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
- Publication number
- CN112725362B CN112725362B CN202011387072.8A CN202011387072A CN112725362B CN 112725362 B CN112725362 B CN 112725362B CN 202011387072 A CN202011387072 A CN 202011387072A CN 112725362 B CN112725362 B CN 112725362B
- Authority
- CN
- China
- Prior art keywords
- trichothecene
- gnat1
- gene
- saccharomyces cerevisiae
- myrothecium roridum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229930013292 trichothecene Natural products 0.000 title claims abstract description 35
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 title claims abstract description 34
- 241001099903 Paramyrothecium roridum Species 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 29
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 29
- 239000003053 toxin Substances 0.000 claims abstract description 17
- 231100000765 toxin Toxicity 0.000 claims abstract description 17
- 239000013604 expression vector Substances 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 108700012359 toxins Proteins 0.000 claims description 15
- 241000206443 Telluribacter humicola Species 0.000 claims 1
- 101000888178 Homo sapiens Guanine nucleotide-binding protein G(t) subunit alpha-1 Proteins 0.000 abstract description 23
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 102100039261 Guanine nucleotide-binding protein G(t) subunit alpha-1 Human genes 0.000 abstract description 5
- 230000003321 amplification Effects 0.000 abstract description 4
- 239000002299 complementary DNA Substances 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 244000000004 fungal plant pathogen Species 0.000 abstract 1
- -1 novel trichothecene compound Chemical class 0.000 abstract 1
- 239000013598 vector Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 101150103547 Gnat1 gene Proteins 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229960001031 glucose Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000980781 Cladosiphon Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 101100393190 Mus musculus Gnat1 gene Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000001147 anti-toxic effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 description 2
- 229940041033 macrolides Drugs 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 241000223600 Alternaria Species 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000223195 Fusarium graminearum Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000223251 Myrothecium Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000002505 Pogostemon cablin Species 0.000 description 1
- 235000011751 Pogostemon cablin Nutrition 0.000 description 1
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 1
- 101150001810 TEAD1 gene Proteins 0.000 description 1
- 101150074253 TEF1 gene Proteins 0.000 description 1
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000032537 response to toxin Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 101150073388 rpl3 gene Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 150000003327 trichothecene derivatives Chemical class 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011387072.8A CN112725362B (zh) | 2020-12-01 | 2020-12-01 | 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011387072.8A CN112725362B (zh) | 2020-12-01 | 2020-12-01 | 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112725362A CN112725362A (zh) | 2021-04-30 |
| CN112725362B true CN112725362B (zh) | 2022-11-22 |
Family
ID=75598127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011387072.8A Active CN112725362B (zh) | 2020-12-01 | 2020-12-01 | 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 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725362B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110684035A (zh) * | 2019-09-03 | 2020-01-14 | 郑州轻工业学院 | 一种单端孢霉烯型化合物及其作用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1510573A1 (en) * | 2003-09-01 | 2005-03-02 | Austria Wirtschaftsservice Gesellschaft mit beschränkter Haftung | Method for detoxification of mycotoxins |
| CN101407523A (zh) * | 2007-10-08 | 2009-04-15 | 国鼎生物科技股份有限公司 | 用于抑制肿瘤细胞生长的漆斑霉菌丝体萃取物 |
| CN102154323A (zh) * | 2010-12-17 | 2011-08-17 | 河南省农业科学院 | 降解赤霉病菌毒素基因及其表达 |
| CN109735537B (zh) * | 2018-12-27 | 2022-06-24 | 广东省微生物研究所（广东省微生物分析检测中心） | 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 |
| CN110904125B (zh) * | 2019-11-26 | 2021-05-11 | 广东省微生物研究所（广东省微生物分析检测中心） | 一种露湿漆斑菌A553抗单端孢霉烯自我保护基因mfs1及其应用 |
-
2020
- 2020-12-01 CN CN202011387072.8A patent/CN112725362B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110684035A (zh) * | 2019-09-03 | 2020-01-14 | 郑州轻工业学院 | 一种单端孢霉烯型化合物及其作用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112725362A (zh) | 2021-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110904125B (zh) | 一种露湿漆斑菌A553抗单端孢霉烯自我保护基因mfs1及其应用 | |
| Kemppainen et al. | Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N | |
| KR20120088035A (ko) | 에탄올?저항성 효모 유전자 및 이의 용도 | |
| Cao et al. | The Magas1 gene is involved in pathogenesis by affecting penetration in Metarhizium acridum | |
| Li et al. | The barley powdery mildew effectors CSEP0139 and CSEP0182 suppress cell death and promote B. graminis fungal virulence in plants | |
| WO2016029802A1 (zh) | 虫草素的合成基因簇的鉴定和应用 | |
| CN110157756A (zh) | 一种通过改造糖多孢红霉菌sace_0303基因提高红霉素产量的方法 | |
| CN104630258A (zh) | 一种酿酒酵母基因表达系统及其构建与应用 | |
| CN112608931B (zh) | 一种深海真菌FS140抗胶霉毒素自我保护基因GliM及其应用 | |
| CN111019945B (zh) | 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 | |
| CN112725362B (zh) | 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 | |
| CN113881579A (zh) | 木霉菌长链抗菌肽合成及抑菌活性提高的方法 | |
| CN109136253B (zh) | 一种通过糖多孢红霉菌sace_5754基因途径提高红霉素产量的方法 | |
| CN112680469B (zh) | 抗胶霉毒素自我保护基因GliK在协助宿主细胞抵抗胶霉毒素中的应用 | |
| Xuan et al. | MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize | |
| Wang et al. | Regulation of conidiation, dimorphic transition, and microsclerotia formation by MrSwi6 transcription factor in dimorphic fungus Metarhizium rileyi | |
| CN104561082A (zh) | 一株能够利用木糖的酵母Candida jeffriesii的表达系统 | |
| Wang et al. | Adaption to stress via Pbs2 during Metarhizium rileyi conidia and microsclerotia development | |
| CN112695044B (zh) | 一种深海真菌FS140抗胶霉毒素自我保护基因mfs-get及其应用 | |
| CN103146716A (zh) | 源于稻瘟病菌的真菌致病性基因Movma11及用途 | |
| CN112852847A (zh) | 一种重组酿酒酵母菌株及其构建方法与应用 | |
| CN104561081A (zh) | 一株能够利用木糖的酵母Candida amazonensis的表达系统 | |
| Ying et al. | Functional characterization of a NEM1-like gene in Magnaporthe oryzae | |
| CN112592954B (zh) | 基因GliT作为筛选标记基因在抗性筛选中的应用 | |
| CN113106106B (zh) | 源于希金斯炭疽菌的真菌致病性基因ChAtg8及应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Address before: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |