA片段；
图2为重组质粒pRHRKACS1的质粒图谱；
图3为菌落PCR验证电泳图；1.DNA分子量标记DL2000；2. RKACS1的cDNA片段；3-7为转化子；
图4重组质粒pRHRKACS1转化红冬孢酵母YM25235阳性克隆验证；1.DNA分子量标记DL2000；2.野生型菌株特异性基因条带；3.RKACS1的cDNA片段；4.转化子验证；
图5过表达菌株YM25235/pRHRKACS1与对照菌株YM25235的类胡萝卜素含量比较结果；
图6过表达菌株YM25235/pRHRKACS1与对照菌株YM25235的油脂含量比较结果。
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，使用常规方法。
实施例1：从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离乙酰CoA合成酶基因RKACS1的核苷酸序列
采用生工生物工程（上海）股份有限公司的UNlQ-10柱式Trizol总RNA抽提试剂盒（产品编号:SK1321）提取红冬孢酵母YM25235的总RNA，然后按照TaKaRa公司试剂盒PrimeScript ® RT reagent KitWith gDNA Eraser(Perfect Real Time)进行反转录合成cDNA，取1μL cDNA为模板进行聚合酶链式反应，根据转录组测序中发现的RKACS1序列，设计特异性引物RKACS1-F和RKACS1-R（引物1和引物2），以上述得到的cDNA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：RKACS1-F：5’-GATCACTCACCATGGTGACCGAACACACCTACGAC -3’（SEQ ID NO:3）（下划线为上游载体末端同源序列）
引物2：RKACS1-R：5’-CCGGTCGGCATCTACCTACGCCTTGGCGAACTT -3’（SEQ ID NO:4）（下划线为下游载体末端同源序列）；
PCR扩增体系如下（50μL）：
| Template cDNA | 1µL |
| Foward Primer (RKACS1-F) | 2µL |
| Reverse Primer (RKACS1-R) | 2µL |
| dNTPs Mix (10 mM) | 1µL |
| 2×phanta Max Buffer | 25µL |
| Phanta Max Super-Fidelity Polymerase | 1µL |
| ddH<sub>2</sub>O | 加至 50µL |
扩增条件：94℃预变性5min，再用94℃变性30s，60℃退火30s，72℃延伸2min，共30个循环，最后72℃彻底延伸10min；反应后取产物2μL，在浓度为1%的琼脂糖凝胶中进行电泳分析，结果如图1所示；扩增得到大小约为2000bp的片段,命名为RKACS1；将pRH2034经BamHⅠ、EcoRⅤ两个限制性内切酶进行双酶切；将以上两个片段用琼脂糖凝胶DNA回收试剂盒（北京索莱宝科技有限公司）回收，将回收后的两个片段进行连接，获得重组质粒pRHRKACS1（图2），连接方式采用ClonExpress II One Step Cloning Kit试剂盒进行，连接体系如下（20µL）：
| Exnase ™ II | 2µL |
| <i>RKACS1</i>片段 | 79ng |
| 线性化pRH2034 | 200ng |
| 5×CEIIBuffer | 4µL |
| ddH<sub>2</sub>O | 加至 20µL |
使用移液器轻轻吹打混匀，短暂离心将反应液收集至管底，然后37℃反应30min；降至4℃或立即置于冰上冷却。
将上面获得的连接产物转入大肠杆菌DH5α中扩增，在含壮观霉素（100µg/mL）的LB固体平板上培养12~16h，挑取平板上生长的白色菌落，通过菌落PCR验证阳性克隆（如图3所示），将验证阳性的克隆子接入LB液体培养基（含100µg/mL壮观霉素）中培养12~16h，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（昆明硕擎生物科技有限公司），测序结果显示所扩增得到的片段大小为1977bp，与转录组序列一致，核苷酸序列如SEQ ID NO:1所示。
实施例2：RKACS1基因过表达对红冬孢酵母YM25235中类胡萝卜素合成的影响
1、农杆菌介导转化红冬孢酵母YM25235
利用农杆菌介导法将重组质粒pRHRKACS1转化至红冬孢酵母YM25235中，以含潮霉素B（HygromycinB）终浓度为150µg/mL的YPD培养基筛选转化子，然后按照上海生工生物工程股份有限公司DNA提取试剂盒说明书中步骤提取酵母转化子的基因组DNA，然后进行PCR验证，结果见图4所示；
2、RKACS1基因过表达的红冬孢酵母YM25235中类胡萝卜素含量分析
将含pRHRKACS1的过表达菌株在28℃条件下培养，提取类胡萝卜素，以野生型红冬孢酵母YM25235菌株为对照，利用紫外-可见分光光度计在445nm下测定总类胡萝卜素的含量（mg/g干菌体），含量如图5所示；由图可知，过表达菌株YM25235/pRHRKACS1的总类胡萝卜素合成量较野生型红冬孢酵母YM25235菌株明显提高，野生型红冬孢酵母YM25235菌株的类胡萝卜素合成量为5.23±0.25mg/g，而过表达菌株YM25235/pRHRKACS1类胡萝卜素合成量为9.57±0.57mg/g，即过表达菌株YM25235/pRHRKACS1类胡萝卜素合成量是对照菌的1.83倍；结果显示乙酰CoA合成酶基因RKACS1的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素含量的增加，RKACS1基因能够促进总类胡萝卜素的合成。
