CN111019945B - 一种露湿漆斑菌A553单端孢霉烯合酶基因Tri12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Tri12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Tri12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Tri12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Tri12启动子Tri12P。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Tri12启动子及其应用。
背景技术
大环单端孢霉烯是一类大环内酯抗生素，具有抗肿瘤、抗真菌、抗疟疾和植物毒性等生物活性。露湿漆斑菌(Myrothecium roridum)A553是一株从广藿香中分离得到的内生真菌，从其发酵液中分离得到了3个大环单端孢霉烯，其中1个为新的大环单端孢霉烯，活性测试表明它们均具有很强的抗肿瘤活性。因此大环单端孢霉烯在抗肿瘤、抗真菌感染和抗疟疾等药物开发方面具有良好的研究和开发前景。
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、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的相关研究少有报道。
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Tri12启动子，所述的露湿漆斑菌A553单端孢霉烯合酶基因Tri12启动子，其核苷酸序列如SEQ IDNO.1所示。
本发明的露湿漆斑菌A553单端孢霉烯合酶基因Tri12启动子Tri12P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Tri12，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Tri12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Tri12启动子核心区域Tri12P(即露湿漆斑菌A553单端孢霉烯合酶基因Tri12启动子，其核苷酸序列如SEQID NO.1所示)。
本发明利用同源重组法将Tri12启动子核心区域Tri12P替换酵母载体YEp352-TEF1-HYRB的TEF1启动子元件。首先设计针对Tri12启动子核心区域Tri12P扩增的上下游引物，其引物序列为Tri12P-F：5'-AGCTCGGTACCCGGGGATCCGGTAAGAAGAGAAGATCAAAGG-3'；Tri12P-R：5'-GGTGAGTTCAGGCATCTTGATCTGATGCCTATGGTTT-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ClonExpress II 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Tri12启动子核心区域Tri12P(其核苷酸序列如SEQ ID NO.1所示)取代TEF1启动子，构建得到YEp352-Tri12P-HYRB载体，将其电转入酿酒酵母BJ5464细胞中，利用含有50μg/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(阳性对照)的酿酒酵母BJ5464相比，含有重组载体YEp352-Tri12P-HYRB的酿酒酵母生长速度一致，阳性菌落数相近。证明Tri12启动子核心区域Tri12P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Tri12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Tri12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Tri12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Tri12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，而转录组测序及文献调研结果也表明，编码单端孢霉烯流出泵的生物合成基因Tri12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Tri12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Tri12启动子序列获得；其中A为Tri12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Tri12启动子核心区域Tri12P扩增产物的电泳图；
图2为重组载体YEp352-Tri12P-HYRB的构建；其中A为YEp352-TEF1-HYRB载体图谱；B为YEp352-Tri12P-HYRB载体图谱；C为Tri12启动子核心区域Tri12P的菌落PCR扩增产物的电泳图；
图3为50μg/mL潮霉素抗性的YPD平板对不含有启动子(A)、含有YEp352-TEF1-HYRB质粒(B)和YEp352-Tri12P-HYRB质粒(C)酿酒酵母的筛选；
图4为Tri12启动子核心区域Tri12P的酿酒酵母菌落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，配制方法：将培养基各成分混合，搅拌溶解，灭菌，即得。
实施例1露湿漆斑菌(Myrothecium roridum)A553单端孢霉烯合酶基因Tri12启动子序列的获得
Tri12启动子Tri12P的扩增：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，利用真菌DNA提取试剂盒提取genome DNA。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Tri12基因(Tri12基因序列见SEQ ID NO.2所示)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Tri12P。设计Tri12基因序列中特异性反向引物sp1、sp2和sp3(表1)，并利用Genomewalking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3.2kb(图1A)，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Tri12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Tri12启动子核心区域Tri12P，大小为999bp(图2B，其核苷酸序列如SEQ ID NO.1所示)。
