CN108624600A - 锌指转录因子基因RkMsn4的用途 - Google Patents
锌指转录因子基因RkMsn4的用途 Download PDFInfo
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Abstract
本发明公开了一种锌指转录因子基因RkMsn4的用途，即其在常温下提高类胡萝卜素产量中的应用，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；该基因是转录因子基因，调控红冬孢酵母产生类胡萝卜素，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的产量，为大规模商业化生产类胡萝卜素奠定基础。
Description
技术领域
本发明属于生物技术领域中的微生物基因工程领域，涉及一种锌指转录因子基因RkMsn4的新用途，具体涉及从红冬孢酵（Rhodosporidium kratochvilovae）YM25235中克隆出锌指转录因子基因RkMsn4将该基因连接到载体上转入到酵母菌中，促进酵母菌中类胡萝卜素合成，为类胡萝卜素的大规模生产提供参考。
背景技术
类胡萝卜素（carotenoid）是一类由植物和某些光合微生物生成的呈红色、橘黄色或者黄色的亲脂性异戊二烯类色素，一般在波长300～600 nm之间有吸收峰。在自然界中已鉴定出天然类胡萝卜素的种类超过700种，广泛存在于食物中，如蔬菜、水果和海鲜。
在自然状态下，类胡萝卜素的典型结构特点是高度不饱和，其最显著特征为含有3～15 个共轭双键，共轭双键存在的位置决定类胡萝卜素的功能特性，包括在光合生物中可起到光吸收和光保护的作用；可作为细胞膜的抗氧化保护剂；具有抗氧化和抗癌特性；对眼睛具有保护功能等。但类胡萝卜素除上述功能外，还可作为一些派生物的重要底物。根据类胡萝卜素种类和降解位点、降解方式的不同，可生成多种衍生物，其在生物体中具有重要的功能，如可作为维生素、植物激素及芳香类化合物等。因此利用类胡萝卜素降解生成活性物质，尤其是香气物质已经成为近年来食品、果酒、化妆品等学科的研究热点之一。
由于天然类胡萝卜素目前在市场上占据着很重要的位置，其着色作用、保护人眼功能、营养补充、抗氧化、抗癌、抗心脑血管疾病、提高免疫力方面的卓越表现，证明了它广阔的市场前景，随着人们日益增长的对安全色素、天然健康食品保健品的需求，花样繁多的产品必然会应运而生。丰富国内资源，加强技术开发，完善产业链，是打破国外垄断的必由之路。就目前市场状况而言，大部分的产品都是脂溶性的产品、粉剂、颗粒剂等，有些类胡萝卜素的水分散液颜色很鲜亮，但是高浓度的水溶性天然类胡萝卜素产品却很少，这与类胡萝卜素本身的性质是息息相关的；另外在生物工程方面，能够作为高含量生产原料的生物仍然不够广泛，未来如果能通过基因技术、物种发现、杂交培育等技术，得到高含量的原料，必然大大缓解天然类胡萝卜素的生产发展局限；在大多数的类胡萝卜素软胶囊的制作原料中都使用了大豆油，这种油脂有潜在的转基因的威胁，未来需要找到安全、优质、健康、廉价的替代品；在生产工艺技术与成本方面，分离提纯技术、抗氧化防护技术、简化工艺流程都有待解决的问题。
发明内容
本发明的目的是提供一种锌指转录因子基因RkMsn4的新用途，即其在常温下提高类胡萝卜素产量中的应用，其核苷酸序列如SEQ ID NO:1 所示，该基因是从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，该基因序列长为2625bp（碱基），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，共编码874个氨基酸；其参与类胡箩卜素代谢途径，该基因使酵母菌大量产生类胡萝卜素。
本发明中锌指转录因子基因RkMsn4可以与酵母表达载体连接所构建的重组载体；进一步可以将重组载体转入到酵母菌中，促进酵母菌中类胡萝卜素合成。
本发明所述红冬孢酵母YM25235基因RkMsn4与载体连接后转化至微生物细胞体内能提高类胡萝卜素产量，具有产物特异性高、生产周期短、生产不受场地、气候、季节的影响等优点，可以用于商业化生产，具有实际应用价值；本发明应用基因工程技术特异性提高微生物细胞类胡萝卜素的产量，具有操作简单、成本低、可行性高等优点，有助于通过基因工程手段对微生物菌株进行改造来提高类胡萝卜素含量，为大规模商业化生产类胡萝卜素打下基础。
附图说明
图1为锌指转录因子RkMsn4基因全长cDNA序列扩增电泳检测示意图；
