CN111534535B - 一种构建麦角硫因生产菌的方法 - Google Patents
一种构建麦角硫因生产菌的方法 Download PDFInfo
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- CN111534535B CN111534535B CN202010587928.XA CN202010587928A CN111534535B CN 111534535 B CN111534535 B CN 111534535B CN 202010587928 A CN202010587928 A CN 202010587928A CN 111534535 B CN111534535 B CN 111534535B
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Abstract
本发明公开了一种圆红酵母工程菌及其构建方法,该圆红酵母工程菌能够表达外源的egt1酶,从而提高生产麦角硫因的能力,发酵后麦角硫因产量接近1.5g/L,且工程菌遗传性状稳定,具有工业化应用前景。
Description
技术领域
本发明属于基因工程领域,涉及一种构建麦角硫因生产菌的方法,尤其涉及一种构建用于生产麦角硫因的圆红酵母菌的方法。
背景技术
麦角硫因(L-ergothionine,EGT),化学名为2-巯基组氨酸三甲基内盐,结构式如下:
麦角硫因是至今唯一为人们所知的天然2-硫代咪唑氨基酸。麦角硫因具有抗氧化、抗炎症、延长细胞生存周期或抗细胞衰老活性,改善神经细胞生成等多种生理功效。同时在多种疾病模型中包括阿兹海默、糖尿病等的并发症中,具有较好的保护细胞和抗击损伤的功效,市场应用前景广阔。
目前,麦角硫因可以经化学合成、食用真菌提取和微生物发酵生产获得。有多种微生物被证实具有麦角硫因的合成能力,包括分枝杆菌、链霉菌、霉菌和酵母等多类微生物。有许多专利技术涉及利用微生物发酵生产麦角硫因,比如CN102978121B公开了食用菌白香蘑菌催化组氨酸底物可生成麦角硫因,底物转化率可达70%,未报道产物产率;CN103184246A公开了大型丝状真菌花脸香蘑摇瓶发酵10天,麦角硫因产量51mg/L;WO2017150304A1公开了青紫链霉菌Streptomyces lividans经7天发酵,麦角硫因产量900mg/L;WO2015180492A1公开了糙皮侧耳在75L发酵罐中经14天发酵,麦角硫因产量352mg/L;CN103734022公开了食用菌糙皮侧耳经7~15天发酵,麦角硫因产量最高产量达143.7mg/L;CN107250347公开了基因工程曲霉包括米曲霉、酱油曲霉和黑曲霉,经基因工程改造后,发酵产量可达438mg/L;大肠杆菌经基因工程改造,异源表达分枝杆菌的麦角硫因基因合成簇,麦角硫因产量640mg/L;CN106661585公开了大肠杆菌经基因工程改造,异源表达分枝杆菌的麦角硫因基因合成簇,工程菌发酵麦角硫因产量12mg/L;CN105296559A公开了糙皮侧耳食用菌经过多重发酵配方成分的调整,发酵至少6天,麦角硫因产量最高315.7mg/L;CN201910664772.8A公开了以枯草芽孢杆菌168为宿主表达外源基因,构建的基因工程菌麦角硫因产量达到568.4mg/L。
文献(Takusagawa,S.,Y.Satoh,I.Ohtsu and T.Dairi(2018)."Ergothioneineproduction with Aspergillus oryzae."Bioscience,Biotechnology,and Biochemistry:1-4.)公开了米曲霉Aspergillus oryzae经基因工程改造,麦角硫因产量231mg/L。此外,目前微生物发酵麦角硫因的最高产量为文献(Tanaka,N.,Y.Kawano,Y.Satoh,T.Dairi and I.Ohtsu(2019)."Gram-scale fermentative production ofergothioneine driven by overproduction of cysteine in Escherichia coli."Scientific reports9(1):1895-1895.)报道,该研究利用基因工程手段,在大肠杆菌中异源表达分枝杆菌来源的麦角硫因的合成,研究获得工程菌,进行发酵罐培养,通过添加IPTG诱导打开合成开关,并持续性补加前体物质组氨酸,进行长达216h即9天的发酵,最终可获得1.3g/L麦角硫因产量。但作为一种条件致病菌以及转基因菌株,其产品应用前景令人担忧。寻找天然来源且具有食源安全属性的微生物,并开发合适的生产工艺,对解决麦角硫因高效生产尤为重要。文献(van der Hoek,S.A.,B.Darbani,K.E.Zugaj,B.K.Prabhala,M.B.Biron,M.Randelovic,J.B.Medina,D.B.Kell and I.Borodina(2019)."Engineeringthe yeast<;em>;Saccharomyces cerevisiae<;/em>;for the production ofL-(+)-ergothioneine."bioRxiv:667592.)公开了酿酒酵母Saccharomyces cerevisiae经基因工程改造,对外源基因进行表达,可合成麦角硫因,产量可达到630mg/L。
发明内容
为了探索利用食源安全性的微生物通过发酵法生产麦角硫因的工业化途径,本发明利用基因工程技术来对天然圆红酵母(或称圆红冬孢酵母,Rhodotorula toruloides)进行遗传改造,构建出了高产麦角硫因的菌株。具体而言,本发明包括如下技术方案:
一种EGT1基因表达盒,其选自SEQ ID NO:1、SEQ ID NO:2或者SEQ ID NO:3。
其中SEQ ID NO:1包含启动子pGPD、来源于粗糙链孢霉Neurospora crassa的酶egt1的编码基因NcEGT1和酿酒酵母来源的CYC1t终止子,并在首尾分别添加XbaI和PmeI酶切位点,用于后续的质粒构建,本文中命名为Pgpd-NcEGT1-ScCYC1t;
SEQ ID NO:2包含启动子pGPD、来源于黑麦麦角菌Claviceps purpurea的酶egt1的编码基因CpEGT1和酿酒酵母来源的CYC1t终止子,并在首尾分别添加XbaI和PmeI酶切位点,用于后续的质粒构建,本文中命名为Pgpd-CpEGT1-ScCYC1t;
SEQ ID NO:3包含启动子pGPD、来源于胶红酵母Rhodotorula mucilaginosa的酶egt1的编码基因RmEGT1和酿酒酵母来源的CYC1t终止子,并在首尾分别添加XbaI和PmeI酶切位点,用于后续的质粒构建,本文中命名为Pgpd-RmEGT1-ScCYC1t。
根据本发明的第二个方面,提供了一种EGT1基因表达质粒,其通过将上述的基因表达盒克隆在Puc57质粒上而获得。对应于基因表达盒名称,分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
根据本发明的第三个方面,提供了一种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒),其通过包括下述步骤的方法构建:
1)使用限制性内切酶XbaI/PmeI对如权利要求2所述EGT1基因表达质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t进行酶切,分别凝胶回收3.7kb、3.6kb、4.9kb片段,得到EGT1基因表达片段;
2)使用限制性内切酶XbaI/PmeI对核苷酸序列为SEQ ID NO:4的质粒pZPK-PGPD-Hyg-Tnos进行酶切,凝胶回收8.3kb片段,得到质粒骨架;
3)将步骤1)中得到的EGT1基因表达片段与步骤2)中得到的质粒骨架相连接,得到麦角硫因功能基因表达质粒。与质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t相对应地,麦角硫因功能基因表达质粒分别为pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1质粒。
文献(Lin,X.,et al.(2014)."Functional integration of multiple genesinto the genome of the oleaginous yeast Rhodosporidium toruloides."FEMS YeastResearch 14(4):547-555.)中描述了上述质粒pZPK-PGPD-Hyg-Tnos。
根据本发明的第四个方面,提供了一种构建麦角硫因生产菌的方法,包括下述步骤:将上述的麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)转化入能够产生麦角硫因的菌株中,得到阳性克隆株。
上述菌株可以为圆红酵母(或称圆红冬孢酵母,Rhodotorula toruloides)。
在一种实施方式中,上述方法是,将麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)通过农杆菌介导转染圆红酵母,得到阳性转化子。
例如,上述方法可以具体包括如下步骤:
A.将上述的麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)转化入农杆菌感受态细胞,得到农杆菌工程菌;优选农杆菌是根癌农杆菌;
B.将农杆菌工程菌菌液与圆红酵母菌液相混合,进行抗性筛选,经表型及PCR验证,获得阳性转化子,从而实现农杆菌介导转染圆红酵母。
根据本发明的第五个方面,提供了一种麦角硫因生产菌,其通过构建麦角硫因生产菌的方法得到。
在一种实施方式中,上述麦角硫因生产菌为圆红酵母,优选原始圆红酵母菌为中国普通微生物保藏管理中心保藏的Rhodotorula toruloides 2.1389。
上述麦角硫因生产菌可以通过发酵法生产麦角硫因。
本发明通过基因工程手段,对天然圆红酵母进行遗传改造,获得具有食源安全性的麦角硫因高产菌种。经发酵验证,麦角硫因产量可达到约1.5g/L,构建的工程菌株经多次传代,遗传性状相对稳定,具有较好的工业应用潜力。
附图说明
图1是本发明构建的质粒pZPK-PGPD-Hyg-Tnos的图谱结构示意图。
具体实施方式
egt1是个多功能酶,可以催化底物组氨酸生成麦角硫因前体S-(组氨酸甜菜碱)-2-)基-L-半胱氨酸S-氧化物(S-(hercyn-2-yl)-L-cysteine S-oxide)。研究发现,通过在可产生麦角硫因的菌株中表达egt1基因,能够促进麦角硫因的产生。
在本文中,为了描述简便,有时会将某种蛋白比如egt1与其编码基因(DNA)名称混用,本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如,对于egt1,用于描述酶功能或类别时,指的是蛋白质;在作为一种基因描述时,指的是编码该酶的基因。
我们筛选出三种微生物来源的egt1酶,分别来源于粗糙链孢霉Neurosporacrassa、黑麦麦角菌Claviceps purpurea和胶红酵母Rhodotorula mucilaginosa,其编码基因分别为NcEGT1、CpEGT1和RmEGT1。
NcEGT1基因,根据NCBI数据库,Genbank accession:XP_956324.3序列,经密码子优化,可以获得如下碱基序列:
atgccatccgctgagtcgatgaccccgtcctccgctttgggccagctcaaagccaccggccagcacgtcctctcgaagctccagcagcaaacttcgaacgccgacatcatcgacattcgccgcgtcgctgttgagatcaacctcaagaccgagatcacctcgatgttcagaccgaaggacggtccgagacaactcccgactttgctcttgtacaatgagcgcggcctccaattgtttgagcgcattacctatttggaggagtactacctcactaatgatgagattaagatcctcaccaagcacgctaccgaaatggcctcgtttatcccgtccggcgccatgatcatcgagctcggttcgggtaacttgcgcaaggttaacttgctcctcgaggccctcgataacgctggtaaggccatcgactactacgctctcgacttgtcgcgcgaggaattggagcgcactctcgctcaagtcccgtcgtacaagcacgttaagtgccacggtctcctcggcacctacgacgatggccgcgactggctcaaggccccagagaacatcaacaaacagaagtgcatcctccacttgggctcctccattggcaactttaaccgctccgacgctgccacttttttgaagggcttcactgacgtcctcggtccgaacgacaaaatgctcatcggtgtcgatgcttgcaatgacccggcccgcgtctaccatgcctacaacgacaaagtcggcatcacccacgaattcatcttgaacggcttgcgcaacgccaacgagattatcggcgagaccgcctttatcgaaggcgactggagagtcatcggcgaatacgtctacgacgaggagggtggtcgccaccaagccttctatgccccaactcgcgatactatggtcatgggcgagctcatcagatcccacgatcgcatccagatcgagcagtccctcaagtactccaaggaggagtccgagcgcctctggtcgactgctggtctcgaacaagtctccgaatggacctacggcaacgaatacggtctccatttgctcgccaaatccagaatgtcgttctccctcatcccgtcggtctatgcccgctcggctctcccaactctcgacgattgggaagccttgtgggccacttgggacgtcgtcaccagacagatgttgccgcaagaggaactcctcgagaagccgatcaagctccgcaacgcttgcatcttctacctcggccacattccaaccttcctcgacatccagttgaccaagaccaccaaacaagccccgtccgagccagctcacttctgcaagatcttcgagcgcggtatcgacccggatgtcgataatccggagctctgccacgctcactccgagatcccggacgagtggccaccagtcgaggagatcctcacctatcaagagaccgttcgctcgcgcttgcgcggtttgtacgcccacggtattgctaacatcccgcgcaacgttggtagagctatttgggtcggtttcgagcacgagctcatgcacatcgagaccctcctctacatgatgttgcagtcggacaagaccctcattccgacccatattccacgcccggacttcgataagttggcccgcaaggctgaatcggagagagtcccgaaccagtggttcaagatcccggcccaagagatcactatcggtctcgacgacccggaggacggttccgacatcaataaacactacggctgggataacgagaagccaccgcgcagagttcaagtcgccgcctttcaagcccaaggtagaccaatcactaacgaggagtacgcccaatacttgctcgagaagaacatcgacaaactcccagcctcgtgggctcgcttggacaatgagaacatctccaacggcaccaccaattccgtttccggtcaccactccaaccgcacctccaagcagcagctcccgtcctcgttcttggagaaaaccgccgtcagaaccgtttatggtttggttccactcaagcacgccctcgactggccggtcttcgcttcgtatgacgaactcgctggttgcgccgcttacatgggcggtcgcatcccgactttcgaagagacccgctccatctatgcctatgccgacgccctcaagaaaaagaaggaagctgaacgccagttgggtcgcaccgtcccagctgttaacgcccacctcaccaataacggcgttgaaatcaccccgccgtcgtcgccgtcgtccgaaaccccggccgaatcctcctccccgtcggattcgaacaccactctcatcaccactgaggacttgttctcggacctcgacggcgccaacgtcggcttccataactggcacccgatgccaatcacctcgaagggtaacactctcgtcggtcaaggtgagctcggcggtgtctgggaatggacctcgtcggtcctccgcaagtgggagggcttcgagccgatggagctctatccgggctacactgctgacttcttcgacgaaaagcacaatatcgtcctcggcggttcgtgggctacccacccaagaatcgccggccgcaagtcctttgtcaactggtaccagcgcaactacccgtatgcttgggtcggcgccagagttgtcagagacctctaa;
CpEGT1序列参考文献van der Hoek,S.A.,et al.(2019)."Engineering theyeast Saccharomyces cerevisiae for the production of L-(+)-ergothioneine."bioRxiv:667592.其为
atgactgccgttaagcaaattcctgaaagaaaggtgttgatagattcaaatcataagtctccatcaaaaccgggtaaacatcctaattctgtcattgatatcaggtctaataaggacgatttaaatttacgtcatgccctagtctcatcttttaatccacacgatggaaaacctaggtggctacctactatgttattgtacgacgaaaaaggtttacaattgtttgaagatataacttacttagatgagtattatttgactggctacgaaattgaattattgaagaaacattcagcagaaattgcagctgctattcctgatggttctatggtcatcgaattgggctctggtaatttgagaaagatctgtttgttgttacaagcctttgaggattcacataagtctatcgactactatgcattagatttatcacaaaaggaattagaaagaactttgagccatgttcctgactttaaatatgtctcttgtcatggactgctaggtacatatgatgatggtgttacatggttgaaacaaccaggtatagtcaataagactaagtgcatcatccatcttggttcgtctattgggaattttcatagaaatgaagctgccgatttcctgcagacatttgctgatgtaatgaaaccagacgactctatggttattggtcttgattcatgcggtaatccagagatgtctcgcattcaaagattcattttgaacggcttatccaatgctaatagcgtttatggcaaggaaatattctatgttccagattggagagtaattggtgaatatgtttacgatgatgaaggtggcagacaccaggcttttatttcacctttgaaagaagtcactgctttagggtctgttattaaagcccatgaaagaattaaaattgaacaatctttgaagtactctaaggcctcagctgacgatttatggagaaatgctggctttcgagaaactcaaacttggacgagaaacggtgaatatggactacatatgttgcaaagagctgatccgcccttctctaaggctccttctttgtatgcagctaatactcttccctctctttctgattggagagcattgtggtgtgcctgggatattgtcactagagctatgttgccacaacaggaattgactgagaaacctatagagttaagacatgcctacatcttttaccttggtcatattcctaccttcttagacatccagttaaccaaaacatcagcatgggctccaacctctccagtttcttatcatgccattttcgagcgcggcattgatcccgatgttgataacccagaaaagtgtcatgatcactcagagattccagatgaatggccaccagtcgaagaaattattgcttatcaagatagggtgcgtgttagattgacagaactgtataaacagggtgtgcacacaattacaagaaaggctgctagagctatctgggtttcatttgaacatgaagctatgcatttggaaaccttgttgtatatgatgctacaaagtgataaagtgttgccacctccacacactggcgttccagactttgaaagaatggcaactaaggctttcgaagctcgtacgcaaaatatgtggttcgaaattccagaacagactattagtcttggaacagatgatccagaagatggggatgaagacgttcattttggatgggacaacgaaaaaccagttagaagagttaaggttcacgcgttgcaagctcaaggaagaccaattacaaatgaggaatacgcattatatatttaccataccaactcttctaaactgccagcatcttggagttcgtccccttcatcttctctgtctaacggcgtgtctcatcccagctcccataacaagcatattccaactgatttgcctcattccttcttgcaaggtaagtttgttagaaccgtatatggtttgatacctttatctttggcgttggattggcctgttcaagcttcttatgatgaattagctgactgtgcattatggatgggtggaagaattccaaccttagaggaagccagatcaatctatgcctttgttgaatctaaaacgcaaatagcaacaggtaacacattggtcaagaaagttcctgctgttaatggacacttggttaataacggagttgaggaaactccaccacatgaatcctcttcggcagttgagaattctttattcatcgacttagccggtttgaacgtgggttttaaaagttggaatcctgaacctgttacatcttctggtacgtctttggctggacaatcctctatgggtggtgtatgggagtggacctcttctgttttaagaccacatgaagggttccacccaatggagttgtatcctggttatacagccgatttctttgatgaaaaacataatattgttctcggaggatcatgggctactcatccaagaatagcgggtagaaaaagctttgttaactggtatcaaagaaactatccgtacgcctgggctggtgccagacttgttaaagatgcttga;
RmEGT1,使用NcEGT1蛋白序列,在NCBI数据库blastp工具,限定胶红酵母Rhodotorula mucilaginosa基因组,进行序列比对,获得如下egt1序列:
