CN110923258A - 一种真姬菇的遗传转化方法 - Google Patents
一种真姬菇的遗传转化方法 Download PDFInfo
- Publication number
- CN110923258A CN110923258A CN201911233767.8A CN201911233767A CN110923258A CN 110923258 A CN110923258 A CN 110923258A CN 201911233767 A CN201911233767 A CN 201911233767A CN 110923258 A CN110923258 A CN 110923258A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- spores
- hypsizigus marmoreus
- node
- liquid
- 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.)
- Pending
Links
- 230000002068 genetic effect Effects 0.000 title claims abstract description 18
- 238000011426 transformation method Methods 0.000 title claims abstract description 9
- 239000005746 Carboxin Substances 0.000 claims abstract description 14
- GYSSRZJIHXQEHQ-UHFFFAOYSA-N carboxin Chemical compound S1CCOC(C)=C1C(=O)NC1=CC=CC=C1 GYSSRZJIHXQEHQ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 241000589158 Agrobacterium Species 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 8
- 230000009466 transformation Effects 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 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 claims description 6
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 claims description 5
- 229960004261 cefotaxime Drugs 0.000 claims description 5
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
- 229930189077 Rifamycin Natural products 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 229960003292 rifamycin Drugs 0.000 claims description 2
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical compound OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 244000252132 Pleurotus eryngii Species 0.000 abstract description 5
- 235000001681 Pleurotus eryngii Nutrition 0.000 abstract description 5
- 239000003550 marker Substances 0.000 abstract description 5
- 230000001131 transforming effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 14
- 241001534815 Hypsizygus marmoreus Species 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 210000001938 protoplast Anatomy 0.000 description 6
- 239000012634 fragment Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 101150054900 gus gene Proteins 0.000 description 2
- 239000000819 hypertonic solution Substances 0.000 description 2
- 229940021223 hypertonic solution Drugs 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000221198 Basidiomycota Species 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003127 anti-melanomic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940051921 muramidase Drugs 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种真姬菇的遗传转化方法,包括收集节孢子和转化农杆菌等。本发明利用来源于杏鲍菇中的萎锈灵抗性基因作为筛选标记,以农杆菌为介导,为通过遗传方法改良真姬菇品种奠定了基础,具有良好的市场应用前景。
Description
技术领域
本发明属于分子生物学领域,特别涉及一种真姬菇的遗传转化方法。