图2 为重组质粒pRHRkMsn4限制性酶切电泳示意图；1、DNA 分子量标记DL10000；2、空质粒pRH2304的BamH Ⅰ、EcoR Ⅴ双酶切；3，4、重组质粒pRHRkMSN4的BamH Ⅰ、EcoRⅤ双酶切；5、RkMSN4基因的PCR产物；6、DNA 分子量标记DL5000；
图3为重组质粒pRHRkMsn4的质粒图谱；
图4为锌指转录因子基因RkMsn4转化红冬孢酵母YM25235后转基因酵母中类胡萝卜素含量变化结果。
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，均采用常规试剂和使用常规方法。
实施例1：红冬孢酵母（Rhodosporidium kratochvilovae）YM25235锌指转录因子RkMsn4基因的克隆
采用OMEGA试剂盒E.Z.N.A Fungal RNA Kit提取红冬孢酵母YM25235总RNA，反转录试剂盒Takara First Strand cDNA Synthesis Kit合成cDNA。根据红冬孢酵母YM25235的转录组序列设计特异性引物（引物1和引物2）进行PCR扩增；反应所用引物、组份和扩增条件如下：
引物1：RkMsn4-F：5’-AATGGATCCTATGGCGCCTGCTCCCCGT-3’（SEQ ID NO:3）
引物2：RkMsn4-R：5’-ATCGATATCTCACGGCATCGCCCACACCT-3’（SEQ ID NO:4）；
（GGATCC为BamH I酶切位点，GATATC为EcoRV酶切位点）；
PCR扩增体系如下（50 μL）：
PCR扩增条件为：95℃预变性5min，95℃ 变性30s、64℃退火 30s、72℃ 延伸3min进行30个循环，最后72℃延伸10min；反应完后取产物1μL，然后在浓度为1%的琼脂糖凝胶中，进行电泳分析（图1）；经凝胶成像系统成像确认2700bp大小的片段，然后用百泰克生物技术有限公司多动能DNA纯化回收试剂盒回收目的片段，然后将PCR扩增得到的目的基因连接到pMD18-T上，连接产物转化至大肠杆菌DH5α感受态细胞中，用含有氨苄青霉素（Amp+）的LB固体平板进行筛选，挑取平板上的转化子进行菌落PCR筛选阳性克隆，然后送去上海生工测序；测序结果表明，获得一段2625bp长的序列，编码874个氨基酸，命名为RkMsn4，序列组成如SEQ ID NO:1所示的核苷酸序列和氨基酸序列。
实施例2：重组表达质粒pRH2034RkMsn4的构建
采用实施例1中测cDNA为模板进行PCR扩增，反应所以引物组合、反应组分和扩增条件如下：
RkMsn4-F：5’-AATGGATCCTATGGCGCCTGCTCCCCGT-3’（SEQ ID NO:3）
RkMsn4-R：5’-ATCGATATCTCACGGCATCGCCCACACCT-3’（SEQ ID NO:4）
（GGATCC为BamH I酶切位点，GATATC为EcoRV酶切位点）
PCR扩增体系如下（50 μL）：
PCR扩增条件为：95℃预变性5min，95℃ 变性30s、 64℃退火 30s、72℃ 延伸3min进行30个循环，最后72℃延伸10min；将上述PCR获得的基因片段和表达载体pRH2034用BamH I和 EcoRV核酸内切酶进行双酶切，用百泰克生物技术有限公司多动能DNA纯化回收试剂盒回收酶切片段，并用T4连接酶在16℃连接过夜，连接产物转化大肠杆菌DH5α，用含有壮观霉素（Spe+）的LB固体平板进行筛选，挑取平板上的转化子进行菌落PCR，筛选阳性克隆，同时进一步对转化子进行双酶切验证分析，结果显示，如图2第3、4泳道所示，用EcoRV和BamH I双酶切，重组质粒产生两条带，小分子条带与泳道5中该基因的PCR产物大小一致，大分子条带与泳道2中用相同酶切质粒pRH2034后产生的条带大小一致，这表明所构建的重组质粒正确，进一步测序分析也证明这一点。将重组表达质粒命名为pRH2034RkMsn4，该质粒图谱如图2所示。
实施例3：RkMsn4基因对红冬孢酵母类胡萝卜素合成的影响
1、农杆菌介导转化红冬孢酵母YM25235
采用农杆菌介导转化法将重组质粒pRH2034RkMsn4转化至红冬孢酵母YM25235菌株，以含潮霉素B（HygromycinB）终浓度为150 µg/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，后进行PCR验证。
2、RkMsn4基因与红冬孢酵母类胡萝卜素含量关系的分析
将转基因菌株YM25235/pRHRkMsn4及对照菌株YM25235/pRH2304在30℃培养120h，提取转基因菌株的类胡萝卜素，利用液相色谱对类胡萝卜素进行分离，两株转基因菌株类胡萝卜素含量结果对比如图4、表1；结果表明，转基因菌株YM25235/pRHRkMsn4的类胡萝卜素含量相较于对照菌株YM25235/pRH2304的类胡萝卜素含量明显增加，提高了1.64倍。因此，在红冬孢酵母YM25235菌株中过表达RkMsn4基因会引起类胡萝卜素增加；