atgccagacgccgcctcaaccgccgctgttcagcctccgcccttcatcctcgacctgcggaaccgctcgccgccgacctcgccgagccaggtcgcctccgatgccgctcgctcgtcctctccgtcgtccctgggcgaatcggagggcactacctcgaccaatgctacccccgaggccccctccgatccgctccgcgagcagatcatcgccggcttgatcggatcgcccaagcccaccgttccaggcaagacggaaaaggatcgcgcgtatgcgtaccgccgcacgatcccgaccatgacactgtacagcgagcgtggcctgtctatctacgaggaaatcaccaagaccaaggtatgctcggccctttccgtccgctttggttcacgaggggccaatgctttctcagcaggccctcgcgcgccgtcggccccacttgccctacggcgtgcgggctctccttcggactaggtggctggcaagcatcccctttgcagacgcactgacctcgtctttgcgtttcgattcaggcctactacccattcgaagccgaaaaggagattctcgaaaagtatggagacgaaattgcctgccgcatgtttggactgccgtcggccttgctcgtcccggacgacgtagtccgtggcaaagacgaccagtacgaaccctctccggcttcaaacatcggcgcgaagaaggagaagtggtgagccggcgttcggaactataacggctcctcatccgcggtacaggcgctgacgcactgcttacaggggcgacgtcgctgtcggtctccacaactacggtgtcaacggctctgcgaacctcgcgcagaacaatgtcgtcgccacgcagggcctcgcggtcgagcttggctcgggctcgctcgacaagacccgtcatctcctccgctctatggccaagctgttgcagtctcgcgacgagggcagctgcccggtctctcctctgcgctccattgactacaaagcagtgcgtttggcgaaacctccgcgagtataggaaatcaatactgacctcccacgtctgccgctgaacagctcgaccttgaagcggcgtcgctctactcgacactgtcctcgcttgcatcggtcgaaggcgattgcgtcacgacggcggtcgacggacagccaaacgcgacgaaacgccgcgtttcggtctcgggtcttcacgccacctacgacgagggcctcgccttcttgaaggcgcagaacgatgctggcagcccgtcgagcgtcttctcagacctccccgacttgcccacgagtccaaagtcggttccgactaccttgtccagtgtcctggaggacgacagcgacgaaggcgagaccgctcaagcgtctcgcggcgagaacgctgcggaacgccgcgcgacttcgatcatgtggctcggttcctcgtgcggcaactacacccgcgaggaagcggtgcagttcctccgcaacatcgagctgcgcgagggcgataccatgctgatcggcatcgacggctgcgcggacgaacctcgtatcgagactgcctacaacgacccgcaggtgagtctgatgtcctgccaaactgaccagtatgagctgctctgacccttgtttgccgctgatcctccagggcgtcacccgcgcgttcattctcgagggcatcgatgtcgcaggccgcaccctcggcggagacgcggccgaagtgctgcagcagaagaactttgactatgtcaaccgctggaacgccgagctcggaaggcacgaggtgagctgccttccgctgtccagaggagagcagcctgagttgcaccggcagccctgttctgacccgtccccgcgacaccctcgcaggcttacgtccgcgccaacaaagatctcacaatcccgatcacgggcgcagacgacgtcaccgaggtcaagctcgaagagggagagtaagtcccgcacttttgcgcgttgagccggtgccatcacaagagcccttcctgacgtttcctctcgcaccaggctcctcaacattgaagtctcttacaagtacacctatgcggaggcggcggcgctcttccacttggcgggcttccggctcatccagcactggaccgactcgtcgcgctcgcattacctctacctggtcgaaaagccccgcatgtggttcccgtcgacgaccgagagcgctgccaagatgctcggtatcgaggtcgagcccgaggagaaggagaccgactacggtgtcccgactctggaaaagtgggaagaaatgtggcgggcgtgggacggcttgatggtgaggatgccctcgtcggcgaaggaattccgcaagcgatgagtggacgctcacctgaaaccgcttcttctaccagctcgagatcatccccaagtcgctgcacttccagaagccgatcccgctccgccacatcccgctcttctacgtcgggcacatacccgctttccgggatattcacctcgcgaggtacttcaacgagccgctgaccgaaccggcaaagtttgccgacatcttcgagcgcggaatcgatccctgtgtcgacgaccccgagaccgtcacgcactggcactcggaagtcccgaaggacgaagcgtgctggccttctctccaggagattacggcgtacgaagcgtccgttcgcgatcgcgtccgcaaggtgtacgccgaacacgagggcaagtggacgaccaagctcgcccgtgtgctgatgatgacttttgagcacgagatgtgcgtccttcctgcttcgggctgattcggtacccatgtcatcactcgactcactcatcctcttgggtccccttgggccccacaggatgcactgggagacctccgtctacatctgtctccaagccgcgagctcgctcaaccttccgccgggaacggcgattcctgatttccgctcgctggcacgtcaagcaaaacgtgatctgcatcagaacggcggcggacagcgactctccttccccgctcaggaggtcacagtcggtcacgacgacgacgataccatcgacgaccagacgccgtttgaccctgcccgagaatacggctgggatgtcgagcaccctcgccgccagcttcgcgtcgacgcattcgagatcgaggtcctcccgatctcgaacggagagtacaaaacctggcttaccgagacgcagcaactctcgaacaaggcgctcatcccgtcgagctggacggacgagactggcgaactctgcgtgaaaacgttgttcggcttggtccccctcactctcgccgaggagtggcccgtggctgcgagcgcggaacagctcgagaagtttgccaaggtgggtatcctttgctcgagcaagaatagggaccgaagaagactgactcgtaactttctgcactcgacaggccaaaggaggacgcttgccgacgttcggcgagctgtcggcgttcaaccagcacaaccctagctctacgcctctcgccaacatcggcctcgcaaacctgcatccggttgccccttcggtacccgggaaagcgcgtgacggcagccagctcccgatcaccgacggaggcctgtggcaatggacctcgacggtcctcgagccctgggctggttacagcggatcggttctttaccctggttactccagtgacgtgagtcgagtgcgactgtgaatgaagtgagatggggcaactgacattaagcgccctctcttccctctagttcttcgacggcaaacaccacatcgtgctgggcgcgagctacgcgagtcctcgtcgcttggcccgcccttctttcctgaactggtaccagaagaactgtgcgtttctggcgctcttccaaggaccgttgccgccagatccccgtactgacatgctctgccgcagaccctttcatgctcggcggcgctcgtgtggcgtacgatgtgtga。
在本发明构建的工程菌中,egt1基因以基因表达盒的形式存在。在本文中,术语“egt1基因表达盒”、“基因表达盒”和“表达盒”表示相同的意义,可以互换使用。
以上述NcEGT1、CpEGT1和RmEGT1为目的基因,可以构建出三种egt1基因表达盒,分别为Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQ ID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)。三个表达盒均在首尾分别设计添加XbaI和PmeI酶切位点,用于后续的质粒构建。
将上述基因表达盒元件序列送苏州金唯智生物技术公司,进行合成,分别获得Puc57-Pgpd-NcEGT1-ScCYC1t,Puc57-Pgpd-CpEGT1-ScCYC1t,Puc57-Pgpd-RmEGT1-ScCYC1t质粒,用于后续的酶切连接克隆构建。
通过在合适的宿主比如某些圆红酵母中表达egt1基因,提高了圆红酵母生产麦角硫因的能力。构建的圆红酵母工程菌在通过发酵生产麦角硫因时,不需要在培养液中添加组氨酸甜菜碱、组氨酸或者半胱氨酸等就能够产生大量的麦角硫因。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
本文中的全基因合成、引物合成及测序皆由苏州金唯智生物技术公司完成。
本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。比如感受态细胞转化方法及感受态制备方法均参照《分子克隆实验指南》(第三版)第1章96页进行。必要时可以通过简单试验确定具体实验条件。
主要培养基及溶液:
LB培养基:5g/L酵母提取物,10g/L胰蛋白胨,10g/L氯化钠。(LB固体培养基另加20g/L琼脂粉。)
YEB培养基:牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,蔗糖5g/L,MgSO4·7H2O0.5g/L,pH 7.4。(YEB固体培养基另加15g/L琼脂粉。)
YPD培养基:1wt%酵母膏,2wt%蛋白胨,2wt%葡萄糖。
农杆菌介导酵母转化溶液及培养基:
MM溶液:3.625g KH2PO4,5.125g K2HPO4,1.250g MgSO4·7H2O,0.375g NaCl,0.165g CaCl2·2H2O,0.0062g FeSO4·7H2O,1.250g(NH4)2SO4,溶解于ddH2O中定容至1L;
M-100微量元素溶液:30mg H3BO3,70mg MnCl2·4H2O,200mg ZnCl2,20mgNa2MoO4·2H2O,50mg FeCl3·6H2O,200mg CuSO4·5H2O,溶解于ddH2O中定容至500ml。
M-100盐溶液:16g KH2PO4,4g Na2SO4,8g KCl,2g MgSO4·7H2O,1g CaCl2,8ml M-100微量元素溶液,溶解于ddH2O中定容至1L。
IM液体培养基:80ml MM溶液,0.36g葡萄糖,1ml甘油,溶解于ddH2O中定容至200ml。灭菌冷却到50℃左右后添加下列成分:8ml 1M MES,4ml 10mM AS,MES和AS的终浓度分别为40mM和200μM。
IM固体培养基:160ml MM盐溶液,0.36g葡萄糖,2ml甘油,溶解于ddH2O中定容至400ml,最后加入6g琼脂粉。灭菌冷却到50℃左右后添加下列成分:16ml 1M MES,8ml 10mMAS,MES和AS的终浓度分别为40mM和200μM。
麦角硫因检测材料和方法:
麦角硫因标准品,购自中国医药(集团)上海化学试剂公司。
HPLC检测条件为:安捷伦高效液相色谱仪1260infinity II,依利特ODS-BP色谱柱,柱温40℃,流动相:A,磷酸二氢铵(配置方法,称取1.1503g磷酸二氢铵+400mL纯化水,氨水调pH至5.0,再加100mL纯化水);B:乙腈。A:B=99:1,流速1mg/min,进样量10μl,检测波长258nm。
胞外产物检测:发酵菌液进行10000rpm,4min离心,取上清,用2μm过滤器进行过滤处理后,样品进行HPLC检测。
胞内产物检测:发酵菌液进行10000rpm,4min离心,收集菌体,称取100mg湿菌体,转移至干净的1.5ml离心管中,加1ml无菌水,涡旋,重悬菌体,然后于90℃进行温浴30min,温浴结束,12000rpm,10min离心,取上清,用2μm过滤器进行过滤处理后,样品进行HPLC检测。
实施例1:原始菌种筛选
根据检索到的文献报道,筛选能够产生麦角硫因的食源安全性微生物。自中国普通微生物保藏管理中心购买多株圆红酵母进行初步筛选对比,评估各菌株麦角硫因产量。
将购买的菌种接种在酵母培养基中,进行摇瓶发酵。酵母种子培养基组成:葡萄糖20g/L,酵母粉10g/L,蛋白胨20g/L。
将甘油管种子,接种种子培养基,30℃,250rpm,17h培养,OD600>16时可转接。
酵母摇瓶发酵培养基:硫酸铵0.5g/L,酵母粉10g/L,蛋白胨10g/L,谷氨酸3g/L,生物素0.6mg/L,盐酸硫胺素0.1g/L,硫酸镁5g/L,七水硫酸亚铁0.278g/L,一水硫酸锰0.085g/L,葡萄糖25g/L,磷酸二氢钾2.5g/L。
摇瓶发酵:使用250ml三角瓶,装液量50ml,接种比例2%,于摇床30℃,250rpm,培养72h。
摇瓶发酵后,评估各菌发酵后的胞内和胞外麦角硫因产量。结果见表1。
表1、不同圆红酵母发酵生产麦角硫因能力的对比
圆红酵母菌种 | 总产量mg/L |
Rhodotorula toruloides 2.2424 | 120 |
Rhodotorula toruloides 2.1003 | 230 |
Rhodotorula toruloides 2.1609 | 187 |