3、RKACS1基因过表达的红冬孢酵母YM25235中油脂含量分析
将含pRHRKACS1的过表达菌株在28℃条件下培养，提取油脂，以野生型红冬孢酵母YM25235菌株为对照，测定油脂的含量（%干菌体），含量如图6所示；由图可知，过表达菌株YM25235/pRHRKACS1的油脂合成量较野生型红冬孢酵母YM25235菌株明显提高，野生型红冬孢酵母YM25235菌株的油脂合成量为4.27±0.51%，而过表达菌株YM25235/pRHRKACS1油脂合成量为5.73±0.24%，即过表达菌株YM25235/pRHRKACS1油脂合成量相较于对照菌株提高了34.19%；结果显示乙酰CoA合成酶基因RKACS1的过表达能引起红冬孢酵母YM25235菌株中油脂含量的增加，RKACS1基因能够促进油脂的合成；
总之，乙酰CoA合成酶基因RKACS1与红冬孢酵母中类胡萝卜素和油脂合成有关。
序列表
<110> 昆明理工大学
<120> 乙酰CoA合成酶基因RKACS1及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1977
<212> DNA
<213> 红冬胞酵母YM25235(Rhodosporidium kratochvilovaeYM25235)
<400> 1
atgaccgaac acacctacga cacgccgtcg caccccatca gcaagcgcaa cgatggcacg 60
ggccaccctg tccacgccaa cacggacgag tacgtcgagc tgtacaagga gtcgatcgac 120
tcgcccaagc agttctggga ccgcatggcc aaggagcacc tctactggca ccgcccgtac 180
tcgaccgtca ccgccggctc tttcgaggca ggagacgtac agtggttccc cgagggcggc 240
ctgaacgtcg cgtacaactg cgtcgaccgc tgggcgtaca agcacccgaa caagacggcc 300
atcatctggg aggcggacga gcccggcgag catgtcgagc tcacgtacga gcagctgttc 360
caggaggtct gcaagactgc caacatcctc aagagctacg gcgtcaagaa gggcgacacc 420
gtcgccatct acctccccat ggtccccgag gcggcaatcg ccttcctcgc gtgcgcccgc 480
ctcggcgcga tccactcggt cgtcttcgcc ggcttctccg ccgagtcgct ccgcgaccgc 540
gtcaacgacg cccagtcgcg cgtcgtcatc accaccgacg agggcaagcg cggcggcaag 600
acgatcgcga ccaagtcgat cgtcgacgcg gcgctctccg agtgccccgt cgtcgagcat 660
gtcctcgtcc tcaagcgcac tggcggcgac gtcaagtgga ccgagggccg cgaccactgg 720
tggcacgagg agaaggagaa ggtccagccg tactgccccg tcgagatcgt ctcggccgag 780
gacccgctct tcatcctcta cacctcgggc tcgaccggca agcccaaggg tgtcgtccac 840
tcgtccgccg gctacctcct cggcgcgttc atgacgctca agtacgtctt cgacgtgcac 900
cccgaggacc gctacgcgtg catggcagac gtcggctgga tcaccggcca cacctacatc 960
gtttacggcc cgcttgcgaa cggcgtcacc accaccatct tcgagtcgac gccggtctac 1020
ccgaccccgt cgcgcttctg ggagacggtc gcgaagcaca agctcacgca gttctacacc 1080
gcgccgaccg ccatccgtct cctccgccgc ctcggcgagg agcacaccaa gggccacgac 1140
ctgtcgacgc tccgcaccat cggctcggtc ggcgagccga tcaaccccga ggcttgggag 1200
tggtactggg agcacgtcgg caagaaggag tgcgccgtcg tcgacacgta ctggcagacc 1260
gagaccggct ccatcatcat cacccccctt cccggcgcga ccaagaccaa gcccggcgcg 1320
gcgacgctgc cgttcttcgg catcgacccg gtcctcctcg acccgacgac gggcgaggag 1380
atcaagggca acgaggtcga gggcgtgctc tgcgtgcgca agccgtggcc gtcgatcgcg 1440
cgcaccgtct acggcgacca caagcggttc ctcgacacgt acatgaaccc gtacccgggc 1500
tactacttca ccggcgacgg cgccgggcgc gaccacgacg ggtactactg gatccgcggc 1560
