CN112725362B - 一种露湿漆斑菌a553抗单端孢霉烯自我保护基因gnat11及其应用 - Google Patents
一种露湿漆斑菌a553抗单端孢霉烯自我保护基因gnat11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抗单端孢霉烯自我保护基因GNAT1及其应用。该基因的核苷酸序列如SEQ ID NO.1所示。本发明基于转录组测序结果，以cDNA文库为模板，扩增获得露湿漆斑菌A553的抗单端孢霉烯自我保护基因GNAT1。本发明将扩增获得的GNAT1插入酿酒酵母表达载体，然后转化至酿酒酵母能够显著提升酿酒酵母对单端孢霉烯毒素的耐受性，从而为后期植物抵抗植物病原真菌提供分子生物学策略，同时为后期通过异源表达提升单端孢霉烯毒素的产量并获得新型单端孢霉烯类化合物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抗单端孢霉烯自我保护基因GNAT11及其应用。
背景技术
单端孢霉烯毒素是一类主要由露湿漆斑菌Myrothecium roridum产生的具有抗肿瘤活性及抗真菌活性的大环内酯。单端孢霉烯对植物细胞也有显著的毒性，是露湿漆斑菌对大豆等经济作物的主要致病因子。单端孢霉烯主要靶向植物细胞中普遍保守的Rpl3蛋白的合成而产生细胞毒性。单端孢霉烯毒素对宿主菌也存在毒性，因此宿主菌中也存在对单端孢霉烯的抗性基因，以减弱其对宿主菌的毒性。因此，十分有必要了解宿主菌对单端孢霉烯霉烯的抗性机制，这对于通过代谢工程异源表达单端孢霉烯毒素并大规模制备尤为关键。
发明内容
本发明的第一个目的是提供一种露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT1，所述的露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT1，其核苷酸序列如SEQ IDNO.1所示。
本发明的露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT1通过以下方法获得的：通过转录组测序结果预测编码抗单端孢霉烯自我保护基因GNAT1的序列，在其上下游设计特异性引物，其含有同源臂的引物序列为GNAT1-F:5'CAATCTCAGCGctcgagGGCGGCGGTGGAGGCAGTagtaaaggagaagaacttttcactg-3'；GNAT1-R:5'-TGATGCGGCCCGTCGACTTATTTGTATAGTTCATCCATGC-3'，以由露湿漆斑菌A553提取的总RNA反转录获得的cDNA文库为模板，通过PCR扩增获得产物并纯化回收片段GANT1。然后利用同源重组法将GNAT1基因插入到酵母载体YEp352-TEF1-CYC1的表达盒内部。已构建的YEp352-TEF1-CYC1载体采用内切酶Sal I和XbaI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DH5α，氨苄青霉素抗性平板结合菌液PCR及测序筛选出阳性克隆。构建得到YEp352-TEF1-GNAT1载体，采用电转法转入酿酒酵母BJ5464感受态细胞中，利用尿嘧啶缺陷型的SD培养基平板进行筛选和验证。以含有YEp352-TEF1-CYC1质粒的酿酒酵母BJ5464-D作为阴性对照。结果表明，转入GANT1功能基因的酿酒酵母在含有DON毒素的平板上比阴性对照生长速度更快，菌落数明显更多，这说明基因GNAT1具有体外抵抗外源单端孢霉烯DON毒素的功能。
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抗单端孢霉烯自我保护基因GNAT11。
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抗单端孢霉烯自我保护基因GNAT1在协助宿主细胞抵抗单端孢霉烯毒素中的应用。
所述的宿主细胞优选为露湿漆斑菌Myrothecium roridum A553或酿酒酵母Saccharomyces cerevisiae BJ5464。
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抗单端孢霉烯自我保护基因GNAT1，然后通过对GNAT11蛋白的表达纯化及酶活力分析验证其体外功能。
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A553分离自广藿香，本课题组前期对该菌株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。转录组测序及文献调研结果也表明，该菌株自有的GNAT1类基因可能同Tri12共同协助露湿漆斑菌发挥抵抗单端孢霉烯毒素毒性的功能。本发明从露湿漆斑菌A553的cDNA文库中获得了抗单端孢霉烯自我保护基因GNAT11序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中，在含有单端孢霉烯DON毒素的平板上进行了抗毒功能验证，从而为进一步解析露湿漆斑菌对单端孢霉烯的抗毒机制及单端孢霉烯的生物合成奠定了分子生物学基础。
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.Cytotoxic trichothecene macrolides from the endophyte fungusMyrothecium roridum,Journal of Asian Natural Products Research,2016,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 GNAT1基因序列的获得：以A553 cDNA文库为模板，基因GNAT1扩增产物的电泳图；
图2为重组载体YEp352-TEF1-GNAT1的构建；其中A为YEp352-TEF1-CYC1载体图谱；B为YEp352-TEF1-GNAT1载体图谱；C为基因GNAT1的菌落PCR扩增产物的电泳图；
图3为三种酿酒酵母在YPD平板(A)和YPD-DON平板(100μM)(B)培养36h的效果图。BJ5464-D-为酿酒酵母BJ5464-D(YEp352-TEF1-CYC1)；GNAT1为酿酒酵母BJ5464-D(YEp352-TEF1-GNAT1)。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。
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固体培养基组成为：葡萄糖20g/L、Do supplement 0.62g/L(-Leu/-Trp/-Ura，Clontech)、无氨基酵母氮源YNB 6.7g/L(普博欣)、亮氨酸0.06g/L、色氨酸0.04g/L和琼脂粉20g/L，溶剂为水，配制方法将各成分混合均匀，灭菌制得。单端孢霉烯DON毒素购买于Sigma。
本实施例中所用的YPD固体培养基的配方为：酵母粉10g/L、蛋白胨20g/L、葡萄糖20g/L和琼脂粉20g/L，溶剂为水，配制方法将各成分混合均匀，灭菌制得。
实施例1露湿漆斑菌Myrothecium roridum A553抗单端孢霉烯自我保护基因GNAT1序列的克隆及表达载体构建
基因GNAT1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30℃培养48h，挑取新鲜的菌丝体，提取总RNA，再用All-in-one RTMaster Kit(Abcam,Canada)逆转录获得cDNA。根据转录组测序结果预测编码酰基转移酶具有自我保护功能的基因GNAT1，设计上下游引物GNAT1-F和GNAT1-R，其中GNAT1-F:5'ATCTAATCTAAGTCTAGAATGGCGTTCAAAATCCGCC-3'；GNAT1-R:5'-GATGCGGCCCGTCGACTTACGCTGAGATTGATGGTTCACGTAT-3'，并以cDNA为模板扩增GANT1片段(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，菌液PCR验证阳性克隆并测序，获得目的基因GNAT1序列(其核苷酸序列如SEQ ID NO.1所示)。
实施例2抗单端孢霉烯自我保护基因GNAT1的功能验证
利用同源重组法将基因GNAT1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2A，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 ofSaccharomyces cerevisiae to produce(+)-valencene and its relatedsesquiterpenes,RSC Adv.,2019,9,30171,DOI:10.1039/c9ra05558d)。将以GNAT1-F和GNAT1-R为引物扩增得到的GNAT1片段加入Sal I和Xba I双酶切后的YEp352-TEF1-CYC1载体中，然后使用ClonExpress II One Step Cloning Kit C112(Vazyme)将两个产物重组连接，得到同源重组的产物。具体的反应体系和条件如下：
表1同源重组反应体系及条件：
将同源重组的产物转化至DH5α中，Amp平板筛选，结合菌液PCR和测序获得阳性克隆。结果表明基因GNAT1成功插入YEp352-TEF1-CYC1载体中(图2A)。得到YEp352-TEF1-GNAT1载体(载体图谱见图2B)。
采用CRISPR/Cas9对酿酒酵母的毒素抗性相关基因BJ5464 pdr5、pdr10和pdr15进行基因敲除。毒素敏感型酿酒酵母Saccharomyces cerevisiae BJ5464-D(relevantgenotype:Δpdr5Δpdr10Δpdr15)感受态细胞对毒性化合物更加敏感，由本实验室制备(具体制备方法参见文献：Wolfgang Schweiger,Jayanand Boddu,Sanghyun Shin,Brigitte Poppenberger,Franz Berthiller,Marc Lemmens,Gary J.Muehlbauer,andGerhard Adam.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-GNAT1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感受态细胞中(1500V，5ms)，然后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-GNAT1质粒以及YEp352-TEF1-CYC1质粒的酿酒酵母BJ5464-D细胞，然后将两种酿酒酵母细胞接种于相应缺陷型的SD培养基中(配制ΔLeu-ΔTrp-ΔUra的三缺型SD培养基(100mL)：称取2g(2％，w/v)无水葡萄糖，用蒸馏水定容至80mL，115℃高温高压灭菌20min。