CN112575013A - 基因gnatb作为筛选标记基因在抗性筛选中的应用 - Google Patents
基因gnatb作为筛选标记基因在抗性筛选中的应用 Download PDFInfo
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Abstract
本发明公开了基因GNATB作为筛选标记基因在抗性筛选中的应用及其应用。基因GNATB的核苷酸序列如SEQ ID NO.1所示。本发明的新型筛选标记基因GNATB能够高效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。此外，乙酰转移酶基因GNATB可开发成一种高效表达且高抗的筛选标记基因，用来筛选和维持培养成功转染放线菌酮抗性基因的真核细胞，应用于分子生物学、微生物学和医药等领域。
Description
技术领域
本发明属于基因工程领域，具体涉及基因GNATB作为筛选标记基因在抗性筛选中的应用。
背景技术
筛选标记基因可使转化子获得自身所不具备的新的遗传特性；是遗传转化载体所必备的基本元件；是利用特定的选择培养基筛选转化子的一类特殊标记基因。抗性筛选是将抗性基因转入受体菌中使其在一定药物浓度下生长而表现抗药性的一类筛选方式。常用的抗性筛选标记基因分为抗生素抗性基因和除草剂抗性基因两类。抗生素类药物对细胞生长的抑制作用主要在于影响细胞壁的形成和细胞膜的功能、抑制核酸或蛋白质的生物合成。常用的抗生素包括潮霉素、遗传霉素、寡霉素、博来霉素及苯菌灵。
放线菌酮(Cycloheximide)是灰色链霉菌(Streptomyces griseus)的一种产物，一种在真核生物中对蛋白质生物合成过程有抑制效应的化合物，它通过干扰蛋白质合成过程中的易位步骤而阻碍翻译过程。放线菌酮抗性由许多不同的基因产生，高水平的抗性常由编码L29核糖体亚基的cyh2基因座发生突变产生，在生物科研领域用于mRNA的合成，阻止肽链的进一步延伸。此外由于放线菌酮的生物合成研宄却是一片空白，这就极大的阻碍了通过组合生物合成对高活性放线菌酮类似物进行进一步改造和利用。据报道放线菌酮的有效成分环己酰亚胺(CHX)能结合核糖体并抑制eEF2介导的易位。令人惊讶的是，环己酰亚胺允许在停止任何进一步延伸之前进行一个完整的易位循环。推测环己酰亚胺需要E位点结合的脱酰基tRNA用于活性。目前尚未有放线菌酮抗性基因的相关报道，也尚未有深海真菌次级代谢产物生物合成基因作为筛选标记的相关研究。
发明内容
本发明的第一个目的是提供基因GNATB作为筛选标记基因在抗性筛选中的应用，所述的基因GNATB，其核苷酸序列如SEQ ID NO.1所示。
本发明根据深海真菌G.pallid FS140基因组测序结果显示，GNATB基因编码乙酰转移酶，通过成功导入酿酒酵母进行验证，发现乙酰转移酶GNATB能够高效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析GNATB基因与放线菌酮的作用机制奠定基础；提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。推测作用机制为GNATB基因能乙酰化放线菌酮的有效成分环己酰亚胺(CHX)抑制蛋白质合成，阻断真核翻译的延伸期，具体的作用机制有待进一步深入研究。
本发明抗放线菌酮的新型筛选标记基因GNATB通过以下方法获得的：通过转录组测序结果预测编码乙酰转移酶基因GNATB的序列，在其上下游设计特异性引物，其引物序列为GNATB-F:5'-ATGTCCTACCAGTCTACAATCTACACCT-3'；GNATB-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，获得目的基因GNATB，其核苷酸序列如SEQ IDNO.1所示。
本发明利用同源重组法将GNATB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GNATB基因的上下游引物，其引物序列为YEp352-GNATB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-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GNATB(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-GNATB载体，将其电转入酿酒酵母BJ5464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相比，含有重组载体YEp352-TEF1-GNATB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GNATB能有效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。
优选，所述的基因GNATB作为筛选标记基因在放线菌酮抗性筛选中的应用。
本发明的第二个目的是提供基因GNATB在协助宿主细胞在抵抗抗生素中的应用。
所述的抗生素为放线菌酮。
所述的宿主细胞优选为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae BJ5464。
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G.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基因作为分子筛选标记的相关报道。因此本发明从深海真菌FS140的cDNA文库中获得了筛选标记GNATB基因序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中通过放线菌酮药物筛选进行功能验证，为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础，同时可开发成一种高效表达且高抗的筛选标记基因应用于分子生物学、微生物学和医药等领域。
本发明的深海真菌G.pallida FS140，其公开于文献：Zhang-Hua Sun,JiangyongGu,Wei Ye,Liang-Xi Wen,Qi-Bin Lin,Sai-Ni Li,Yu-Chan Chen,Hao-Hua Li,Wei-MinZhang.Geospallins A–C:New Thiodiketopiperazines with Inhibitory Activityagainst 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是筛选标记GNATB基因序列的获得：以FS140 cDNA文库为模板，基因GNATB扩增产物的电泳图；
图2为实验所使用放线菌酮结构式及酿酒酵母BJ5464和酿酒酵母YEp352-TEF1-GNATB(GNATB)在YPD平板和含不同浓度的放线菌酮的YPD平板中培养30h的效果图。
