CN112626046B - 一种能抗b型和d型单端孢霉烯族毒素的酰基转移酶gantb及其应用 - Google Patents
一种能抗b型和d型单端孢霉烯族毒素的酰基转移酶gantb及其应用 Download PDFInfo
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Abstract
本发明公开了一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB及其应用。抗单端孢霉烯族毒素新型酰基转移酶GANTB，其核苷酸序列如SEQ ID NO.1所示。因此本发明从深海真菌FS140的cDNA文库中获得了抗单端孢霉烯族毒素新型酰基转移酶GANTB序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中进行了抗毒功能验证，从而为后期提高酿酒酵母抗单端孢霉烯族毒素的能力，提升单端孢霉烯族毒素的异源表达水平并获得新型单端孢霉烯族毒素奠定分子生物学基础。
Description
技术领域
本发明属于微生物领域，具体涉及一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB及其应用。
背景技术
真菌毒素是产毒真菌在生长过程中产生的一类次生代谢物质，危害比较大的主要包括单端孢霉烯族毒素、黄曲霉毒素和玉米赤霉烯酮等。特别是单端孢霉烯族毒素，被认为是最危险的自然发生粮食污染物，已成为国际研究热点。单端孢霉烯族毒素是一大类由镰刀菌产生的化学结构相似的毒性物质的统称，其基本结构为四环的倍半萜，根据取代基的不同，可以分为A、B、C、D四种类型。是一类全球性的谷物污染物，在多种谷物小麦、玉米、燕麦、大麦、黑麦、谷物制品面包、啤酒、谷物类早餐和词料中均有检出。迄今为止已经发现了大概170种单端孢霉稀族毒素，而A型和B型单端孢霉稀族毒素污染率最高，包括DON、3-ADON、15-ADON、NIV、T-2和DAS毒素等，其中污染率和含量最高的是DON,因此,阻止和减少DON毒素进入人和动物的食物链以防止其危害为主要研究内容。
脱氧雪腐镰刀菌烯醇(DON),属B族单端孢霉烯族毒素，是世界上分布最广泛的真菌毒素之一,DON毒素可以通过污染小麦,大麦,玉米等原料进入食品和饲料,家畜和家禽食用污染的饲料还会使不毒素进入牛奶,肉和禽蛋中从而间接影响人类健康,因此,阻止和减少不毒素进入人和动物的食物链以防止其危害为主要研究内容。目前DON毒素的防控技术主要包括田间污染控制,食品和饲料的脱毒及抑制毒素在胃肠道的吸收。MR4，是本课题组从广藿香植物中分离的露湿漆斑菌Myrothecium roridum A553(GenBank:KJ813720)中分离得到的杆孢菌素E异构体，属D型单端孢霉烯族毒素,具有强抗肿瘤活性，但对于人体和农产品的危害未知，有待于进一步研究。
由于传统的物理和化学消除方法存在效果不稳定、营养成分损失大、影响饲料适口性，且难以规模化生产等缺点而不能广泛应用到实际生产中。微生物及生物酶降解具有解毒效率高、特异性强、对饲料和环境没有污染等特点和优势，从而备受研究者的关注。近年来已有报道降解玉米赤霉烯酮的微生物菌株，也有一些降解呕吐毒素的降解酶的研究报道，这些报道大多数是单一或混合菌株降解一种毒素，目前还没有关于单一的酶同时抗B型和D型单端孢霉烯族毒素的研究报道。本发明公开了一种能同时抗脱氧雪腐镰刀菌烯醇DON和杆孢菌素E两种单端孢霉烯族毒素的酰基转移酶GANTB，为有效控制和消除脱氧雪腐镰刀菌烯醇DON和杆孢菌素E对粮食和饲料的污染和帮助宿主抵抗毒素危害，改善动物生产性能和保障人类食品安全有非常重要的意义。
发明内容
本发明的第一个目的是提供一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB，所述的酰基转移酶GANTB，其由核苷酸序列如SEQ ID NO.1所示的序列编码而成。
本发明能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB通过以下方法获得的：通过转录组测序结果预测编码酰基转移酶基因GANTB的序列，在其上下游设计特异性引物，其引物序列为GANTB-F:5'-ATGTCCTACCAGTCTACAATCTACACCT-3'；GANTB-R:5'-CTACACTATATCCTTATCGCAACCCACC-3'，以由深海真菌FS140转录组反转录而得的cDNA文库为模板，通过PCR扩增获得产物并纯化回收片段，获得酰基转移酶基因GANTB，其核苷酸序列如SEQ ID NO.1所示。