灭菌后冷却，在超净工作台中按1:10的比例加入10×酵母氮源(YNB)溶液和10×DO Supplement溶液，4℃保存备用)，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，用无菌水将其稀释成10-1，再以相同的方式稀释成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平板和含有100μM单端孢霉烯DON毒素的YPD-DON平板上进行点板，在30℃培养并实时观察拍照。培养36小时的平板结果显示(图3)，酿酒酵母BJ5464、酿酒酵母BJ5464-D(YEp352-TEF1-CYC1)、酿酒酵母BJ5464-D(YEp352-TEF1-GNAT1)在不添加任何毒素的YPD平板上生长状况没有显著差异；但在含有100μM DON毒素的YPD平板上，阴性对照BJ5464-D(YEp352-TEF1-CYC1)几乎观察不到菌落，10-3和10-4两个梯度的菌液几乎没有生长。而导入GNAT1功能基因的酿酒酵母BJ5464则生长良好，与野生酿酒酵母BJ5464生长状态几乎一致。说明来源于露湿漆斑菌A553的GNAT1功能基因能增加酿酒酵母BJ5464-D对单端孢霉烯毒素的抵抗力，这为后期通过分子生物学手段提高作物对禾谷镰刀菌等致病菌的抗性奠定了可靠的分子生物学基础。
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抗单端孢霉烯自我保护基因GNAT1及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 744
<212> DNA
<213> 露湿漆斑菌A553(Myrothecium roridum )
<400> 1
atggcgttca aaatccgccc ggctgaggca gccgatgtgc ctgctatcgc tagtgctcac 60
tacagtgcca tgacccctct caacgacttc tatgggacac tgtttgcgat ggatctgcag 120
gaggcattcc aggccgcagt ggaagcggcc ctccaaaatg cagccaccac cttcttggtg 180
gctgagcatc ccgacagtgg agtcgttggt tttgttcgct accagattga agatgcatcg 240
gacagccaag aggagcaaca gaacaaggac gttggcacac tgcaccaagc aacaacgacg 300
cccgcggcac cttcaccctg ggcacgaaag ccgcatctgg aagaaatatg gcagggattg 360
caggctcgca cggacgagat ggatgcctgt gaacagcaag ccttgcaagg ccgaagatac 420
ctcagcataa ggcatttgat gatccattcg gactggcaaa ggcaaggctt gggctcgaag 480
ctgctcaaag aagtccttgc gagaagcgaa gcagaacgca tcccctgctg gattgtatcg 540
tcagctgaat cacaagagct gtatgccaag cttggctttc aggaacttgg cgtttggacc 600
atcgacaacg cgtactgggc gaaacgcttg gtcgaccacg agaagagcct tggaatcaca 660
gggaacgaag gtctcgtgga ccaatacgca ggtgtcatcg aggtcgagaa gtgtttcata 720
cgtgaaccat caatctcagc gtaa 744
Claims (5)
1.一种露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT11，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所述的露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT11。
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；所述的宿主细胞为酿酒酵母Saccharomyces cerevisiae BJ5464。
4.权利要求1所述的露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT11在协助宿主细胞抵抗单端孢霉烯毒素中的应用；所述的宿主细胞为露湿漆斑菌Myrothecium roridumA553或酿酒酵母Saccharomyces cerevisiae BJ5464。
5.一种表达盒，其特征在于，含有权利要求1所述的露湿漆斑菌A553抗单端孢霉烯自我保护基因GNAT11。
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| TW200913987A (en) * | 2007-09-20 | 2009-04-01 | Golden Biotechnology Corp | Extract of Myrothecium sp. mycelium used to inhibit growth of tumor cell |
| CN105441331A (zh) * | 2015-11-20 | 2016-03-30 | 山东省农业科学院植物保护研究所 | 一株露湿漆斑菌及其应用 |
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