表1目的基因Tri12特异性反向引物序列
实施例2 Tri12启动子核心区域Tri12P的功能验证
首先，利用酶切连接法将潮霉素抗性基因hygromycin-B(GenBank Accession:XM_003071606)插入到酵母载体YEp352-TEF1-CYC1的Xba I和Sal I酶切位点之间(YEp352-TEF1-CYC1为早期构建质粒，携带有组成型启动子TEF1和终止子CYC1，为现有技术中的已知产品：Xiaodan Ouyang,Yaping Cha,Wen Li,Chaoyi Zhu,Muzi Zhu,Shuang Li,Min Zhuo,Shaobin Huang and Jianjun Li.Stepwise engineering of Saccharomyces cerevisiaeto produce(+)-valencene and its related sesquiterpenes,RSC Adv.,2019,9,30171,DOI:10.1039/c9ra05558d)，构建阳性对照质粒YEp352-TEF1-HYRB(YEp352-TEF1-HYRB载体图谱见图2A)。
其次，利用同源重组法将Tri12启动子核心区域Tri12P(其核苷酸序列如SEQ IDNO.1所示)，通过替换元件TEF1的方式插入到酵母载体YEp352-TEF1-HYRB中。首先设计针对Tri12启动子核心区域Tri12P(SEQ ID NO.1)扩增的上下游引物Tri12P-F(5'-AGCTCGGTACCCGGGGATCCGGTAAGAAGAGAAGATCAAAGG-3')和Tri12P-R(5'-GGTGAGTTCAGGCATCTTGATCTGATGCCTATGGTTT-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 promoter并回收产物，然后使用ClonExpress II One Step Cloning KitC112(Vazyme)将两个产物重组连接并转化至大肠杆菌DH5α感受态细胞中筛选阳性克隆。采用引物Tri12P-F和Tri12P-R进行菌落PCR验证，结果表明Tri12P成功插入YEp352-HYRB载体中，并通过测序予以确认(图2C)，得到YEp352-Tri12P-HYRB载体(YEp352-Tri12P-HYRB载体图谱见图2B)。
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-Tri12P-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50μg/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。没有启动子的阴性对照平板上(不添加质粒载体)没有菌落产生，说明50μg/mL潮霉素抗性浓度可以用来筛选酿酒酵母BJ5464。与阳性对照(含有YEp352-TEF1-HYRB质粒载体)相比，含有YEp352-Tri12P-HYRB质粒载体的酿酒酵母生长速度一致，而菌落数相近(图3)，证明Tri12启动子核心区域Tri12P能有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。挑取含有重组质粒载体YEp352-Tri12P-HYRB的酿酒酵母进行菌落PCR，所采用引物为Tri12P-F和Tri12P-R。扩增得到Tri12启动子核心区域Tri12P，筛选得到阳性克隆，并通过测序得到验证(图4)。
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Tri12启动子及其应用
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 999
<212> DNA
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ggtaagaaga gaagatcaaa gggcggagaa ggaggtttaa agaaagtcaa tatggagaac 60
gaaaaaaaaa aacaagaaaa gaagaaagaa gagagaagaa ataaaacaac ggtctcagcc 120
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catttcccag tcaaagttgt tgctccaaga gatgtaccaa tctgtttgac gtctttggcc 240
cgatacaaga agccaccgaa ttttcactaa ctcatcaccg aggagtataa cggttaagct 300
agtgaagtac ggtggatttt cgcatgcgct ccgtgtctgt tgcgttaggg cctcatgatg 360
gctgtgacgt gtacaggctt acgagatggc aagacatgtt cccccatcac tcctctgata 420
agtcgactat aacacaggga cccccgacta gcgcatttta atgttcacgg tccccggttt 480
tggggagaca accttggaaa agcgcagttt gaagtaagac ctgtctcacc gcgcttggga 540
attaaggccc tcgataagaa gcggcggcgg ggcctcgacc aggaatcgac taccgacagg 600
cctgaaggag cctgcatatc caaccagcca gaaggcatcg gccccttgtc cagaggagca 660
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cattctagac catcggctag aaaaaaaata taatgggtat tcgcgtcctc atctaagact 840
tgcactctcg ctccaacaca ctccattcag ccatctcgtg ctacctgagt ccatccagtt 900
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aaagccctga ccgccgacgt caatgagttg cccgatggct actatcggag ccctcgtttg 120
atcggcactt tcatctccat tgcgctgaac ctggtcgcca cctactttgc tttccaagcc 180
tcagcagctg ccatctcaaa catcgccgca gatgtcggac ccagcaagaa catcagcctg 240