cgcgtcgacg acgtgatcaa cgtctcgggc caccgcctct cgaccgccga aatcgagagc 1620
gccctcatcc accacaacgg cgtagccgag acggccgtcg tcggcatccc cgacgagctc 1680
accggccagg ccgtcgtcgc ctacgtcgcc ctcaagcccg agtttgcggc cgagaacccc 1740
gacgagccgg cgctgctgaa ggaattggtg ttgcaggtcc gcaagaccat cggtccgttc 1800
gcggccccga agaaactcgt gcttgtgggt gatctgccga agacccggag cggcaagatc 1860
gtccggcggg cactgcgcaa gatcgcgagc ggcgagggcg accagctcgg cgatctttcg 1920
acactggcgg agcctgccat catcgacgag atcaaggaga agttcgccaa ggcgtag 1977
<210> 2
<211> 658
<212> PRT
<213> 红冬孢酵母YM25235(Rhodosporidium kratochvilovae YM25235)
<400> 2
Met Thr Glu His Thr Tyr Asp Thr Pro Ser His Pro Ile Ser Lys Arg
1 5 10 15
Asn Asp Gly Thr Gly His Pro Val His Ala Asn Thr Asp Glu Tyr Val
20 25 30
Glu Leu Tyr Lys Glu Ser Ile Asp Ser Pro Lys Gln Phe Trp Asp Arg
35 40 45
Met Ala Lys Glu His Leu Tyr Trp His Arg Pro Tyr Ser Thr Val Thr
50 55 60
Ala Gly Ser Phe Glu Ala Gly Asp Val Gln Trp Phe Pro Glu Gly Gly
65 70 75 80
Leu Asn Val Ala Tyr Asn Cys Val Asp Arg Trp Ala Tyr Lys His Pro
85 90 95
Asn Lys Thr Ala Ile Ile Trp Glu Ala Asp Glu Pro Gly Glu His Val
100 105 110
Glu Leu Thr Tyr Glu Gln Leu Phe Gln Glu Val Cys Lys Thr Ala Asn
115 120 125
Ile Leu Lys Ser Tyr Gly Val Lys Lys Gly Asp Thr Val Ala Ile Tyr
130 135 140
Leu Pro Met Val Pro Glu Ala Ala Ile Ala Phe Leu Ala Cys Ala Arg
145 150 155 160
Leu Gly Ala Ile His Ser Val Val Phe Ala Gly Phe Ser Ala Glu Ser
165 170 175
Leu Arg Asp Arg Val Asn Asp Ala Gln Ser Arg Val Val Ile Thr Thr
180 185 190
Asp Glu Gly Lys Arg Gly Gly Lys Thr Ile Ala Thr Lys Ser Ile Val
195 200 205
Asp Ala Ala Leu Ser Glu Cys Pro Val Val Glu His Val Leu Val Leu
210 215 220
Lys Arg Thr Gly Gly Asp Val Lys Trp Thr Glu Gly Arg Asp His Trp
225 230 235 240
Trp His Glu Glu Lys Glu Lys Val Gln Pro Tyr Cys Pro Val Glu Ile
245 250 255
Val Ser Ala Glu Asp Pro Leu Phe Ile Leu Tyr Thr Ser Gly Ser Thr
260 265 270
Gly Lys Pro Lys Gly Val Val His Ser Ser Ala Gly Tyr Leu Leu Gly
275 280 285
Ala Phe Met Thr Leu Lys Tyr Val Phe Asp Val His Pro Glu Asp Arg
290 295 300
Tyr Ala Cys Met Ala Asp Val Gly Trp Ile Thr Gly His Thr Tyr Ile
305 310 315 320
Val Tyr Gly Pro Leu Ala Asn Gly Val Thr Thr Thr Ile Phe Glu Ser
325 330 335
Thr Pro Val Tyr Pro Thr Pro Ser Arg Phe Trp Glu Thr Val Ala Lys
340 345 350
His Lys Leu Thr Gln Phe Tyr Thr Ala Pro Thr Ala Ile Arg Leu Leu
355 360 365
Arg Arg Leu Gly Glu Glu His Thr Lys Gly His Asp Leu Ser Thr Leu
370 375 380
