CN109735537B - 一种露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子及其应用 - Google Patents
一种露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子及其应用 Download PDFInfo
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Abstract
本发明公开了一种露湿漆斑菌(Myrothecium roridum)A553单端孢霉烯合酶基因Tri5启动子及其应用。该启动子的核苷酸序列如SEQ ID NO.1所示。本发明通过染色体步移技术获得Tri5基因的上游启动子序列并进行启动子核心区域预测,获得露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子Tri5P。本发明的启动子能够高效启动潮霉素抗性基因hph的表达,且启动效率与组成型启动子TEF1相近,从而为后期通过转录调控和异源表达以提高单端孢霉烯毒素产量并获得更多高活性的新型单端孢霉烯毒素奠定分子生物学基础。
Description
技术领域
本发明属于基因工程领域,具体涉及一种露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子及其应用。
背景技术
露湿漆斑菌(Myrothecium roridum)A553是从广药植物广藿香中分离得到的真菌,从中分离得到了具有很强抗肿瘤活性的新型化合物单端孢霉烯毒素。从露湿漆斑菌A553中共获得单端孢霉烯毒素3种,其中1种为新型单端孢霉烯毒素。单端孢霉烯毒素具有抗肿瘤、抗真菌、抗疟疾和植物毒活性,因此在抗肿瘤、抗真菌感染和抗疟疾等药物开发方面具有良好的研究前景。
启动子作为结构基因和功能基因转录调控的必要元件,能够募集转录因子与RNA聚合酶精确地起始转录。近年来,由于丝状真菌在新型、高活性次级代谢产物的发掘以及活性酶的研究和开发方面发展迅速,且丝状真菌中启动子对其内源基因的转录活性较高,因此很多不同种属丝状真菌的启动子相继被发掘,如里氏木霉(Trichoderma reesei)的cbh1、tef1启动子,gpdA启动子,木霉属(Trichoderma sp.)pki1启动子,曲霉属的glaA启动子等。但目前植物内生真菌次级代谢产物生物合成基因的启动子尚未被发掘。禾谷镰刀菌中单端孢霉烯毒素的生物合成基因簇已经被报道,而且已经证实Tri5是单端孢霉烯毒素生物合成的关键基因(Brown,D.W.,R.B.Dyer,S.P.McCormick,D.F.Kendra,andR.D.Plattner.2004.Functional demarcation of the Fusarium core trichothecenegene cluster.Fungal Genet.Biol.41:454–462.)。
发明内容
本发明的第一个目的是提供一种露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子,所述的露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子,其核苷酸序列如SEQ ID NO.1所示。
本发明的露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子Tri5P通过以下方法获得的:通过转录组测序获得编码单端孢霉烯合酶的单端孢霉烯毒素生物合成基因Tri5,其核苷酸序列如SEQ ID NO.2所示。在其上游序列中设计特异性反向引物sp1、sp2和sp3,采用Genome walking试剂盒的通用正向引物AP3进行三轮巢式PCR扩增,每一轮扩增产物稀释后作为下一轮扩增的模板。最后一轮的扩增产物进行TA克隆,转化至大肠杆菌感受态细胞,涂布于氨苄青霉素抗性平板筛选出阳性克隆,菌液PCR验证阳性克隆并测序,获得目的基因Tri5上游启动子序列,并利用启动子预测软件分析上游启动子序列,获得Tri5启动子核心区域Tri5P(即露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子,其核苷酸序列如SEQ IDNO.1所示)。
本发明利用同源重组法将Tri5启动子核心区域Tri5P替换酵母载体YEp352-TEF1-HYRB的TEF1启动子元件。首先设计针对Tri5启动子核心区域Tri5P扩增的上下游引物,其引物序列为Tri5P-f0:5'-AGCTCGGTACCCGGGGATCCCAATGTTCGCACTCTTAGTCAAG-3';Tri5P-R:5'-GGTGAGTTCAGGCATCAGGGCTCAACTCAAGATCAAAGT-3',通过PCR扩增获得产物并纯化回收片段。对已构建的YEp352-TEF1-HYRB载体采用BamH I和Xba I双酶切,然后使用ClonExpressII One Step Cloning Kit C112(Vazyme)将片段和酶切载体重组连接并转化至大肠杆菌感受态细胞,涂布于氨苄青霉素抗性平板筛选出阳性克隆。经过此轮分子克隆,目的基因Tri5启动子核心区域Tri5P(其核苷酸序列如SEQ ID NO.1所示)取代TEF1启动子,构建得到YEp352-Tri5P-HYRB载体,将其电转入酿酒酵母BJ5464细胞中,利用40μg/mL潮霉素抗性的YPD平板进行筛选和验证。与转入YEp352-TEF1-HYRB质粒的酿酒酵母BJ5464相比,含有重组载体YEp352-Tri5P-HYRB的酿酒酵母生长速度一致,阳性菌落数相近。证明Tri5启动子核心区域Tri5P能启动潮霉素抗性基因hph的表达,且启动效率与组成型启动子TEF1相近。