ttttcgacgg tgtggaccac tggccaggca atctgcatcc tgctgatggg caggctcacg 300
gatcgcttcg gtcgccggcc attcttcatg ttgaccaacg cattgggctt gattggaggc 360
atcgtcgcat gtactgcgaa gagcatggag accctgattg gtgcccaggt cctgctgggt 420
atctcagctg gacagggagg cgcgagtcct ctcttcgttg gcgagctcat gagcaacaag 480
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cccgtaattg gacagacttt gggtaaccaa ggcaactggc ggtggattta ttacatctac 600
atcatcaaaa tgacaattgc ctcactgctt gccttcttct tctaccatcc tccttccttc 660
gtccagcttc acggcaagaa gatcagcaag cgacaagagc tcgccaaggt cgattggatt 720
ggagtattcc tgcttactgc tggtttaacc ctcttccttc ttggcgtctc gtggggtggc 780
cagccggata acgcatggga ctccagcaaa atcttgggat tgcttatctc tggtggcatt 840
accctcgttt tgtttgttct attcgagtgc tttgccaaag tggagcgtcc catcgtccct 900
atgcacttct tccgggatct acgaggcttc acttgcttgg tcgtcgtgag cagcaccatg 960
ggcgtcatga accttgccct gttcatcatg taccctcagc aagttgtcaa catcttcggt 1020
tctaccgcaa gcggatggag agagattgcc tggatgtcat caaccgcagc ctttggaatc 1080
tgggctggta ttctcatcct gggaagcttg ttccacgtca tccgtcgcat ccgatggcag 1140
ctctttgcag tggcggcttg ggaattggct ttcctgggtg ctatgtcttc ggtggaccgc 1200
catcacaaga ccgctgccat tgtgttctct ttcttcacag gcttcgtggt cggtttcgca 1260
caggacgtga ctatgctcgt ggtgcagtac attgtcgatg acgatgacct cggcgcggca 1320
ttcggtattg ttgctggtag tcgttccgtt ttcggatcta tccttaccgc tgctttcatc 1380
gccatctaca ctaacaagtt tcctggacag ctccagtcga agctcatccc tgctgttagc 1440
gaggctgggc ttccggaatc ctccattccg ggacttctca gcgccatcgc gtctggtgcc 1500
ccacaagcca tcagtgctgt tcctggaatg acgccagccc tcacaaaggt cactgcagat 1560
gcaattgctg acagctacgc ggcctcgtat gcctatgttt actacttcgc catggccctg 1620
ggagtcctcg ccatgggcgc aggtcttttc tctcgagatt tcgatcgcta tatgacgaac 1680
cacgtctcgc accagatcta caagaagtcg gacgcggata aagacccact ggaatcaagc 1740
tcttcctcca attccagcag ggaacaggtg tacgcagagg agaagtaa 1788
<210> 3
<211> 3271
<212> DNA
<213> 露湿漆斑菌 A553(Myrothecium roridum A553)
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accttgggta cgagctcgga tcactagtaa cggccgccag tgtgctggaa ttgcccttat 60
caaacgaggg ctccgatagt agccgtcggc cacatcctca cgagtcggca gtttcgggca 120
atatgggcac tgtcgcgaag ggcacatcag gaaagactgg cgctaatgaa gcacaccttc 180
cggaccacct ggctggccct gggtccagga tttcccccag tcttaagtgc taggattaat 240
atcgcccgta tcggggctcc gccgttttct tggcagtttg gctaagcaca gctctccagc 300
gagtagttaa ggtcaccttc cggtatataa tcgttacgag gaagagattg cagctttccc 360
tttttgccac agcattgaag gagagctaaa agaaacattg gaactggagt attccctcca 420
agctcgataa catcatgctg ccacaggttg ttttgagctc gttgagctcc atcagtgaag 480
ccatctcgag ctggacctca ggctaccact ctgttagcaa tggacaatgc ccgccgctgc 540
ccaaaggaga tttgatttat gatgtgtata tgggataccc tgagaatttt atgtgggaca 600
agaggcgttg cgttgcctac gtcagataag aagcccacgg attgcatttt ctattccaca 660
tgtcactgtg ataataacta acattcacag caacctttac aatgccacag ttagcatttt 720
tggcccatac cagccgaagg tactggagac ccttgagttt ccaggcctga gccatcctgg 780
aaacacagcc atcgacaacc ctttgcacac gagtgggctg gtcctacgac ctgactctta 840
ccgtgctgaa actctggaga tagttgttga caatggcgac gctttcttct ccaacgggct 900