表1：
。
序列表
<110> 昆明理工大学
<120> 锌指转录因子基因RkMsn4的用途
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ccgcgcgagt cgcccaccga gcctgccgcg cccgtagcgt cgacctccgc catgcccgtc 300
gacatactgt acccgccgta cccaccgccc gtcgcctcgt cctcgtccgc cgttcctgcg 360
ccgacctact cgccatatga cacaggcgcg caggacctgc ctgtcccccc agccagcttc 420
ctcgacctcg tctcttcgca accgtaccct gcgccctccc cgtccctccc cgccttctcg 480
cccaacatgg cgagcctgta tcccatccca ttgaggaccg gcagcacagc ctcgcccgcc 540
tcgagcggca tgtccggctc gccgttctcg cgcgccacgt acgcctcgac gaccggcaca 600
tcgcccgaca tggcgatgca gaaggagctg ccgctcacgc tccctgacgt gatggcttcc 660
atctcggcga cgacgagcgc gccgcgccct caaccgccgc aggagcagca agcgatgccg 720
acgctcgccg cgctcgaccc gtacggcgcc gcgccgcagc cttcgtccgc attcaaggcc 780
ggcgtcacgc agcacgatac gctcgcgccc ggcctgtcgc gcacgggcag cttcaccaag 840
gacgaagtgc tcgcgtccga ggtcctgcag gacctgatgc gcacgccgtt cggcgcgccg 900
taccccgcgc cgccagcgag cacgagcggc gtgccctggc acggcgcgaa ggcggcgcag 960
cgcgcggcgg agcagcccga gtcggcgcag gcgaacgaag ctgtcagcct ggtcgactcg 1020
tccggcggcg actgggcttt cagcctcggc ggcggcgcgg ttctgcccat cggcgggttc 1080
ggcgggcccg tctcaaacaa gctggaggag acgccagcgg cgcagcagct cgccgagtac 1140
ttcaataagg gcggcgtcgg cggcatcacg gcgctcgacc tcggcttcac ggtcgagccg 1200
agcctgtggc ctgagtggat cacgatggag ccgaagccgg tgcacgacga catgaagcgg 1260
tggtggctgc cagagcagaa gttctgcctc ggctatctct acccttggca tgtcccgccc 1320
cttcccgttc tctccggata cgcccgcaag gctacggagc agctcctcgc agctgtgccc 1380
gtcctgcatg gtccgtccgc ggtcatgacc gagctaccga cgcacacggc gtttgcgctg 1440
accgtcgccg gcggggcgta cgagcccgag ggccagagct tcagtaacga gatgctcgtc 1500
gagaagaggg tgttcctcgt gcgcggcttc caggagaagg acaagagctg ggaggaccgc 1560
ttcgcgtcac tccagtcgct cctgctctac cagcttctcg gcatcttcca ccgcgacgag 1620
cagcagcgcc tgctctccca ctccttccat tcggcgctcg tctacatgtt tcgcgcgctc 1680
gacctcccga accgcgtaac gcagaccgcg ctcgtcaagc cgcgggcaga catgcatggt 1740
ggggagctcg agaaggcgtg gaaggagtgg atcgaggtcg aaacccggcg gcgagtcgcg 1800