本发明的第二个目的是提供一种表达载体,含有上述的露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子。
本发明的第三个目的是提供一种宿主细胞,含有上述的表达载体。
所述的宿主细胞优选为酿酒酵母(Saccharomyces cerevisiae)BJ5464。
本发明的第四个目的是提供上述的露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子在启动下游基因在宿主细胞中表达的应用。
所述的宿主细胞优选为露湿漆斑菌(Myrothecium roridum)A553或酿酒酵母(Saccharo myces cerevisiae)BJ5464。
所述的下游基因优选为单端孢霉烯合酶基因Tri5或潮霉素抗性基因hph。
本发明的第五个目的是提供一种表达盒,该表达盒含有上述的露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子。
与现有技术相比,本发明具有以下有益效果:
本发明所涉及的露湿漆斑菌(Myrothecium roridum)A553分离自广药植物广藿香,本课题组前期对该菌进行了转录组测序并对单端孢霉烯毒素生物合成相关基因进行了注释。鉴于目前关于露湿漆斑菌(Myrothecium roridum)A553的启动子尚未见报道,而转录组测序及荧光定量PCR结果也表明,编码单端孢霉烯合酶的生物合成基因Tri5的表达水平较高,预示该基因的启动子具有较高的转录活性。因此本发明采用Genome Walking试剂盒,利用通用引物和反向特异性引物,利用TAIL-PCR原理获得露湿漆斑菌A553单端孢霉烯合酶基因Tri5的上游启动子序列,启动子核心区域预测获得启动子核心区域并进行功能验证,从而为后期通过转录调控和异源表达以提升单端孢霉烯毒素的表达水平并获得新型单端孢霉烯毒素奠定分子生物学基础。
本发明的露湿漆斑菌(Myrothecium roridum)A553,其公开于文献:Hong-XinLiu,Wei-Zhen Liu,Yu-Chan Chen,Zhang-Hua Sun,Yu-Zhi Tan,Hao-Hua Li&Wei-MinZhang(2016):Cytotoxic trichothecene macrolides from the endophyte fungusMyrothecium roridum,Journal of Asian Natural Products Research,DOI:10.1080/10286020.2015.1134505。该菌种本申请人也持有,保证自发明的申请日起20年内向公众提供。
附图说明
图1为露湿漆斑菌A553单端孢霉烯毒素生物合成基因Tri3、Tri4、Tri5、Tri6、Tri11、Tri12的表达水平;
图2为露湿漆斑菌Tri5启动子序列获得;其中A为Tri5启动子的染色体步移扩增产物的电泳图,G1为第一次巢式PCR扩增产物,G2为第二次巢式PCR扩增产物,G3为第三次巢式PCR扩增产物;B为Tri5启动子核心区域Tri5P扩增产物的电泳图;
图3为重组载体YEp352-Tri5P-HYRB的构建;其中A为YEp352-TEF1-HYRB载体图谱;B为YEp352-Tri5P-HYRB载体图谱;C为Tri5启动子核心区域Tri5P的菌落PCR扩增产物的电泳图;
图4为40μg/mL潮霉素抗性平板对含有YEp352-TEF1-HYRB或YEp352-Tri5P-HYRB酿酒酵母的筛选;
图5为Tri5启动子核心区域Tri5P的菌落PCR扩增产物的电泳图。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
本实施例中Genome walking试剂盒购买自Takara生物工程有限公司(大连,中国);TA克隆试剂盒购买自全氏金生物工程有限公司(北京,中国)。
本实施例中所用的YPD固体培养基的配方为:每升含有酵母粉10g、蛋白胨20g、葡萄糖20g和琼脂粉30g,余量为蒸馏水。
实施例1露湿漆斑菌(Myrothecium roridum)A553单端孢霉烯合酶基因Tri5启动子序列的获得