本发明利用同源重组法将GANTB基因插入到酵母载体YEp352-TEF1-CYC1的表达盒内部。首先设计含有同源臂的GANTB基因的上下游引物，其引物序列为YEp352-GANTB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GANTB-R:5'-TACATGAT GCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3(下划线序列为同源臂片段)，通过PCR扩增获得产物并纯化回收片段。对已构建的YEp352-TEF1-CYC1载体采用内切酶Sal I和Xba I双酶切，然后使用Clo nExpress 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GANTB(其核苷酸序列如SEQ ID NO.1所示)插入到启动子TEF1和终止子CYC1之间，构建得到YEp352-TEF1-GA NTB载体，将其电转入酿酒酵母BJ5464-D细胞中，利用尿嘧啶缺陷型的SD培养基平板进行筛选和验证。与转入YEp352-TEF1-CYC1质粒(阴性对照)的酿酒酵母BJ5464-D相比，含有重组载体YEp352-TEF1-GANTB的酿酒酵母生长速度明显加快，相同培养时间内菌落密度更高，证明功能基因GANTB能有效协助酿酒酵母抵抗单端孢霉烯族毒素，为在酿酒酵母内重构单端孢霉烯族毒素生物合成通路奠定基础，利于下一步分离纯化酰基转移酶GANTB，明确其对真菌毒素降解效果，为开发成经济、可行的降毒试剂、防控真菌毒素添加剂等提供新思路。
本发明的第二个目的是提供一种表达载体，含有上述的能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB。
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能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB在协助宿主细胞抵抗单端孢霉烯族毒素(B型和/或D型单端孢霉烯族毒素)中的应用。
所述的宿主细胞优选为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae B J5464。
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能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB。
与现有技术相比，本发明具有以下有益效果：
本发明所涉及的深海真菌Geosmithia pallida FS140分离自南海沉积物，本课题组前期对该菌株进行了转录组测序并对基因进行了注释。鉴于目前尚未有关于深海真菌Geosmithia pallida FS140抗单端孢霉烯族毒素研究。众多研究表明，能够转化降解单端孢霉烯族毒素的微生物广泛存在于自然界中，但目前得到实际应用的却不多，唯一商品化应用的是基于菌株BBSH797的饲料添加剂产品，在食品工业领域还没有得到这方面的应用。这主要的原因可能是还没有找到一种高效、安全的，可用于食品工业的脱毒微生物，以及对微生物脱毒作用的机理，特别是微生物产生的脱毒酶类还不是很了解，这些方面还需要深入的研究。此外，目前还没有关于单一的酶同时抗B型和D型单端孢霉烯族毒素的研究报道。因此本发明从深海真菌FS140的cDNA文库中获得了抗单端孢霉烯族毒素酰基转移酶基因GANTB序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中进行了抗毒功能验证，从而为后期提高酿酒酵母抗单端孢霉烯族毒素的能力，提升单端孢霉烯族毒素的异源表达水平奠定分子生物学基础。此外，有利于下一步分离纯化酰基转移酶基因GANTB，明确其对真菌毒素是否具有降解效果，为开发成经济、可行的降毒试剂、防控真菌毒素添加剂等提供新思路。
本发明的深海真菌Geosmithia pallida FS140，其公开于文献：Zhang-Hua Sun,Jiangyong Gu,W ei Ye,Liang-Xi Wen,Qi-Bin Lin,Sai-Ni Li,Yu-Chan Chen,Hao-HuaLi,Wei-Min Zhang.Geospalli ns A–C:New Thiodiketopiperazines with InhibitoryActivity against Angiotensin-Converting Enzyme from a Deep-Sea-Derived FungusGeosmithia pallida FS140.Marine Drugs,2018,16(12),464.https://doi.org/10.3390/md16120464。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为实验所使用化合物结构式：其中，A为B型单端孢霉烯族毒素脱氧雪腐镰刀菌烯醇(DON)的结构式；B为D型单端孢霉烯毒素杆孢菌素E异构体(MR4)的结构式。
图2为深海真菌FS140 GANTB基因序列的获得：以FS140 cDNA文库为模板，基因GANTB扩增产物的电泳图(后三个泳道)；
图3为重组载体YEp352-TEF1-GANTB的构建；其中A为YEp352-TEF1-CYC1载体图谱；B为YEp352-TEF1-GANTB载体图谱；C为基因GANTB的菌落PCR扩增产物的电泳图；
图4为两种酿酒酵母在无毒YPD平板、YPD-DON平板(250μM)和YPD-MR4平板(200μM)培养24h的效果图。A、酿酒酵母BJ5464-D(YEp352-TEF1-CYC1)；B，酿酒酵母BJ5464-D(YEp352-TEF1-GANTB)。10-2、10-3、10-4分别代表OD600约为0.01、0.001、0.0001的5μL菌液样品。
图5为利用生物信息学在线软件工具TMHMM Server,v.2.0对基因GANTB的蛋白质跨膜螺旋预测结果。
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。
本实施例中所用的SD固体培养基的配方为：每升含有葡萄糖20g、Do supplement0.62g(-Leu/-Trp/-Ura，Clontech)、无氨基酵母氮源YNB 6.7g(普博欣)、亮氨酸0.06g、色氨酸0.04g和琼脂粉20g，余量为蒸馏水，其配制方法是将各成分混合均匀，灭菌制得。
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20g，余量为蒸馏水，其配制方法是将各成分混合均匀，灭菌制得。
本实施例中所用的毒素：B型单端孢霉烯族毒素脱氧雪腐镰刀菌烯醇(DON)购买自Sigma；D型单端孢霉烯族毒素杆孢菌素E异构体(MR4)为本课题组分离自内生真菌Myrothecium roridum A553，具体结构如图1所示。
实施例1深海真菌Geosmithia pallida FS140抗单端孢霉烯族毒素自我保护基因序列的获得
基因GANTB的扩增：将深海真菌Geosmithia pallida FS140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RNA提取试剂盒提取RNA，再用All-in-oneRT Master Kit逆转录获得cDNA。根据转录组测序结果预测编码抗单端孢霉烯酰基转移酶GANTB序列，设计引物YEp352-GANTB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GANTB-R:5'-TACATGATGCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3(下划线序列为同源臂片段)，以cDNA文库为模板扩增，获得PCR产物(图2)。回收产物并用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，以通用引物M13-F(5′-GTAAAACGACGGCCAGT-3′)和M13-R(5′-CAGGAAACAGCTATGAC-3′)进行菌液PCR验证阳性克隆并测序，获得目的基因GANTB序列(其核苷酸序列如SEQ ID NO.1所示,atgtcctaccagtctacaatctacaccttcatcttcggctctcgcaccatacaatacaactacaaagtcagcaaacccaaaccaacagacagaaaaaaaaaaacataccttgcatgcatcccaaccatccacacactcaatctccctcagcaaccgaaaccccatcttctcatagatcaccagattgggcatccccttggaactctccaggtaacacggcaccccctccgcatccgcacgactggtcacaacctcaaccagtctcctcccgacccccatcccgcgcatctccgcactcaccgcaaccacattgcagaagtagtaaccccggtcatccgtccagagatcctgatgggtgcgcgcctgcagatccttccagatccagtaccggcggacattaagcccgccgcggccccagaaccggaggttgtacaggagctggcggacggagagtagccagtcctgggaccaggacgtccaagaaggggactgggaaggtggatgcggggcgaaccaccagcagacgccgacgatgggggatgagggtcgggggtgtttgtcagtggcagtggggcggtatttggcgatgtagatgggggcgttgcagctgaggccgtggaggaaatgggcggttatggaggctgcgttgcgttgggggttgtactacttgcattaacgggttgttttttttttttaaggagaagaggacgtactttggatgggtcatcaaagacccagaggaagtacgggtcgtcggcgaaggtcttctggatgcaggctacggcatcggggatgtcttctttggtcaatgggaggatatcgatagccatttttgagggagttcaatctctactggctctgcgggtgggttgcgataaggatatagtgtag)。通过生物学在线软件工具TMHMM Server,v.2.0对基因GANTB编码的蛋白质跨膜螺旋进行预测(图5)，结果表明GANTB基因有1个跨膜组分，其功能可推断为参与毒素转运的基因，在保护宿主免受毒性作用方面发挥着重要作用。