ttcatcgttt tcctcatcga cctcgagcac gcggcggcga ccgagacgcc gcagctcctc 1860
gcgctcagcg acctcgacct cgaccttccc gcctccgagc gcgcgtggaa ggcagagaac 1920
gccgccgaat ggctcgatcg cagtacctcg cccctctacc ccgaaaccat ctccttcctc 1980
gccgccatcc gcgccctcct ctcgactacc ccgcccgagc ccttctccgc cgcaggcgtt 2040
ctcctcgccg aactcggccg cctctcgtcc ttcccgctcc tcatcctctc gcggacgctc 2100
tcgtacctcg agcgcaagac gcaggaggcg ctcgcgcaaa tcgacccgtt caagagcctc 2160
ctcggcggcc tcggcgtcct tgagggccgc gagaccgaga atcgcgccgt cctcgagcgc 2220
attcgtcgcg gccgcgaagt cctgcgccgc ctgcctggcg ggattgcgcg cgggggtgga 2280
gagggctggt tcaacgagat catcccctcg gccaaggatt tcgtcccagc gtcggactcg 2340
cccgcagcgt cgagttcgag cgcgggcacc tccccgcggg acgcgttctc ctcctcctcc 2400
gcaagcggga cggcatcgac atttccctcg ccgcaagacg cgcccgcctc gctcgaggag 2460
ctcttcgccg agttcgacga gcagccgtac cgcccgttcc atggcgcggg cggcgcgacg 2520
cccggcgaga cgtacgagca ggcgcaggag cgcctgcgca agcatgccga gcggcgcgtg 2580
cgcgatgcgc agtccgcact gcccgaggtg tgggcgatgc cgtga 2625
<210> 2
<211> 874
<212> PRT
<213> 红冬孢酵YM25235(Rhodosporidium kratochvilovae YM25235)
<400> 2
Met Ala Pro Ala Pro Arg Gly Ser Ala Ser Gly Gln Thr Phe Pro Cys
1 5 10 15
Pro Asp Cys Asp Lys Val Phe Ser Arg Lys Glu Tyr Met Ala Arg His
20 25 30
Tyr Arg Ser Arg His Ser Lys Glu Lys Pro Phe Gln Cys Glu Tyr Cys
35 40 45
Glu His Ala Phe Ser Arg Ser Asp Leu Leu Arg Arg His His Lys Thr
50 55 60
Cys Ser Gln Ala Lys Ala Ala Arg Gly Glu Gly Asp Ser Pro Asp Leu
65 70 75 80
Pro Arg Glu Ser Pro Thr Glu Pro Ala Ala Pro Val Ala Ser Thr Ser
85 90 95
Ala Met Pro Val Asp Ile Leu Tyr Pro Pro Tyr Pro Pro Pro Val Ala
100 105 110
Ser Ser Ser Ser Ala Val Pro Ala Pro Thr Tyr Ser Pro Tyr Asp Thr
115 120 125
Gly Ala Gln Asp Leu Pro Val Pro Pro Ala Ser Phe Leu Asp Leu Val
130 135 140
Ser Ser Gln Pro Tyr Pro Ala Pro Ser Pro Ser Leu Pro Ala Phe Ser
145 150 155 160
Pro Asn Met Ala Ser Leu Tyr Pro Ile Pro Leu Arg Thr Gly Ser Thr
165 170 175
Ala Ser Pro Ala Ser Ser Gly Met Ser Gly Ser Pro Phe Ser Arg Ala
180 185 190