单端孢霉烯毒素生物合成基因表达水平分析:将植物内生真菌露湿漆斑菌(Myrothecium roridum)A553接种于YPD培养基平板,于37℃培养72h,挑取新鲜的菌丝体,利用植物RNA提取试剂盒提取RNA,利用All-in-one RT Master Kit逆转录获得cDNA。采用Hiseq2000进行转录组测序,通过前期转录组测序得到的目的基因序列设计单端孢霉烯毒素生物合成相关基因Tri3、Tri4、Tri5、Tri6、Tri11、Tri12引物,引物序列为:针对Tri3基因(FP:5′-ATGCTACTACAGGCAAATTTCAT-3′,RP:5′-TCCGATATACGTCGCGAATCTAGC-3′),针对Tri4基因(FP:5′-ATGGATGCTCCGAAATCCAATG-3′,RP:5′-GCCGATATATCTC GCATACCG-3′),针对Tri5基因(FP:5′-ATGAGAGGACCAGACAGGAAGATCA-3′,RP:5′-CGAGAACCTCCTGAGCGGCCTGG-3′),针对Tri6基因(FP:5′-ATGGCCACCAG AGATGCGCCAG-3′,RP:5′-TGTCTTGCTGCCTTTCTTGTG-3′),针对Tri11基因(FP:5′-ATGA TGGCCTCCGTGGACCAAC-3′,RP:5′-CTCAGCGTATGCGAGCATG-3′),针对Tri12基因(FP:5′-ATGGCTGTTCCAGAGGATGTG-3′,RP:5′-CTTCTCCTCTGCGTACACCTG-3′),以cDNA为模板进行荧光定量PCR,分析这6个基因的相对内参基因GAPDH的表达水平,并通过琼脂糖凝胶电泳进一步验证目的基因的表达水平,结果见图1。如图1所示,Tri5的相对表达水平远高于Tri3等其他合成相关基因的表达水平,暗示Tri5的启动子具有更高的转录活性,因此对Tri5的启动子序列进行扩增和研究。
Tri5启动子Tri5P的扩增:采用Genome walking试剂盒,利用TAIL-PCR技术扩增得到露湿漆斑菌A553单端孢霉烯合酶基因Tri5基因的上游序列,通过TA克隆测序获得上游序列的核苷酸序列,利用启动子分析软件获得该启动子的核心区域Tri5P。设计Tri5基因序列中特异性反向引物(表1),并利用Genome walking试剂盒的通用正向引物AP3进行巢式PCR扩增,通过3次巢式PCR反应对M.roridum基因组扩增,得到对应的扩增产物G-1、G-2和G-3,最后一次巢式PCR扩增产物G-3的目的条带大小约为2.5kb(图2A),并将最后一次巢式PCR获得的目的条带利用pEASY-T1试剂盒进行TA克隆,转化至大肠杆菌感受态细胞,涂布于氨苄青霉素抗性平板筛选出阳性克隆,利用M13-F(5′-GTAAAACGACGGCCAGT-3′)和M13-R(5′-CAGGAAACAGCTATGAC-3′)引物进行菌液PCR验证阳性克隆并测序,获得目的基因Tri5上游启动子序列(其核苷酸序列如SEQ ID NO.3所示),并利用启动子预测软件(http://www.fruitfly.org/seq_tools/promoter.html)分析上游启动子序列,获得Tri5启动子核心区域Tri5P,大小为1458bp(图2B,其核苷酸序列如SEQ ID NO.1所示)。
表1目的基因Tri5特异性反向引物序列
实施例2Tri5启动子核心区域Tri5P的功能验证
利用同源重组法将Tri5启动子核心区域Tri5P插入到酵母载体YEp352-TEF1-HYRB中(YEp352-TEF1-HYRB为早期构建质粒,携带有组成型启动子TEF1启动的潮霉素B抗性基因hph,为现有技术中的已知产品,YEp352-TEF1-HYRB载体图谱见图3A)。首先设计针对Tri5启动子核心区域Tri5P(SEQ ID NO.1)扩增的上下游引物Tri5P-f0(5'-AGCTCGGTACCCGGGGATCCCAATGTTCGCACTCTTAGTCAAG-3')和Tri5P-R(5'-GGTGAGTTCAGGCATCAGGGCTCAACTCAAGATCAAAGT-3'),通过PCR扩增获得产物。对载体YEp352-TEF1-HYRB采用BamH I和Xba I双酶切切除TEF1并回收产物,然后使用ClonExpress II One Step Cloning Kit C112(Vazyme)将两个产物重组连接并转化至DH5α中筛选阳性克隆。采用引物Tri5P-f0和Tri5P-R进行菌落PCR验证,结果表明Tri5P成功插入YEp352-HYRB载体中,并通过测序予以确认(图3C),得到YEp352-Tri5P-HYRB载体(YEp352-TEF1-HYRB载体图谱见图3B)。
制备酿酒酵母(Saccharomyces cerevisiae)BJ5464的感受态细胞,将YEp352-TEF1-HYRB质粒载体(阳性对照)以及YEp352-Tri5P-HYRB质粒载体分别电转入酿酒酵母BJ5464细胞中(1500V,5ms),均匀涂布于含有40μg/mL潮霉素抗性的YPD平板中,在30℃培养2d,利用菌落PCR筛选阳性克隆,并进一步测序验证。不添加质粒载体的阴性对照平板上并无菌落产生,说明40μg/mL潮霉素抗性浓度足以筛选酿酒酵母BJ5464。与阳性对照(含有YEp352-TEF1-HYRB质粒载体)相比,含有YEp352-Tri5P-HYRB质粒载体的酿酒酵母生长速度一致,而菌落数相近(图4),证明Tri5启动子核心区域Tri5P能启动潮霉素抗性基因hph的表达,且启动效率与组成型启动子TEF1相当。挑取含有重组质粒载体YEp352-Tri5P-HYRB的酿酒酵母进行菌落PCR,所采用引物为Tri5P-f0和Tri5P-R。扩增得到Tri5启动子核心区域Tri5P,筛选得到阳性克隆,并通过测序得到验证(图5)。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东省微生物研究所(广东省微生物分析检测中心)