Rhodotorula toruloides 2.1389 | 385 |
Rhodotorula toruloides 2.499 | 281 |
Rhodotorula toruloides 2.278 | 80 |
Rhodotorula toruloides 2.107 | 34 |
由表1可见,不同的圆红酵母亚种生产麦角硫因的能力是有差别的,其中Rhodotorula toruloides 2.1389最优。下述实施例中将该菌株作为遗传改造的出发菌。
实施例2:构建质粒pZPK-PGPD-Hyg-Tnos
圆红酵母的遗传操作方法可参考下述文献:Lin,X.,et al.(2014)."Functionalintegration of multiple genes into the genome of the oleaginous yeastRhodosporidium toruloides."FEMS Yeast Research 14(4):547-555.
参考该文献,根据公开序列,确定质粒pZPK-PGPD-Hyg-Tnos完整序列,并送苏州金唯智生物科技有限公司进行合成,获得质粒pZPK-PGPD-Hyg-Tnos(SH205),其结构如图1所示,其碱基序列为SEQ ID NO:4,8378bp。
以此为质粒骨架,进行后续的质粒构建。
实施例3:构建EGT1基因表达盒和表达质粒
对于三种微生物来源的目的基因NcEGT1、CpEGT1和RmEGT1,设计以pGPD为启动子来控制该功能基因的表达;以酿酒酵母来源的CYC1t终止子来控制该功能基因的表达终止;并且在首、尾分别设计添加XbaI酶切位点(tctaga)和PmeI酶切位点(gtttaaac),以便用于后续的质粒构建。设计的基因表达盒分别为Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQ ID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)
将设计的基因表达盒序列送苏州金唯智生物技术公司进行合成,并将上述的基因表达盒克隆在Puc57质粒上,获得EGT1基因表达质粒,用于后续的酶切连接克隆构建。对应于基因表达盒名称,分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
分别获得Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)质粒。
实施例4:构建麦角硫因功能基因表达质粒
4.1在收到上述Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t和Puc57-Pgpd-RmEGT1-ScCYC1t三个质粒后,分别使用XbaI/PmeI限制性内切酶进行酶切,分别凝胶回收3.7kb,3.6kb和4.9kb片段。
4.2骨架质粒pZPK-PGPD-Hyg-Tnos经XbaI/PmeI酶切后,回收8.3kb片段。将该片段与上述3种基因表达盒的酶切片段分别进行连接,获得三种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒)pZPK-NcEGT1、p ZPK-CpEGT1和p ZPK-RmEGT1,用于转化宿主。连接方法为:使用购买自NEB公司的T4连接酶体系,参照使用说明,按载体和基因表达盒摩尔比例1:5添加到T4连接酶体系,16℃,连接2h,将连接产物转化DH5a大肠杆菌,使用卡那霉素进行克隆筛选,经过菌落PCR、质粒酶切和质粒测序验证后,获得上述三种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒)pZPK-NcEGT1、p ZPK-CpEGT1和p ZPK-RmEGT1。
实施例5:制备根癌农杆菌工程菌
5.1根癌农杆菌菌株AGL-1感受态的制备:从含50μg/ml羧苄青霉素(Carb)的YEB平板上挑取菌株AGL-1(上海康朗生物科技有限公司)的单菌落到5ml YEB液体培养基中(含50μg/ml Carb),于28℃、200rpm摇床培养过夜。取2ml菌液到50ml YEB(含50μg/ml Carb)中,于28℃、200rpm摇床培养至菌液在600nm处的OD吸收值为0.5左右。取出菌液,冰上冰浴30min后,4℃,8000rpm离心5min收集菌体。用10ml ddH2O洗一次。4℃,8000rpm离心5min再次收集菌体。弃上清液,加入2ml终浓度为10%无菌甘油,每份100μl分装,即为根癌农杆菌菌株AGL-1感受态。
5.2质粒转化至根癌农杆菌AGL-1:将农杆菌感受态置于冰上10min。加1~2μg实施例4中构建的EGT1基因转化质粒pZPK-NcEGT1、pZPK-CpEGT1和pZPK-RmEGT1,用移液器轻轻吹打混匀,转移至0.2cm无菌电转杯中,冰上放置5min,2.5kv电击,加入1ml液体YEB,于28℃,150rpm培养3h。8000rpm离心2min收集菌体。去掉上清后用100μl液体YEB重悬菌体,并均匀涂布在YEB平板上(含50μg/ml Carb和50μg/ml Kan),于28℃培养2~3天。挑取转化子,用3ml液体YEB(含50μg/ml Carb和50μg/ml Kan)于28℃、220rpm摇菌过夜。PCR验证转化子,并将其菌液保存于-80℃(含15%的甘油),得到农杆菌工程菌AGL-1/pZPK-NcEGT1、AGL-1/pZPK-CpEGT1和AGL-1/pZPK-RmEGT1。
实施例6:农杆菌介导转染圆红酵母
6.1农杆菌工程菌培养:将实施例5中构建的农杆菌工程菌AGL-1/pZPK-NcEGT1、AGL-1/pZPK-CpEGT1、AGL-1/pZPK-RmEGT1单菌落接种于4-5ml的LB培养基(含抗生素Amp和Kan)中,30℃过夜培养。测定OD值,一般需要达到OD600=4.2以上,计算稀释倍数,应稀释(据实计算)倍,可达到OD=0.6。取一定量的菌液于1.5ml离心管中,12000rpm离心30s,将菌体沉底,加入灭菌蒸馏水悬浮,再次离心沉底。最后用计算好的水体积将菌悬浮至OD=0.6。备用。
6.2圆红酵母宿主培养:将圆红酵母Rhodotorula toruloides 2.1389单菌落接种于4-5ml的YPD培养基中过夜培养,约15-16h。测定OD600值,计算稀释倍数,应稀释(据实计算)倍,稀释OD至0.6。按需要,取一定量的菌液于1.5ml离心管中,13000rpm离心30s,将菌体沉底,加入灭菌蒸馏水悬浮,再次离心沉底。最后用计算好的水体积将菌悬浮至OD=0.6。备用。
6.3平板培养转染:将上述稀释好的农杆菌工程菌液和酵母菌液混合后,涂布平板:分别取100ul混合后,涂在IM平板上,24℃培养2-3天。
6.4抗性筛选:IM平板上获得的菌落,使用YPD液体培养基洗脱刮下,将菌悬液涂布到YPD+潮霉素抗性平板上,30℃培养4天。待有转化子出现后,挑选克隆子,进行YPD+潮霉素抗性平板上进行划线传代培养,需连续传代3代,对连续传代抗性表型稳定的克隆,进行基因组抽提,进行特定的靶点序列PCR验证,并经表型验证,获得阳性转化子。
实施例7:工程菌摇瓶发酵考察
按照实施例1的方法,对实施例6中获得的阳性转化子进行摇瓶发酵考察。摇瓶发酵后,评估各工程菌发酵后的胞内和胞外麦角硫因产量。结果见表2。
表2、不同圆红酵母工程菌摇瓶发酵生产麦角硫因能力的对比
由表2可见,三种不同微生物来源的EGT1基因在同一个圆红酵母亚种Rhodotorulatoruloides 2.1389中表达来促进麦角硫因生产的能力是有差别的,其中NcEGT1最优,且不同微生物来源EGT1基因的表达都能提高麦角硫因产量。
实施例8:工程菌发酵罐发酵考察
圆红酵母划线平板上,使用接种环取单菌落接种子培养基(葡萄糖20g/L,蛋白胨10g/L,酵母提取物5g/L,pH7.0)试管,4ml装液量,30℃,230rpm培养过夜;按5%v/v的接种量,接种到种子培养基摇瓶,250ml三角瓶装液50ml,30℃,230rpm摇床培养过夜,准备种子。将种子液按10%v/v的接种比例,转接发酵罐培养基(葡萄糖60g/L,蛋白胨10g/L,玉米浆粉10g/L,硫酸亚铁10mg/L,pH6.5)。
发酵罐搅拌转速为400~800rpm,溶氧偶联,溶氧控制40%,温度为30℃,空气流量3L/min,补加氨水和组氨酸,控制pH6.5,发酵可以一直持续到120h。其间,在12-24h,以20ml/h的速率流加500g/L的葡萄糖溶液;24h-72h,以10ml/h的速率流加500g/L的葡萄糖溶液;72h-96h,以3-5ml/h的速率流加500g/L的葡萄糖溶液;24-96h,以5-8ml/h的速率流加100g/L的玉米浆溶液。发酵结束后,评估各工程菌发酵后的胞内和胞外麦角硫因产量。结果见表3。
表3、不同圆红酵母工程菌发酵罐发酵生产麦角硫因能力的对比
由表3可见,与摇瓶发酵类似,三种不同微生物来源的EGT1基因在同一个圆红酵母亚种Rhodotorula toruloides 2.1389中表达来促进麦角硫因生产的能力是有差别的,不同微生物来源EGT1基因的表达都能提高麦角硫因产量,其中NcEGT1仍是最优,发酵后麦角硫因产量接近1.5g/L,具有工业化应用前景。
实施例9:工程菌遗传稳定性评估
将上述菌株Rhodotorula toruloides 2.1389,Rhodotorula toruloides2.1389-NcEGT1,Rhodotorula toruloides 2.1389-CpEGT1,Rhodotorula toruloides2.1389-RmEGT1,进行无抗种子平板划线传代培养,传代后对其进行摇瓶发酵验证,评估产量,判断工程菌的遗传稳定性。
上述实验结果表明,本发明方法构建的基因工程菌株经过6次传代,产物产量仍高于出发菌株Rhodotorula toruloides 2.1389,表明工程菌的遗传性状相对稳定。
序列表
<110> 浙江华睿生物技术有限公司
<120> 一种构建麦角硫因生产菌的方法
<130> SHPI2010310
<150> CN202010194710.8
<151> 2020-03-19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3667
<212> DNA
<213> 人工序列()
<400> 1
tctagagatc tcttctccgg ctttccgtcc ctcgagtacc gtcgcggcgg ctgttttgca 60
tagcggagta agtcatgcgg tcctgtgcaa gcaagtgtcg tcgagaccgg cgactaaccc 120
cggaggccga gcgggtagtc ctccctgcgc aacaacgcga tttcatcagt gggccgtttc 180
gccagaagtt gtcccactcg cagggtgctc acatgaggat ctgaggcggg atcaggggtc 240
ggatgttctt gatccggcgg tcatcgaggt cgttgctggt cgtcgcacat gtccatcatg 300
acgagttgag tcagtctgtc agtcaggctc tgaagcgggg cggtaggctg cgtggctcac 360
agcgccgtct tgacgttggc ttgaggtgtc tgagtgccgt ctgcgcgcat cttgccctgg 420
atccgagtgt agttggttgg tcaatcagga tcttgcgaaa aagttgcgcg tgcgtgggcg 480
ccacgctcgt tcaagcgctc gttgattcat tcattgcctc caaacagggg ggaaccaggc 540
agcgtaaacc ccgcggagtt gtgcccgtcg ggttgccagc ggaacgtcag ccggacaatc 600
tcgcctcgct cgctcgtcac cctagacgca gaactagaga agcgaatcta ccctggcttg 660
tttgctgagg acgactctgc gccgcgctcc gtcaacgaca ccgctcgcca cctcgctcgt 720
ccagttcacc tctcttcgct caactactac cccaaccgat tcgcttcgct ctagctctac 780
ccgccgcgcg ttcagccagt cgagctacaa gatgccatcc gctgagtcga tgaccccgtc 840
ctccgctttg ggccagctca aagccaccgg ccagcacgtc ctctcgaagc tccagcagca 900
aacttcgaac gccgacatca tcgacattcg ccgcgtcgct gttgagatca acctcaagac 960