Arg Thr Ile Gly Ser Val Gly Glu Pro Ile Asn Pro Glu Ala Trp Glu
385 390 395 400
Trp Tyr Trp Glu His Val Gly Lys Lys Glu Cys Ala Val Val Asp Thr
405 410 415
Tyr Trp Gln Thr Glu Thr Gly Ser Ile Ile Ile Thr Pro Leu Pro Gly
420 425 430
Ala Thr Lys Thr Lys Pro Gly Ala Ala Thr Leu Pro Phe Phe Gly Ile
435 440 445
Asp Pro Val Leu Leu Asp Pro Thr Thr Gly Glu Glu Ile Lys Gly Asn
450 455 460
Glu Val Glu Gly Val Leu Cys Val Arg Lys Pro Trp Pro Ser Ile Ala
465 470 475 480
Arg Thr Val Tyr Gly Asp His Lys Arg Phe Leu Asp Thr Tyr Met Asn
485 490 495
Pro Tyr Pro Gly Tyr Tyr Phe Thr Gly Asp Gly Ala Gly Arg Asp His
500 505 510
Asp Gly Tyr Tyr Trp Ile Arg Gly Arg Val Asp Asp Val Ile Asn Val
515 520 525
Ser Gly His Arg Leu Ser Thr Ala Glu Ile Glu Ser Ala Leu Ile His
530 535 540
His Asn Gly Val Ala Glu Thr Ala Val Val Gly Ile Pro Asp Glu Leu
545 550 555 560
Thr Gly Gln Ala Val Val Ala Tyr Val Ala Leu Lys Pro Glu Phe Ala
565 570 575
Ala Glu Asn Pro Asp Glu Pro Ala Leu Leu Lys Glu Leu Val Leu Gln
580 585 590
Val Arg Lys Thr Ile Gly Pro Phe Ala Ala Pro Lys Lys Leu Val Leu
595 600 605
Val Gly Asp Leu Pro Lys Thr Arg Ser Gly Lys Ile Val Arg Arg Ala
610 615 620
Leu Arg Lys Ile Ala Ser Gly Glu Gly Asp Gln Leu Gly Asp Leu Ser
625 630 635 640
Thr Leu Ala Glu Pro Ala Ile Ile Asp Glu Ile Lys Glu Lys Phe Ala
645 650 655
Lys Ala
<210> 3
<211> 35
<212> DNA
<213> 人工序列(Artificial)
<400> 3
gatcactcac catggtgacc gaacacacct acgac 35
<210> 4
<211> 33
<212> DNA
<213> 人工序列(Artificial)
<400> 4
ccggtcggca tctacctacg ccttggcgaa ctt 33
Claims (3)
1.一种乙酰CoA合成酶基因RKACS1，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的乙酰CoA合成酶基因RKACS1在产类胡萝卜素和油脂中的应用。
3.权利要求1所述的乙酰CoA合成酶基因RKACS1在产油脂中的应用。
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| CN113621630A (zh) * | 2021-08-05 | 2021-11-09 | 昆明理工大学 | 3-酮脂酰-CoA硫解酶基因RkACAA1-1及其应用 |
| CN113652440A (zh) * | 2021-08-05 | 2021-11-16 | 昆明理工大学 | 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用 |
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| CN112410355B (zh) * | 2020-11-23 | 2022-03-25 | 昆明理工大学 | 一种酰基辅酶a氧化酶2基因rkacox2及其应用 |
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| CN113652440B (zh) * | 2021-08-05 | 2023-04-21 | 昆明理工大学 | 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用 |
| CN116606868A (zh) * | 2023-05-12 | 2023-08-18 | 昆明理工大学 | 一种乙酰CoA合成酶基因RkACS2及其应用 |
| CN116606868B (zh) * | 2023-05-12 | 2024-04-16 | 昆明理工大学 | 一种乙酰CoA合成酶基因RkACS2及其应用 |
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