<120> 一种露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1458
<212> DNA
<213> 露湿漆斑菌 A553(Myrothecium roridum A553)
<400> 1
caatgttcgc actcttagtc aaggccacgg tcttcgccgc ggccctgtat gtgttgtcga 60
ttcctgttca agccatttac aatctctact tccatccttt gagacacata cctggaccga 120
agttatggat tgcctttccc atcttgggtc aaatttctcg tgtgagagga gtcttggatt 180
cgtacatgtg cgagcttcac tgcacctatg gtgaagccgt ccgttacggt cctgatgagg 240
tgtcaatcat tacggaacaa gcttggaggg acatctatga tcataagcca aaccagctgg 300
aacgaaacat cctatcgtcg acgcggcgcc cggacatttt tgatgcgaat gaggtggacc 360
atgatcgtta ccgcaaggcc atgtctcatg ccttctctcc gagaggactt caagaacaag 420
ggcctatcgt caagggctac ctcgacctgt tgattgaacg cctccgacaa gtggccgcca 480
aaggagaaaa gaccgacatg gtccagtggt acaactttac ccttttcgac attattggtg 540
atcttgcctt cggccagtcc ttcggaggcc tgcgcgatca agtcctccac ttctccatct 600
ctttcacatt tgaagcattc aagctcctca catacttgga agccggtgca cgctacccgc 660
tcctgttgaa gctgctcgag ctgtttacac caaagagcat cattgaagct cgagacagga 720
aggaggagca tgccgaggct actgtcaagc agaggctgga gaacggatcg ctgcatggtc 780
gtggagactt catggacgcc atgttgaaga accgtggcaa gcctcaaggt ctcaatgaca 840
aagagcttgt tgcaaatgcc agcactttga tcacggctgg aagtgagacc acggccacga 900
ttctttgcgg tgtcacttat tggctgttgc gatctcctga tatctgcgaa aaggtggtgc 960
aggaggtgcg cactgcctat aagaatgaat ccgacattca gatgagcaca accatcacca 1020
agttaccatt cacggttgct tgtatccagg aggccttcag gctgtacccc cctgttccga 1080
gttgcctgca gcgagtcaca ccagagacgg gaatcacgcg catttctggc tacgatatcc 1140
cccctaacgt gagttttgcc tcagactcaa tactatcaac catagagcta actgtccgat 1200
agactaaagt cggcgtccat gcgttggccg cttacacaga tcccatgaac tggtacaagc 1260
cagaactatt cctgcccgaa agatggctcc ccgaggcgaa gaatgaccag acttcccctt 1320
actataatga ccatcgcaat gcgctgcaac ctttctccgt cggaccgcgc tcctgcattg 1380
gtcgggacat ggctggacaa gaaatgcgtc ttattctcgc ccgattgcta tggaactttg 1440
atcttgagtt gagccctg 1458
<210> 2
<211> 1311
<212> DNA
<213> 露湿漆斑菌 A553(Myrothecium roridum A553)
<400> 2
atgagaggac cagacaggaa gatcagacag acagatcaga cagacagatt catcaatcga 60
cagttaagaa gtctccaata tcacttttct gcccagaaat atctatctct ctcagaatct 120