cgagatcacc tcgatgttca gaccgaagga cggtccgaga caactcccga ctttgctctt 1020
gtacaatgag cgcggcctcc aattgtttga gcgcattacc tatttggagg agtactacct 1080
cactaatgat gagattaaga tcctcaccaa gcacgctacc gaaatggcct cgtttatccc 1140
gtccggcgcc atgatcatcg agctcggttc gggtaacttg cgcaaggtta acttgctcct 1200
cgaggccctc gataacgctg gtaaggccat cgactactac gctctcgact tgtcgcgcga 1260
ggaattggag cgcactctcg ctcaagtccc gtcgtacaag cacgttaagt gccacggtct 1320
cctcggcacc tacgacgatg gccgcgactg gctcaaggcc ccagagaaca tcaacaaaca 1380
gaagtgcatc ctccacttgg gctcctccat tggcaacttt aaccgctccg acgctgccac 1440
ttttttgaag ggcttcactg acgtcctcgg tccgaacgac aaaatgctca tcggtgtcga 1500
tgcttgcaat gacccggccc gcgtctacca tgcctacaac gacaaagtcg gcatcaccca 1560
cgaattcatc ttgaacggct tgcgcaacgc caacgagatt atcggcgaga ccgcctttat 1620
cgaaggcgac tggagagtca tcggcgaata cgtctacgac gaggagggtg gtcgccacca 1680
agccttctat gccccaactc gcgatactat ggtcatgggc gagctcatca gatcccacga 1740
tcgcatccag atcgagcagt ccctcaagta ctccaaggag gagtccgagc gcctctggtc 1800
gactgctggt ctcgaacaag tctccgaatg gacctacggc aacgaatacg gtctccattt 1860
gctcgccaaa tccagaatgt cgttctccct catcccgtcg gtctatgccc gctcggctct 1920
cccaactctc gacgattggg aagccttgtg ggccacttgg gacgtcgtca ccagacagat 1980
gttgccgcaa gaggaactcc tcgagaagcc gatcaagctc cgcaacgctt gcatcttcta 2040
cctcggccac attccaacct tcctcgacat ccagttgacc aagaccacca aacaagcccc 2100
gtccgagcca gctcacttct gcaagatctt cgagcgcggt atcgacccgg atgtcgataa 2160
tccggagctc tgccacgctc actccgagat cccggacgag tggccaccag tcgaggagat 2220
cctcacctat caagagaccg ttcgctcgcg cttgcgcggt ttgtacgccc acggtattgc 2280
taacatcccg cgcaacgttg gtagagctat ttgggtcggt ttcgagcacg agctcatgca 2340
catcgagacc ctcctctaca tgatgttgca gtcggacaag accctcattc cgacccatat 2400
tccacgcccg gacttcgata agttggcccg caaggctgaa tcggagagag tcccgaacca 2460
gtggttcaag atcccggccc aagagatcac tatcggtctc gacgacccgg aggacggttc 2520
cgacatcaat aaacactacg gctgggataa cgagaagcca ccgcgcagag ttcaagtcgc 2580
cgcctttcaa gcccaaggta gaccaatcac taacgaggag tacgcccaat acttgctcga 2640
gaagaacatc gacaaactcc cagcctcgtg ggctcgcttg gacaatgaga acatctccaa 2700
cggcaccacc aattccgttt ccggtcacca ctccaaccgc acctccaagc agcagctccc 2760
gtcctcgttc ttggagaaaa ccgccgtcag aaccgtttat ggtttggttc cactcaagca 2820
cgccctcgac tggccggtct tcgcttcgta tgacgaactc gctggttgcg ccgcttacat 2880
gggcggtcgc atcccgactt tcgaagagac ccgctccatc tatgcctatg ccgacgccct 2940
caagaaaaag aaggaagctg aacgccagtt gggtcgcacc gtcccagctg ttaacgccca 3000
cctcaccaat aacggcgttg aaatcacccc gccgtcgtcg ccgtcgtccg aaaccccggc 3060
cgaatcctcc tccccgtcgg attcgaacac cactctcatc accactgagg acttgttctc 3120
ggacctcgac ggcgccaacg tcggcttcca taactggcac ccgatgccaa tcacctcgaa 3180
gggtaacact ctcgtcggtc aaggtgagct cggcggtgtc tgggaatgga cctcgtcggt 3240
cctccgcaag tgggagggct tcgagccgat ggagctctat ccgggctaca ctgctgactt 3300
cttcgacgaa aagcacaata tcgtcctcgg cggttcgtgg gctacccacc caagaatcgc 3360
cggccgcaag tcctttgtca actggtacca gcgcaactac ccgtatgctt gggtcggcgc 3420
cagagttgtc agagacctct aaacaggccc cttttccttt gtcgatatat gtaattagtt 3480
atgtcacgct tacattcacg ccctcctccc acatccgctc taaccgaaaa ggaaggagtt 3540
agacaacctg aagtctaggt ccctatttat tttttttaat agttatgtta gtattaagaa 3600
cgttatttat atttcaaatt tttctttttt ttctgtacaa acgcgtgtac gcatgtaacg 3660
tttaaac 3667
<210> 2
<211> 3565
<212> DNA
<213> 人工序列()
<400> 2
tctagagatc tcttctccgg ctttccgtcc ctcgagtacc gtcgcggcgg ctgttttgca 60
tagcggagta agtcatgcgg tcctgtgcaa gcaagtgtcg tcgagaccgg cgactaaccc 120
cggaggccga gcgggtagtc ctccctgcgc aacaacgcga tttcatcagt gggccgtttc 180
gccagaagtt gtcccactcg cagggtgctc acatgaggat ctgaggcggg atcaggggtc 240
ggatgttctt gatccggcgg tcatcgaggt cgttgctggt cgtcgcacat gtccatcatg 300
acgagttgag tcagtctgtc agtcaggctc tgaagcgggg cggtaggctg cgtggctcac 360
agcgccgtct tgacgttggc ttgaggtgtc tgagtgccgt ctgcgcgcat cttgccctgg 420
atccgagtgt agttggttgg tcaatcagga tcttgcgaaa aagttgcgcg tgcgtgggcg 480
ccacgctcgt tcaagcgctc gttgattcat tcattgcctc caaacagggg ggaaccaggc 540
agcgtaaacc ccgcggagtt gtgcccgtcg ggttgccagc ggaacgtcag ccggacaatc 600
tcgcctcgct cgctcgtcac cctagacgca gaactagaga agcgaatcta ccctggcttg 660
tttgctgagg acgactctgc gccgcgctcc gtcaacgaca ccgctcgcca cctcgctcgt 720
ccagttcacc tctcttcgct caactactac cccaaccgat tcgcttcgct ctagctctac 780
ccgccgcgcg ttcagccagt cgagctacaa gatgactgcc gttaagcaaa ttcctgaaag 840
aaaggtgttg atagattcaa atcataagtc tccatcaaaa ccgggtaaac atcctaattc 900
tgtcattgat atcaggtcta ataaggacga tttaaattta cgtcatgccc tagtctcatc 960
ttttaatcca cacgatggaa aacctaggtg gctacctact atgttattgt acgacgaaaa 1020
aggtttacaa ttgtttgaag atataactta cttagatgag tattatttga ctggctacga 1080
aattgaatta ttgaagaaac attcagcaga aattgcagct gctattcctg atggttctat 1140
ggtcatcgaa ttgggctctg gtaatttgag aaagatctgt ttgttgttac aagcctttga 1200
ggattcacat aagtctatcg actactatgc attagattta tcacaaaagg aattagaaag 1260
aactttgagc catgttcctg actttaaata tgtctcttgt catggactgc taggtacata 1320
tgatgatggt gttacatggt tgaaacaacc aggtatagtc aataagacta agtgcatcat 1380
ccatcttggt tcgtctattg ggaattttca tagaaatgaa gctgccgatt tcctgcagac 1440
atttgctgat gtaatgaaac cagacgactc tatggttatt ggtcttgatt catgcggtaa 1500
tccagagatg tctcgcattc aaagattcat tttgaacggc ttatccaatg ctaatagcgt 1560
ttatggcaag gaaatattct atgttccaga ttggagagta attggtgaat atgtttacga 1620
tgatgaaggt ggcagacacc aggcttttat ttcacctttg aaagaagtca ctgctttagg 1680
gtctgttatt aaagcccatg aaagaattaa aattgaacaa tctttgaagt actctaaggc 1740
ctcagctgac gatttatgga gaaatgctgg ctttcgagaa actcaaactt ggacgagaaa 1800
cggtgaatat ggactacata tgttgcaaag agctgatccg cccttctcta aggctccttc 1860
tttgtatgca gctaatactc ttccctctct ttctgattgg agagcattgt ggtgtgcctg 1920
ggatattgtc actagagcta tgttgccaca acaggaattg actgagaaac ctatagagtt 1980