图3为重组载体YEp352-TEF1-GNATB的构建；其中A为YEp352-TEF1-CYC1载体图谱；B为YEp352-TEF1-GNATB载体图谱；C为基因GNATB的菌落PCR扩增产物的电泳图；
图4是GNATB编码的蛋白质跨膜螺旋进行预测。
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。
本实施例中所用抗生素:放线菌酮购于Dr.Ehrenstorfer。
实施例1抗放线菌酮的新型筛选标记GNATB基因序列的获得
基因GNATB的扩增：将深海真菌G.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-one RT MasterKit逆转录获得cDNA。根据转录组测序结果预测编码乙酰转移酶GNATB基因序列，设计上下游引物YEp352-GNATB-F和YEp352-GNATB-R，其引物序列为YEp352-GNATB-F:5'-TAGCAATCT AATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-R:5'-TACATGATGCGGCCCGT CGACCTACACTATATCCTTATCGCAACCCA-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1)。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GNATB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GNATB编码的蛋白质跨膜螺旋进行预测(图4)，结果表明GNATB基因有1个跨膜组分，其功能可推测乙酰转移酶GNATB乙酰化环己酰亚胺为低活性化合物从而保护宿主免受药物作用方面发挥着重要作用。
实施例2抗放线菌酮的新型筛选标记GNATB的功能验证
利用同源重组法将GNATB基因插入到酵母载体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,Muzi Zhu,Shuang Li,Min Zhuo,Shaobin Huang and Jianjun Li.Stepwise engineering ofSaccharomycescerevisiae to produce(+)-valencene and its relatedsesquiterpenes,RSC Adv.,2019,9,30171,DOI:10.1039/c9ra05558d)。首先设计针对基因GNATB(SEQ ID NO.1)扩增的上下游引物YEp352-GNATB-F和YEp352-GNATB-R，其引物序列为YEp352-GNATB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-R:5'-TACATGATGCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3'(下划线序列为同源臂片段)，以cDNA文库为模板扩增，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-GNATB-F和YEp352-GNATB-R进行菌落PCR验证，结果表明基因GNATB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-GNATB载体(载体图谱见图3B)。
将YEp352-TEF1-GNATB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-GNATB质粒以及YEp352-TEF1-CYC1质粒的酿酒酵母BJ5464细胞。
分别将酿酒酵母BJ5464(YEp352-TEF1-CYC1)、酿酒酵母BJ5464(YEp352-TEF1-GNATB)接种于相应缺陷型的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100μL不同菌株的10-4的稀释液分别在YPD无抗平板、各种浓度(100μg/mL、250μg/mL和500μg/mL)放线菌酮平板涂板，在30℃培养并实时观察。培养24h的平板结果显示(图2)，BJ5464为酿酒酵母BJ5464(YEp352-TEF1-CYC1)、GNATB为酿酒酵母BJ5464(YEp352-TEF1-GNATB)，酿酒酵母BJ5464(YEp352-TEF1-CYC1)和酿酒酵母BJ5464(YEp352-TEF1-GNATB)在不添加抗生素的YPD平板上生长状况近乎一致，但在含有放线菌酮的YPD平板上，阴性对照BJ5464(YEp352-TEF1-CYC1)生长均明显受阻，没有菌落生长。而导入酰基转移酶GNATB基因的酿酒酵母则生长良好，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GNATB基因部分或全部恢复了酿酒酵母BJ5464对添加放线菌酮的耐受性(耐受浓度＞500μg/mL)，有效帮助酿酒酵母在含有放线菌酮的环境下正常生长。
本发明从深海真菌FS140的cDNA文库中获得了筛选标记GNATB基因序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中通过放线菌酮药物筛选进行功能验证，为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础，同时可开发成一种高效表达且高抗的筛选标记基因应用于分子生物学、微生物学和医药等领域。
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> 基因GNATB作为筛选标记基因在抗性筛选中的应用
<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 (5)
1.基因GNATB作为筛选标记基因在抗性筛选中的应用，所述的基因GNATB，其核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述的基因GNATB作为筛选标记基因在放线菌酮抗性筛选中的应用。
3.基因GNATB在协助宿主细胞在抵抗抗生素中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的抗生素为放线菌酮。
5.根据权利要求3或4所述的应用，其特征在于，所述的宿主细胞为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae BJ5464。
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| US4857460A (en) * | 1985-08-27 | 1989-08-15 | Nikka Whisky Distilling Co., Ltd. | Cycloheximide resistant gene |
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