实施例2抗单端孢霉烯族毒素新型酰基转移酶GANTB的功能验证
利用同源重组法将基因酰基转移酶GANTB插入到酵母载体YEp352-TEF1-CYC1中(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，载体图谱见图3A，为现有技术中的已知产品：Xiaodan Ouyang,Yaping Cha,Wen Li,Chaoyi Zhu,MuziZhu,Shuang Li,Min Zhuo,Shaobin Huang and Jianjun Li.Stepwise engineering ofSaccharomyces cerevisiae to produce(+)-valencene and its relatedsesquiterpenes,RSC Adv.,2019,9,30171,DOI:10.1039/c9ra05558d)。首先设计针对基因GANTB(SEQ ID NO.1)扩增的上下游引物YEp352-GANTB-F和YEp352-GANTB-R，其引物序列为YEp352-GANTB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GANTB-R:5'-TACATGATGCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3'(下划线序列为同源臂片段)，通过PCR扩增获得产物。对载体YEp352-TEF1-CYC1采用Sal I和Xba I双酶切并回收产物，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YEp352-GANTB-F和YEp352-GANTB-R进行菌落PCR验证，结果表明基因GANTB成功插入YEp352-TEF1-CYC1载体中(图3C)，并通过测序予以确认，得到YEp352-TEF1-GANTB载体(载体图谱见图3B)。
制备毒素敏感型酿酒酵母Saccharomyces cerevisiae BJ5464-D(relevantgenotype:Δpdr5Δpdr10Δpdr15)的感受态细胞(为现有技术中的已知产品，该菌株对毒性化合物更加敏感：Wolfgang Schwe iger,Jayanand Boddu,Sanghyun Shin,BrigittePoppenberger,Franz Berthiller,Marc Lemmens,Gary J.Muehlbauer,and GerhardAdam.Validation of a Candidate Deoxynivalenol-Inactivating UDP-Glucosyltransferase from Barley by Heterologous Expression in Yeast,MPMI,2010,Vol.23,No.7,DOI:10.1094/MPMI-23-7-0977)。将YEp352-TEF1-GANTB质粒载体以及YEp352-TEF1-CYC1质粒载体(阴性对照)分别电转入酿酒酵母BJ5464-D细胞中(1500V，5ms)，均匀涂布于尿嘧啶缺陷型的SD平板中，在30℃培养2d，利用菌落PCR筛选阳性克隆，获得分别含有YEp352-TEF1-GANTB质粒以及YEp352-TEF1-CYC1质粒的酿酒酵母BJ5464-D细胞。
分别将酿酒酵母BJ5464-D(YEp352-TEF1-CYC1)、酿酒酵母BJ5464-D(YEp352-TEF1-GANTB)接种于Ura-缺陷型的SD培养基中，在30℃培养2d。用分光光度计测量各菌液OD600，将各菌液用无菌水稀释到OD600≈1.0作为原液，再以100μL的原液加900μL的无菌水的方式稀释成10-1，以同样的方式稀释成10-2、10-3、10-4。各取5μL不同菌株的10-2、10-3、10-4的稀释液分别在YPD平板、YPD-DON平板(含有250μM DON毒素)和YPD-MR4平板(含有200μM MR4毒素)点板，在30℃培养并实时观察。培养24h的平板结果显示(图4)，A为酿酒酵母BJ5464-D(YEp352-TEF1-CYC1)、B为酿酒酵母BJ5464-D(YEp352-TEF1-GANTB)在不添加任何毒素的YPD平板上生长状况近乎一致，但在含有250μM DON毒素的YPD平板上和含有200μM MR4毒素的YPD平板上，阴性对照BJ5464-D(YEp352-TEF1-CYC1)生长均明显受阻，没有菌落生长。而导入酰基转移酶GANTB功能基因的酿酒酵母则生长良好，其不同稀释度下的菌体密度与正常酿酒酵母相当，说明来源于深海真菌Geosmithia pallida FS140的GANTB功能基因部分或全部恢复了酿酒酵母BJ5464-D对外源添加毒素的耐受性，有效帮助酿酒酵母在含有毒素的环境下正常生长。