cccaaagaac agctacattc aaaggaattg cctaaaatgg acgagttccc tactgagtat 180
ttcctcggca ccgccgtgcg gttgctggag aacgtcaagt acagagacag caactacacg 240
agggaggagc gtattgagaa cctttcgtac gcctacaaca aggctgcggc ccatttcgcc 300
caggagcgcc aacagagcat cctgaaggtg aaccccaaga ggctcgaggc ttcccttcga 360
accatcgttg gcatggtcgt ctactcctgg gtcaaggtgt ccaaggagct catggccgac 420
ctcagcatcc actacacata cactctcatc ctggacgaca gcgaggatga cccgcacccc 480
aacatgttga ccttcttcga cgacctccaa aacggacgcc aacagaagca cccctggtgg 540
atgctggtca acgagcactt ccccaacgtc ttgaggcact tcggcccatt ctgctcattg 600
aacctcattc gcagcaccct ggacttcttt gagggctgct ggatcgagca gtacaacttc 660
cacggcttcc ccggatctta tgatttcccc gggttcctcc gtcgcatgaa cggcctgggc 720
cactgtgtcg gtggatctct gtggcccaag gagctctttg atgagcagaa gcatttcctt 780
gagatcacca gcgccgtcgc tcagatggag aactggatgg tctgggtcaa cgatttgatg 840
tcgttctaca aggagttcga cgaccctcgc gaccagacca gtctcgtcaa gaactacgtc 900
gtttgcgacg gcatcaccct tacccaggct ctggagaagc tcaccgtgga caccctgacc 960
tcatcggaac agatgatgaa ggtgttctcc gacaaggacc ccaagctcat ggagacgatc 1020
gagtgcttca tgcacggcta catcacctgg cacttgtgcg accaccgcta caggctcagg 1080
gagatctacg agggcaccat gcacattgag actgaggatg ccattaggtt ccgcaagttc 1140
tacggccagg ctgccaaggt cggtgccatt gagcacgagg agtgggcttt ccccaccgtg 1200
gctgagcgta ttgagctccg aaaggccgag gagcaggccg ccaaggacga ccaggtcgtc 1260
ctgaccaacg ccgaatcggc cgtcccccag gccgctcagg aggttctcgc t 1311
<210> 3
<211> 2368
<212> DNA
<213> 露湿漆斑菌 A553(Myrothecium roridum A553)
<400> 3
ctagtaacgg ccgccagtgt gctggaattg ccctttctgt ctgtctgatc tgtctgtctg 60
atcttcctgt ctggtcctct catccgggct cagcttgaga cataaataac attgctacca 120
cgagtctgct cgtcggccgt ccaacctttg ccatcgccaa cggattctag ggcctctcaa 180
aacgcgccgt atagggccgt ataggttacc caggaagttg aagggaagct ggatgccatc 240
acggagtgcc tcattaggta atccgcagac cagaggcgag tcgaatcaaa ttccccgacc 300
actgcccgtt tcggcactaa ccaatcgtgg cagcatggcc ctgagaccct tcctaccatg 360
atcggtgcga tcctcggtgc ttatcatccg acggggcata aggcctaccg cataaggacg 420
tggagagtgg agagtggacc tagagcgtgg atggctttgc gtaggagcag cggaaaatga 480
catagagagg tggccaaatg ccaaaaagaa aacaaaaaaa gccaaaacaa gggaaagatt 540
gatctaaaaa ccacgacgca tgcggatttg agttgcagaa atacccgaca tggacaagga 600
gaagaccttg ttcgaccgtc ctcgacctgc ttgctcaggc cttgtctcag cttattctcc 660