gaatgtctca ggtcctgact atctcctaac catggatctg aagacgaagg aggtgaccaa 960
tcagctgcgt ctcaacaacg gcctatacgc cggctacgct gatgcagagc ttggaagtga 1020
tggaaacacc tacgttcttg gcacctacac cggcaacatc ttgcgcgtca cccctgagaa 1080
gaagctcacc actttttacg tggagaagcc actggcaccg ccccgcctgt acggttttac 1140
gggaatcaca cacgtggggg atgccatcat cgccaacaac aacattattg gccagttcct 1200
ccgctttaat gtccacgatg aagagggaac cccaacagtc atcaagcaaa caccttatca 1260
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cgaggtggat gcgttggttt acgccgacaa aaatgagttg taatgtccaa tgtatgtccc 1620
atgattggct tacttatgag aatacctcgg gtgctaagga atagccggaa attagtgatg 1680
tgccggactt caaaaatgtt acatagacag atagcgagag agatgataat agtgaaatta 1740
ttgctttatc tatgggttcg attctatgag cagggctcat gggtcccgct atgaacactt 1800
cgatggtgca ttcagctcgc tcactgtgcg agtcaccgac aaaacagtag ttagtttgcg 1860
acatgtctca tcacagagag acgatgacaa gcttttttct caacatggaa ttatttcagt 1920
tcgtcgccgc aatatgccgt gcccagcgac gacgagcgaa agttgcttga ttcgccaacc 1980
aatggttttg cggcaacgcc gaacaaggcg attaattaca gcctactgct gttcaaacgg 2040
agggagacag gattcggtag tgcatggcaa gccgggcagt gtaagtcatg cctcaaacgt 2100
cacagcttgg ttcgtgtgca catacggtca agaatgagat ggttatggtc caagcaactt 2160
caatggagct gcttaaatac tggatcaagt atgttctagg cttctactct gcctcatgac 2220
tgtttcagag ctcttctgct aaatggaccg attcaacttt taggtaagga aaggtaagaa 2280
gagaagatca aagggcggag aaggaggttt aaagaaagtc aatatggaga acgaaaaaaa 2340
aaaacaagaa aagaagaaag aagagagaag aaataaaaca acggtctcag ccagaagggc 2400
cttattctta gtaataatgt ctaccgcaaa tgcggtctga catgacacct cccatttccc 2460
agtcaaagtt gttgctccaa gagatgtacc aatctgtttg acgtctttgg cccgatacaa 2520
gaagccaccg aattttcact aactcatcac cgaggagtat aacggttaag ctagtgaagt 2580
acggtggatt ttcgcatgcg ctccgtgtct gttgcgttag ggcctcatga tggctgtgac 2640
gtgtacaggc ttacgagatg gcaagacatg ttcccccatc actcctctga taagtcgact 2700
ataacacagg gacccccgac tagcgcattt taatgttcac ggtccccggt tttggggaga 2760
caaccttgga aaagcgcagt ttgaagtaag acctgtctca ccgcgcttgg gaattaaggc 2820
cctcgataag aagcggcggc ggggcctcga ccaggaatcg actaccgaca ggcctgaagg 2880
agcctgcata tccaaccagc cagaaggcat cggccccttg tccagaggag cacctttcgt 2940
cttggaaccg ttttttggtg gctggatcaa acccttctgg gtaaagaggc aaaacggcga 3000
aggcgctttt ctgcgagcac gaagaaacat gtctagcatt gaagatactg gccattctag 3060
accatcggct agaaaaaaaa tataatgggt attcgcgtcc tcatctaaga cttgcactct 3120
cgctccaaca cactccattc agccatctcg tgctacctga gtccatccag ttctctcata 3180
cagcctggat tcacgattgg cgattggacc acagctcaat tgcgtatcac cgacttccag 3240
aacagcggga aaccataggc atcagatcaa g 3271
Claims (7)
1.一种Tri12启动子，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
2.一种表达载体，其特征在于，含有权利要求1所述的Tri12启动子。
3.一种宿主细胞，其特征在于，含有权利要求2所述的表达载体。
4. 根据权利要求3所述的宿主细胞，其特征在于，所述的宿主细胞为酿酒酵母（Saccharomyces cerevisiae）BJ5464。
5. 权利要求1所述的Tri12启动子在启动下游基因在宿主细胞中表达的应用；所述的宿主细胞为露湿漆斑菌（Myrothecium roridum）A553或酿酒酵母（Saccharomyces cerevisiae）BJ5464。
6.根据权利要求5所述的应用，其特征在于，所述的下游基因为单端孢霉烯合酶基因Tri12或潮霉素抗性基因hph。
7.一种表达盒，其特征在于，含有权利要求1所述的Tri12启动子。
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