本发明从深海真菌FS140的cDNA文库中获得了抗单端孢霉烯族毒素酰基转移酶GANTB序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中进行了抗毒功能验证，从而为后期提高酿酒酵母抗单端孢霉烯族毒素的能力，提升单端孢霉烯族毒素的异源表达水平奠定分子生物学基础。此外，有利于下一步分离纯化酰基转移酶GANTB，明确其对真菌毒素是否具有降解效果，为开发成经济、可行的降毒试剂、防控真菌毒素添加剂等提供新思路。
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> 一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 891
<212> DNA
<213> 深海真菌FS140(Geosmithia pallida)
<400> 1
atgtcctacc agtctacaat ctacaccttc atcttcggct ctcgcaccat acaatacaac 60
tacaaagtca gcaaacccaa accaacagac agaaaaaaaa aaacatacct tgcatgcatc 120
ccaaccatcc acacactcaa tctccctcag caaccgaaac cccatcttct catagatcac 180
cagattgggc atccccttgg aactctccag gtaacacggc accccctccg catccgcacg 240
actggtcaca acctcaacca gtctcctccc gacccccatc ccgcgcatct ccgcactcac 300
cgcaaccaca ttgcagaagt agtaaccccg gtcatccgtc cagagatcct gatgggtgcg 360
cgcctgcaga tccttccaga tccagtaccg gcggacatta agcccgccgc ggccccagaa 420
ccggaggttg tacaggagct ggcggacgga gagtagccag tcctgggacc aggacgtcca 480
agaaggggac tgggaaggtg gatgcggggc gaaccaccag cagacgccga cgatggggga 540
tgagggtcgg gggtgtttgt cagtggcagt ggggcggtat ttggcgatgt agatgggggc 600
gttgcagctg aggccgtgga ggaaatgggc ggttatggag gctgcgttgc gttgggggtt 660
gtactacttg cattaacggg ttgttttttt tttttaagga gaagaggacg tactttggat 720
gggtcatcaa agacccagag gaagtacggg tcgtcggcga aggtcttctg gatgcaggct 780
acggcatcgg ggatgtcttc tttggtcaat gggaggatat cgatagccat ttttgaggga 840
gttcaatctc tactggctct gcgggtgggt tgcgataagg atatagtgta g 891
Claims (6)
1.一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶GANTB，其特征在于，其由如SEQID NO.1所示的核苷酸序列编码。
2.一种能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
3.一种表达载体，其特征在于，含有权利要求2所述的抗能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB。
4.一种宿主细胞，其特征在于，含有权利要求3所述的表达载体；所述的宿主细胞为酿酒酵母Saccha romyces cerevisiae BJ5464。
5.权利要求1所述的能抗B型和D型单端孢霉烯族毒素的基因GANTB在协助宿主细胞抗单端孢霉烯族毒素中的应用；所述的宿主细胞为深海真菌Geosmithia pallid FS140或酿酒酵母Saccharomyces cerevisiae BJ5464；所述的单端孢霉烯族毒素是B型和/或D型单端孢霉烯族毒素。
6.一种表达盒，其特征在于，含有权利要求2所述的能抗B型和D型单端孢霉烯族毒素的酰基转移酶基因GANTB。
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