aagacatgcc tacatctttt accttggtca tattcctacc ttcttagaca tccagttaac 2040
caaaacatca gcatgggctc caacctctcc agtttcttat catgccattt tcgagcgcgg 2100
cattgatccc gatgttgata acccagaaaa gtgtcatgat cactcagaga ttccagatga 2160
atggccacca gtcgaagaaa ttattgctta tcaagatagg gtgcgtgtta gattgacaga 2220
actgtataaa cagggtgtgc acacaattac aagaaaggct gctagagcta tctgggtttc 2280
atttgaacat gaagctatgc atttggaaac cttgttgtat atgatgctac aaagtgataa 2340
agtgttgcca cctccacaca ctggcgttcc agactttgaa agaatggcaa ctaaggcttt 2400
cgaagctcgt acgcaaaata tgtggttcga aattccagaa cagactatta gtcttggaac 2460
agatgatcca gaagatgggg atgaagacgt tcattttgga tgggacaacg aaaaaccagt 2520
tagaagagtt aaggttcacg cgttgcaagc tcaaggaaga ccaattacaa atgaggaata 2580
cgcattatat atttaccata ccaactcttc taaactgcca gcatcttgga gttcgtcccc 2640
ttcatcttct ctgtctaacg gcgtgtctca tcccagctcc cataacaagc atattccaac 2700
tgatttgcct cattccttct tgcaaggtaa gtttgttaga accgtatatg gtttgatacc 2760
tttatctttg gcgttggatt ggcctgttca agcttcttat gatgaattag ctgactgtgc 2820
attatggatg ggtggaagaa ttccaacctt agaggaagcc agatcaatct atgcctttgt 2880
tgaatctaaa acgcaaatag caacaggtaa cacattggtc aagaaagttc ctgctgttaa 2940
tggacacttg gttaataacg gagttgagga aactccacca catgaatcct cttcggcagt 3000
tgagaattct ttattcatcg acttagccgg tttgaacgtg ggttttaaaa gttggaatcc 3060
tgaacctgtt acatcttctg gtacgtcttt ggctggacaa tcctctatgg gtggtgtatg 3120
ggagtggacc tcttctgttt taagaccaca tgaagggttc cacccaatgg agttgtatcc 3180
tggttataca gccgatttct ttgatgaaaa acataatatt gttctcggag gatcatgggc 3240
tactcatcca agaatagcgg gtagaaaaag ctttgttaac tggtatcaaa gaaactatcc 3300
gtacgcctgg gctggtgcca gacttgttaa agatgcttga acaggcccct tttcctttgt 3360
cgatatatgt aattagttat gtcacgctta cattcacgcc ctcctcccac atccgctcta 3420
accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt tttttaatag 3480
ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacaaac 3540
gcgtgtacgc atgtaacgtt taaac 3565
<210> 3
<211> 4932
<212> DNA
<213> 人工序列()
<400> 3
tctagagatc tcttctccgg ctttccgtcc ctcgagtacc gtcgcggcgg ctgttttgca 60
tagcggagta agtcatgcgg tcctgtgcaa gcaagtgtcg tcgagaccgg cgactaaccc 120
cggaggccga gcgggtagtc ctccctgcgc aacaacgcga tttcatcagt gggccgtttc 180
gccagaagtt gtcccactcg cagggtgctc acatgaggat ctgaggcggg atcaggggtc 240
ggatgttctt gatccggcgg tcatcgaggt cgttgctggt cgtcgcacat gtccatcatg 300
acgagttgag tcagtctgtc agtcaggctc tgaagcgggg cggtaggctg cgtggctcac 360
agcgccgtct tgacgttggc ttgaggtgtc tgagtgccgt ctgcgcgcat cttgccctgg 420
atccgagtgt agttggttgg tcaatcagga tcttgcgaaa aagttgcgcg tgcgtgggcg 480
ccacgctcgt tcaagcgctc gttgattcat tcattgcctc caaacagggg ggaaccaggc 540
agcgtaaacc ccgcggagtt gtgcccgtcg ggttgccagc ggaacgtcag ccggacaatc 600
tcgcctcgct cgctcgtcac cctagacgca gaactagaga agcgaatcta ccctggcttg 660
tttgctgagg acgactctgc gccgcgctcc gtcaacgaca ccgctcgcca cctcgctcgt 720
ccagttcacc tctcttcgct caactactac cccaaccgat tcgcttcgct ctagctctac 780
ccgccgcgcg ttcagccagt cgagctacaa gatgccagac gccgcctcaa ccgccgctgt 840
tcagcctccg cccttcatcc tcgacctgcg gaaccgctcg ccgccgacct cgccgagcca 900
ggtcgcctcc gatgccgctc gctcgtcctc tccgtcgtcc ctgggcgaat cggagggcac 960
tacctcgacc aatgctaccc ccgaggcccc ctccgatccg ctccgcgagc agatcatcgc 1020
cggcttgatc ggatcgccca agcccaccgt tccaggcaag acggaaaagg atcgcgcgta 1080
tgcgtaccgc cgcacgatcc cgaccatgac actgtacagc gagcgtggcc tgtctatcta 1140
cgaggaaatc accaagacca aggtatgctc ggccctttcc gtccgctttg gttcacgagg 1200
ggccaatgct ttctcagcag gccctcgcgc gccgtcggcc ccacttgccc tacggcgtgc 1260
gggctctcct tcggactagg tggctggcaa gcatcccctt tgcagacgca ctgacctcgt 1320
ctttgcgttt cgattcaggc ctactaccca ttcgaagccg aaaaggagat tctcgaaaag 1380
tatggagacg aaattgcctg ccgcatgttt ggactgccgt cggccttgct cgtcccggac 1440
gacgtagtcc gtggcaaaga cgaccagtac gaaccctctc cggcttcaaa catcggcgcg 1500
aagaaggaga agtggtgagc cggcgttcgg aactataacg gctcctcatc cgcggtacag 1560
gcgctgacgc actgcttaca ggggcgacgt cgctgtcggt ctccacaact acggtgtcaa 1620
cggctctgcg aacctcgcgc agaacaatgt cgtcgccacg cagggcctcg cggtcgagct 1680
tggctcgggc tcgctcgaca agacccgtca tctcctccgc tctatggcca agctgttgca 1740
gtctcgcgac gagggcagct gcccggtctc tcctctgcgc tccattgact acaaagcagt 1800
gcgtttggcg aaacctccgc gagtatagga aatcaatact gacctcccac gtctgccgct 1860
gaacagctcg accttgaagc ggcgtcgctc tactcgacac tgtcctcgct tgcatcggtc 1920
gaaggcgatt gcgtcacgac ggcggtcgac ggacagccaa acgcgacgaa acgccgcgtt 1980
tcggtctcgg gtcttcacgc cacctacgac gagggcctcg ccttcttgaa ggcgcagaac 2040
gatgctggca gcccgtcgag cgtcttctca gacctccccg acttgcccac gagtccaaag 2100
tcggttccga ctaccttgtc cagtgtcctg gaggacgaca gcgacgaagg cgagaccgct 2160
caagcgtctc gcggcgagaa cgctgcggaa cgccgcgcga cttcgatcat gtggctcggt 2220
tcctcgtgcg gcaactacac ccgcgaggaa gcggtgcagt tcctccgcaa catcgagctg 2280
cgcgagggcg ataccatgct gatcggcatc gacggctgcg cggacgaacc tcgtatcgag 2340
actgcctaca acgacccgca ggtgagtctg atgtcctgcc aaactgacca gtatgagctg 2400
ctctgaccct tgtttgccgc tgatcctcca gggcgtcacc cgcgcgttca ttctcgaggg 2460
catcgatgtc gcaggccgca ccctcggcgg agacgcggcc gaagtgctgc agcagaagaa 2520
ctttgactat gtcaaccgct ggaacgccga gctcggaagg cacgaggtga gctgccttcc 2580
gctgtccaga ggagagcagc ctgagttgca ccggcagccc tgttctgacc cgtccccgcg 2640
acaccctcgc aggcttacgt ccgcgccaac aaagatctca caatcccgat cacgggcgca 2700
gacgacgtca ccgaggtcaa gctcgaagag ggagagtaag tcccgcactt ttgcgcgttg 2760
agccggtgcc atcacaagag cccttcctga cgtttcctct cgcaccaggc tcctcaacat 2820
tgaagtctct tacaagtaca cctatgcgga ggcggcggcg ctcttccact tggcgggctt 2880
ccggctcatc cagcactgga ccgactcgtc gcgctcgcat tacctctacc tggtcgaaaa 2940
gccccgcatg tggttcccgt cgacgaccga gagcgctgcc aagatgctcg gtatcgaggt 3000
cgagcccgag gagaaggaga ccgactacgg tgtcccgact ctggaaaagt gggaagaaat 3060
gtggcgggcg tgggacggct tgatggtgag gatgccctcg tcggcgaagg aattccgcaa 3120