tgtgataggc cttgttgcaa cctttgggct ttagcggcat gtctagcaaa agctcgggca 720
gtaagcttag ctgaaatgat cagatcttag ccatcgtatg agccccgtcc tcgggtcttt 780
ggtgcggagc cctgggtttc ctctcatatt tgaatccact taaacacttc gccgcatcgt 840
tttctcgctg aacgaattat ccctcctgca atttttcctc tttcaatcca aagcatccgg 900
attggctcaa caatgttcgc actcttagtc aaggccacgg tcttcgccgc ggccctgtat 960
gtgttgtcga ttcctgttca agccatttac aatctctact tccatccttt gagacacata 1020
cctggaccga agttatggat tgcctttccc atcttgggtc aaatttctcg tgtgagagga 1080
gtcttggatt cgtacatgtg cgagcttcac tgcacctatg gtgaagccgt ccgttacggt 1140
cctgatgagg tgtcaatcat tacggaacaa gcttggaggg acatctatga tcataagcca 1200
aaccagctgg aacgaaacat cctatcgtcg acgcggcgcc cggacatttt tgatgcgaat 1260
gaggtggacc atgatcgtta ccgcaaggcc atgtctcatg ccttctctcc gagaggactt 1320
caagaacaag ggcctatcgt caagggctac ctcgacctgt tgattgaacg cctccgacaa 1380
gtggccgcca aaggagaaaa gaccgacatg gtccagtggt acaactttac ccttttcgac 1440
attattggtg atcttgcctt cggccagtcc ttcggaggcc tgcgcgatca agtcctccac 1500
ttctccatct ctttcacatt tgaagcattc aagctcctca catacttgga agccggtgca 1560
cgctacccgc tcctgttgaa gctgctcgag ctgtttacac caaagagcat cattgaagct 1620
cgagacagga aggaggagca tgccgaggct actgtcaagc agaggctgga gaacggatcg 1680
ctgcatggtc gtggagactt catggacgcc atgttgaaga accgtggcaa gcctcaaggt 1740
ctcaatgaca aagagcttgt tgcaaatgcc agcactttga tcacggctgg aagtgagacc 1800
acggccacga ttctttgcgg tgtcacttat tggctgttgc gatctcctga tatctgcgaa 1860
aaggtggtgc aggaggtgcg cactgcctat aagaatgaat ccgacattca gatgagcaca 1920
accatcacca agttaccatt cacggttgct tgtatccagg aggccttcag gctgtacccc 1980
cctgttccga gttgcctgca gcgagtcaca ccagagacgg gaatcacgcg catttctggc 2040
tacgatatcc cccctaacgt gagttttgcc tcagactcaa tactatcaac catagagcta 2100
actgtccgat agactaaagt cggcgtccat gcgttggccg cttacacaga tcccatgaac 2160
tggtacaagc cagaactatt cctgcccgaa agatggctcc ccgaggcgaa gaatgaccag 2220
acttcccctt actataatga ccatcgcaat gcgctgcaac ctttctccgt cggaccgcgc 2280
tcctgcattg gtcgggacat ggctggacaa gaaatgcgtc ttattctcgc ccgattgcta 2340
tggaactttg atcttgagtt gagccctg 2368
Claims (1)
1.露湿漆斑菌A553单端孢霉烯合酶基因Tri5启动子在启动潮霉素抗性基因hph在酿酒酵母(Saccharomyces cerevisiae)BJ5464中表达的应用;所述的Tri5启动子的核苷酸序列如SEQ ID NO.1所示。
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