Thr Tyr Ala Ser Thr Thr Gly Thr Ser Pro Asp Met Ala Met Gln Lys
195 200 205
Glu Leu Pro Leu Thr Leu Pro Asp Val Met Ala Ser Ile Ser Ala Thr
210 215 220
Thr Ser Ala Pro Arg Pro Gln Pro Pro Gln Glu Gln Gln Ala Met Pro
225 230 235 240
Thr Leu Ala Ala Leu Asp Pro Tyr Gly Ala Ala Pro Gln Pro Ser Ser
245 250 255
Ala Phe Lys Ala Gly Val Thr Gln His Asp Thr Leu Ala Pro Gly Leu
260 265 270
Ser Arg Thr Gly Ser Phe Thr Lys Asp Glu Val Leu Ala Ser Glu Val
275 280 285
Leu Gln Asp Leu Met Arg Thr Pro Phe Gly Ala Pro Tyr Pro Ala Pro
290 295 300
Pro Ala Ser Thr Ser Gly Val Pro Trp His Gly Ala Lys Ala Ala Gln
305 310 315 320
Arg Ala Ala Glu Gln Pro Glu Ser Ala Gln Ala Asn Glu Ala Val Ser
325 330 335
Leu Val Asp Ser Ser Gly Gly Asp Trp Ala Phe Ser Leu Gly Gly Gly
340 345 350
Ala Val Leu Pro Ile Gly Gly Phe Gly Gly Pro Val Ser Asn Lys Leu
355 360 365
Glu Glu Thr Pro Ala Ala Gln Gln Leu Ala Glu Tyr Phe Asn Lys Gly
370 375 380
Gly Val Gly Gly Ile Thr Ala Leu Asp Leu Gly Phe Thr Val Glu Pro
385 390 395 400
Ser Leu Trp Pro Glu Trp Ile Thr Met Glu Pro Lys Pro Val His Asp
405 410 415
Asp Met Lys Arg Trp Trp Leu Pro Glu Gln Lys Phe Cys Leu Gly Tyr
420 425 430
Leu Tyr Pro Trp His Val Pro Pro Leu Pro Val Leu Ser Gly Tyr Ala
435 440 445
Arg Lys Ala Thr Glu Gln Leu Leu Ala Ala Val Pro Val Leu His Gly
450 455 460
Pro Ser Ala Val Met Thr Glu Leu Pro Thr His Thr Ala Phe Ala Leu
465 470 475 480
Thr Val Ala Gly Gly Ala Tyr Glu Pro Glu Gly Gln Ser Phe Ser Asn
485 490 495
Glu Met Leu Val Glu Lys Arg Val Phe Leu Val Arg Gly Phe Gln Glu
500 505 510
Lys Asp Lys Ser Trp Glu Asp Arg Phe Ala Ser Leu Gln Ser Leu Leu
515 520 525
Leu Tyr Gln Leu Leu Gly Ile Phe His Arg Asp Glu Gln Gln Arg Leu
530 535 540
Leu Ser His Ser Phe His Ser Ala Leu Val Tyr Met Phe Arg Ala Leu
545 550 555 560
Asp Leu Pro Asn Arg Val Thr Gln Thr Ala Leu Val Lys Pro Arg Ala
565 570 575
Asp Met His Gly Gly Glu Leu Glu Lys Ala Trp Lys Glu Trp Ile Glu
580 585 590
Val Glu Thr Arg Arg Arg Val Ala Phe Ile Val Phe Leu Ile Asp Leu
595 600 605
Glu His Ala Ala Ala Thr Glu Thr Pro Gln Leu Leu Ala Leu Ser Asp