gcgatgagtg gacgctcacc tgaaaccgct tcttctacca gctcgagatc atccccaagt 3180
cgctgcactt ccagaagccg atcccgctcc gccacatccc gctcttctac gtcgggcaca 3240
tacccgcttt ccgggatatt cacctcgcga ggtacttcaa cgagccgctg accgaaccgg 3300
caaagtttgc cgacatcttc gagcgcggaa tcgatccctg tgtcgacgac cccgagaccg 3360
tcacgcactg gcactcggaa gtcccgaagg acgaagcgtg ctggccttct ctccaggaga 3420
ttacggcgta cgaagcgtcc gttcgcgatc gcgtccgcaa ggtgtacgcc gaacacgagg 3480
gcaagtggac gaccaagctc gcccgtgtgc tgatgatgac ttttgagcac gagatgtgcg 3540
tccttcctgc ttcgggctga ttcggtaccc atgtcatcac tcgactcact catcctcttg 3600
ggtccccttg ggccccacag gatgcactgg gagacctccg tctacatctg tctccaagcc 3660
gcgagctcgc tcaaccttcc gccgggaacg gcgattcctg atttccgctc gctggcacgt 3720
caagcaaaac gtgatctgca tcagaacggc ggcggacagc gactctcctt ccccgctcag 3780
gaggtcacag tcggtcacga cgacgacgat accatcgacg accagacgcc gtttgaccct 3840
gcccgagaat acggctggga tgtcgagcac cctcgccgcc agcttcgcgt cgacgcattc 3900
gagatcgagg tcctcccgat ctcgaacgga gagtacaaaa cctggcttac cgagacgcag 3960
caactctcga acaaggcgct catcccgtcg agctggacgg acgagactgg cgaactctgc 4020
gtgaaaacgt tgttcggctt ggtccccctc actctcgccg aggagtggcc cgtggctgcg 4080
agcgcggaac agctcgagaa gtttgccaag gtgggtatcc tttgctcgag caagaatagg 4140
gaccgaagaa gactgactcg taactttctg cactcgacag gccaaaggag gacgcttgcc 4200
gacgttcggc gagctgtcgg cgttcaacca gcacaaccct agctctacgc ctctcgccaa 4260
catcggcctc gcaaacctgc atccggttgc cccttcggta cccgggaaag cgcgtgacgg 4320
cagccagctc ccgatcaccg acggaggcct gtggcaatgg acctcgacgg tcctcgagcc 4380
ctgggctggt tacagcggat cggttcttta ccctggttac tccagtgacg tgagtcgagt 4440
gcgactgtga atgaagtgag atggggcaac tgacattaag cgccctctct tccctctagt 4500
tcttcgacgg caaacaccac atcgtgctgg gcgcgagcta cgcgagtcct cgtcgcttgg 4560
cccgcccttc tttcctgaac tggtaccaga agaactgtgc gtttctggcg ctcttccaag 4620
gaccgttgcc gccagatccc cgtactgaca tgctctgccg cagacccttt catgctcggc 4680
ggcgctcgtg tggcgtacga tgtgtgaaca ggcccctttt cctttgtcga tatatgtaat 4740
tagttatgtc acgcttacat tcacgccctc ctcccacatc cgctctaacc gaaaaggaag 4800
gagttagaca acctgaagtc taggtcccta tttatttttt ttaatagtta tgttagtatt 4860
aagaacgtta tttatatttc aaatttttct tttttttctg tacaaacgcg tgtacgcatg 4920
taacgtttaa ac 4932
<210> 4
<211> 8378
<212> DNA
<213> 人工序列()
<400> 4
agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60
ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120
cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180
tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240
cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300
tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360
aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420
gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480
ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540
accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600
gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660
gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720
gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780
gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840
gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900
acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960
ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020
gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080
gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140
cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200
tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260
cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320
cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380
gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440
ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500
gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560
cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620
cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680
gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740
tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800
atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860
agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920
acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980
atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040
ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100
cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160
aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220
aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280
gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340
aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400
gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460
gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520
gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580
tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640
ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700
accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760
acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820
cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880
gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940
gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000
tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060
ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120
gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180
gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240
aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300
catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360
aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420
ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480
tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540
ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600
aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660
gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720
cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780
tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840
gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900
ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960
actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020
aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agccatattc 4920
aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4980
ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 5040
ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 5100
ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5160
agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5220
cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5280
cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5340
gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5400
attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5460
ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5520
tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5580
gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5640
aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5700
tgatgctcga tgagtttttc taatcacagg cagcaacgct ctgtcatcgt tacaatcaac 5760
atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 5820
gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 5880
gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 5940
gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca 6000
acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat tgaattcgag 6060
ctcggtaccc gggcgttctc ctcgctccgc aagcattgga atgaaccttg ctctctagtt 6120
ccctcctccg tgacctcgtt tcgtccttta gacggcacga tggaaggaag aaatctctgc 6180
ggacaagcaa atctgctggc tcgccttgta ggtcgcctac cggagcaagc cttgtgccgc 6240
cgggatgcca acgtcgtttt ttgacgtttg caagacgtag aggacgcttc ggacgacgaa 6300
acaagctgtg aggacatgga agtcgtggga ggaacggcgc agagcggcgc cgcgggagca 6360
taaggcaagc gagatagtcc agaaatcgcg gcgccaagta cagtaattta ttggagcagg 6420
caccagaagc gggcagcagt atgcgcaggc ttggggtcga cgagagacga ctccctcata 6480
ctcggttacc tcgagcaata caatcaatcg aagctgcgcg aatctcggct tgtaagggtc 6540
ggaaaggaac ctcggagatg gccacgtcac atcaccaact tatcgatctc agccgacgtc 6600
gcagagaggg cgagcgaagc ggtgaaggag ggaaacaatc cctcgagagc atgatccgtc 6660
tgaatctgca gcgcaggaag ccgtcacacg cccgcctcga gcgcaggtcg ggtccagccg 6720
ggggacgaaa cgcgcgaggg ctgatttcgt gagcgaagga agccgcatcg acaagttcgc 6780
gtccctttgc cctctttccc atcacccgct ctcgctctac ccgctcagaa caacaccaga 6840
tcagtcacaa tgccggagct cacggcgacg tcggtcgaga agttcctcat cgaaaagttc 6900
gactcggtct cggacctcat gcaactctcg gagggagagg aatcgcgcgc gttctcgttc 6960
gacgtcggag gccgcggata cgtcctccgc gtcaactcgt gtgcggacgg attctacaag 7020
gatcggtacg tctaccgcca ttttgcgtcg gcggcgctcc cgatccccga agtcctcgac 7080
atcggagagt tctcggaatc cctcacgtac tgtatctcgc gccgggcgca aggagtcacg 7140
ctccaagatc tcccggagac ggaactcccg gcggtcctcc aaccggtcgc ggaagcgatg 7200
gacgcgatcg cggccgcgga cctctcgcaa acgtcgggat tcggaccgtt tggaccgcaa 7260
ggaatcggac aatacacgac gtggcgcgac ttcatctgtg cgatcgcgga tccccatgtc 7320
taccactggc aaacggtcat ggatgacacg gtctcggcgt cggtcgcgca agcgctcgac 7380
gagctcatgc tctgggcgga ggactgtccg gaggtccgcc acctcgtcca cgcggacttt 7440
ggatcgaaca acgtcctcac ggacaacgga cgcatcacgg cggtcatcga ctggtcggag 7500
gcgatgtttg gagactcgca atacgaggtc gcgaacatct tcttctggcg cccgtggctc 7560
gcgtgcatgg agcaacagac gcgctacttc gagcgccgcc acccggagct cgcgggatcg 7620
ccgcgcctcc gcgcgtacat gctccgcatc ggcctcgacc aactctacca gtcgctcgtc 7680
gacggaaact tcgacgatgc cgcgtgggcc caaggacgct gcgacgcgat tgtccgctcg 7740
ggagcgggaa ccgtgggacg cacgcaaatt gcgcggcgct cggcggccgt ctggacggat 7800
ggatgtgtcg aagtcctcgc ggattcggga aaccggcgcc cgtcgacgcg cccgcgggcg 7860
aaagaacacc accatcacca tcactaggat cgttcaaaca tttggcaata aagtttctta 7920
agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 7980
aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 8040
agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 8100
gataaattat cgcgcgcggt gtcatctatg ttactagatc gggcctggat cctctagagt 8160
cgacctgcag catgcaagct tggagcttga gcttggatca gattgtcgtt tcccgccttc 8220
agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 8280
tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 8340
tatgtgcatg ccaaccacag ggttcccctc gggatcaa 8378
Claims (8)
1.一种麦角硫因生产菌,其特征在于,是以中国普通微生物保藏管理中心保藏的圆红酵母Rhodotorula toruloides 2.1389为宿主构建的工程菌,其基因组中包含选自下组的egt1基因:来源于粗糙链孢霉的酶egt1的编码基因NcEGT1、来源于黑麦麦角菌的酶egt1的编码基因CpEGT1、来源于胶红酵母的酶egt1的编码基因RmEGT1,egt1基因以基因表达盒的形式存在,NcEGT1基因表达盒是SEQ ID NO:1,CpEGT1基因表达盒是SEQ ID NO:2,RmEGT1基因表达盒是SEQ ID NO:3。
2.如权利要求1所述的麦角硫因生产菌,其特征在于,所述egt1基因是NcEGT1。
3.如权利要求1所述的麦角硫因生产菌,其特征在于,构建工程菌时,NcEGT1基因表达盒、CpEGT1基因表达盒、RmEGT1基因表达盒分别克隆在pUC57质粒上,获得EGT1基因表达质粒,分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
4.如权利要求3所述的麦角硫因生产菌,其特征在于,EGT1基因表达质粒被构建成麦角硫因功能基因表达质粒,所述麦角硫因功能基因表达质粒通过包括下述步骤的方法构建:
1)使用限制性内切酶XbaI/PmeI对EGT1基因表达质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t进行酶切,分别凝胶回收3.7kb、3.6kb、4.9kb片段,得到EGT1基因表达片段;
2)使用限制性内切酶XbaI/PmeI对核苷酸序列为SEQ ID NO:4的质粒pZPK-PGPD-Hyg-Tnos进行酶切,凝胶回收8.3kb片段,得到质粒骨架;
3)将步骤1)中得到的EGT1基因表达片段与步骤2)中得到的质粒骨架相连接,得到麦角硫因功能基因表达质粒。
5.一种构建如权利要求1所述的麦角硫因生产菌的方法,其特征在于,将如权利要求4中所述的麦角硫因功能基因表达质粒转化入圆红酵母Rhodotorula toruloides 2.1389中,得到阳性克隆株。
6.如权利要求5所述的方法,其特征在于,将如权利要求4中所述的麦角硫因功能基因表达质粒通过农杆菌介导转染圆红酵母,得到阳性转化子。
7.如权利要求6所述的方法,其特征在于,包括如下步骤:
A.将如权利要求4中所述的麦角硫因功能基因表达质粒转化入农杆菌感受态细胞,得到农杆菌工程菌;
B.将农杆菌工程菌菌液与圆红酵母Rhodotorula toruloides 2.1389菌液相混合,进行抗性筛选,经表型及PCR验证,获得阳性转化子。
8.如权利要求1或2所述麦角硫因生产菌在发酵法生产麦角硫因中的用途。
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