610 615 620
Leu Asp Leu Asp Leu Pro Ala Ser Glu Arg Ala Trp Lys Ala Glu Asn
625 630 635 640
Ala Ala Glu Trp Leu Asp Arg Ser Thr Ser Pro Leu Tyr Pro Glu Thr
645 650 655
Ile Ser Phe Leu Ala Ala Ile Arg Ala Leu Leu Ser Thr Thr Pro Pro
660 665 670
Glu Pro Phe Ser Ala Ala Gly Val Leu Leu Ala Glu Leu Gly Arg Leu
675 680 685
Ser Ser Phe Pro Leu Leu Ile Leu Ser Arg Thr Leu Ser Tyr Leu Glu
690 695 700
Arg Lys Thr Gln Glu Ala Leu Ala Gln Ile Asp Pro Phe Lys Ser Leu
705 710 715 720
Leu Gly Gly Leu Gly Val Leu Glu Gly Arg Glu Thr Glu Asn Arg Ala
725 730 735
Val Leu Glu Arg Ile Arg Arg Gly Arg Glu Val Leu Arg Arg Leu Pro
740 745 750
Gly Gly Ile Ala Arg Gly Gly Gly Glu Gly Trp Phe Asn Glu Ile Ile
755 760 765
Pro Ser Ala Lys Asp Phe Val Pro Ala Ser Asp Ser Pro Ala Ala Ser
770 775 780
Ser Ser Ser Ala Gly Thr Ser Pro Arg Asp Ala Phe Ser Ser Ser Ser
785 790 795 800
Ala Ser Gly Thr Ala Ser Thr Phe Pro Ser Pro Gln Asp Ala Pro Ala
805 810 815
Ser Leu Glu Glu Leu Phe Ala Glu Phe Asp Glu Gln Pro Tyr Arg Pro
820 825 830
Phe His Gly Ala Gly Gly Ala Thr Pro Gly Glu Thr Tyr Glu Gln Ala
835 840 845
Gln Glu Arg Leu Arg Lys His Ala Glu Arg Arg Val Arg Asp Ala Gln
850 855 860
Ser Ala Leu Pro Glu Val Trp Ala Met Pro
865 870
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial)
<400> 3
aatggatcct atggcgcctg ctccccgt 28
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial)
<400> 4
atcgatatct cacggcatcg cccacacct 29
Claims (2)
1.一种锌指转录因子RkMsn4基因在常温下提高类胡萝卜素产量中的应用，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.根据权利要求1所述的应用，其特征在于：在常温下促进酵母菌生产类胡萝卜素。
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| CN111454854A (zh) * | 2020-05-02 | 2020-07-28 | 昆明理工大学 | 一株产虾青素的红冬孢酵母基因工程菌株 |
| CN111454854B (zh) * | 2020-05-02 | 2022-05-06 | 昆明理工大学 | 一株产虾青素的红冬孢酵母基因工程菌株 |
| CN113430215A (zh) * | 2021-06-03 | 2021-09-24 | 昆明理工大学 | 乙酰CoA合成酶基因RKACS1及其应用 |
| CN113430215B (zh) * | 2021-06-03 | 2023-04-18 | 昆明理工大学 | 乙酰CoA合成酶基因RKACS1及其应用 |
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