背景技术
真姬菇是一种在我国被工厂化大量栽培的食用菌,营养价值高,具有很大的市场价值。另外有报道表明真姬菇的子实体具有抗黑色素瘤物质,具有潜在的药用价值。传统的杂交育种被用来改进栽培品种的口感和缩短生产周期。遗传转化是更快捷有效的改良品种农艺性状的方法。
目前真姬菇遗传转化主要是以原生质体作为转化材料。原生质体的制备需要用融壁酶消化菌丝来去除细胞壁,操作比较繁琐。另外原生质体需要保存在高渗溶液中,比较脆弱而且容易在操作和离心过程中破碎。制备好的原生质体有很多丢失了细胞核,导致在遗传转化后的再生率低。
某些担子菌在特定的培养条件下会产生无性孢子。节孢子是无性孢子的一种,它是由营养菌丝的细胞产生隔膜然后断裂形成的。先前已有研究培养真姬菇的单核菌丝能产生大量的节孢子,但到目前为止没有文献报道真姬菇的节孢子能用于遗传转化。
发明内容
本发明所要解决的技术问题是提供一种真姬菇的遗传转化方法,该方法利用来源于杏鲍菇中的萎锈灵抗性基因作为筛选标记,以农杆菌为介导,为通过遗传方法改良真姬菇品种奠定了基础,具有良好的市场应用前景。
本发明提供了一种真姬菇的遗传转化方法,包括:
(1)挑取含有转化质粒pTSdi-GUS的EHA105农杆菌单克隆,接菌到含有利发霉素和卡那霉素的LB液体培养基中,经摇床培养得到农杆菌菌液用于侵染;
(2)将上述农杆菌菌液与重悬于IM液体培养基的真姬菇节孢子混合进行侵染,随后涂布到含琼脂的IM液体培养基上,遮光培养;最后用吐温-20洗下IM固体培养基上的节孢子,涂布在含有萎锈灵和头孢噻肟霉素的YMG平板上进行筛选,得到真姬菇转化子。
所述步骤(1)中的LB液体培养基的组成为:10g/L胰蛋白胨,5g/L酵母提取物,5g/LNaCl,pH 7.0。
所述步骤(2)中的IM液体培养基的组成为:10mM K2HPO4,10mM KH2PO4,2.5mMNaCl,2mM MgSO4·7H2O,0.7mM CaCl2,9μM FeSO4·7H2O,4mM(NH4)2SO4,10mM葡萄糖,0.5%甘油,0.2mM乙酰丁香酮,40mM 2-(N-吗啉基)乙磺酸,pH 5.6。
所述步骤(2)中的YMG平板的组成为:1%葡萄糖,1%麦芽提取物,0.4%酵母提取物,2%琼脂,pH7.0。
所述步骤(2)中的萎锈灵浓度为2μg/mL,头孢噻肟霉素浓度为300μg/mL。
有益效果
本发明利用来源于杏鲍菇中的萎锈灵抗性基因作为筛选标记,以农杆菌为介导对真姬菇的节孢子成功进行了遗传转化。到目前为止公开报道的真姬菇转化都以原生质体作为转化材料,原生质体的制备比较繁琐,需要保存在高渗溶液中,比较脆弱而且容易在操作和离心过程中破碎,不是理想的转化材料。本发明以节孢子作为转化材料,简化了真姬菇的遗传转化步骤,加快了外源基因导入的效率。另外本发明成功将杏鲍菇中的萎锈灵抗性基因作为筛选标记,比目前食用菌遗传转化常用的细菌来源的潮霉素抗性基因更加安全可靠。本发明为通过遗传方法改良真姬菇品种奠定了基础,具有良好的市场应用前景。
附图说明
图1为构建的转化质粒pTSdi-GUS示意图;
图2为收集的真姬菇节孢子的显微镜观察照片;
图3为在萎锈灵筛选培养基上再生的真姬菇转化子;
图4为真姬菇转化子的分子检测结果;
图5为真姬菇转化子的GUS染色结果。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
一、转化质粒pTSdi-GUS构建
质粒pCambia1301被用来作为空载构建转化质粒。用限制性内切酶XhoI和SacI消化pCambia1301去除潮霉素抗性基因盒,在琼脂糖凝胶上分离pCambia 1301的残留载体骨架并进行纯化回收。通过PCR扩增方法从含有杏鲍菇萎锈灵抗性筛选标记的质粒pTsdi中扩增萎锈灵抗性基因盒,PCR的正向和反向引物中分别加入XhoI和SacI识别位点的接头。PCR扩增片段经限制性内切酶XhoI和SacI消化,用T4 DNA连接酶连接到经过纯化的含有XhoI和SacI接头的pCambia1301片段上,得到转化质粒pSdi-GUS。附图1显示了转化质粒pSdi-GUS的左边界和右边界之间的萎锈灵抗性基因盒以及GUS基因盒,具体序列如SEQ No.1所示。
二、培养基的配置:
LB液体培养基的组成为:10g/L胰蛋白胨,5g/L酵母提取物,5g/L NaCl,pH 7.0。
IM培养基的组成为:10mM K2HPO4,10mM KH2PO4,2.5mM NaCl,2mM MgSO4·7H2O,0.7mM CaCl2,9μM FeSO4·7H2O,4mM(NH4)2SO4,10mM葡萄糖,0.5%甘油,0.2mM乙酰丁香酮,40mM 2-(N-吗啉基)乙磺酸,pH 5.6。
YMG平板的组成为:1%葡萄糖,1%麦芽提取物,0.4%酵母提取物,2%琼脂,pH7.0。
三、节孢子收集和农杆菌介导转化:
①节孢子的收集方法
1.挑取少量真姬菇单核菌丝,接种于YMG固体培养基上,25度恒温培养15天。
2.在平板上加入3毫升0.05%吐温-20,用接种棒刮下培养基表面的菌丝和节孢子。
3.用4层无纺布过滤去掉菌丝,得到的节孢子用4000g离心力离心10分钟。
4.弃去上清,用2毫升IM培养基重悬,在光学显微镜下用血球计数板对节孢子计数,并用IM培养基调整到106个节孢子/mL。
②农杆菌转化方法
1.挑取含有转化质粒pTSdi-GUS的EHA105农杆菌单克隆,接菌到5mL含有50μg/mLrifampicin和50μg/mL kanamycin的LB液体培养基中,置于28℃摇床上以200rpm转速培养1天。
2.菌液转移到10mL离心管中,以3000g离心力离心10min。弃上清,菌体重悬于10mlIM培养基,测量菌液OD600,并用IM培养基调整OD600到0.4左右。
3.置于28℃摇床上以150rpm转速培养6小时,测量菌液OD600,并用IM培养基调整OD600到0.4左右用于侵染。
4.混合1ml用于侵染的农杆菌与1ml重悬于IM培养基的真姬菇节孢子,置于28℃摇床上以80rpm转速侵染20min。
5.将侵染后的农杆菌和节孢子混合物涂布到到含2%琼脂的IM固体培养基上,28度遮光培养2天。
6.用2毫升0.05%吐温-20洗下IM固体培养基上的节孢子,涂布在含有2μg/mL萎锈灵、300μg/mL头孢噻肟霉素的YMG平板上进行筛选。抗性转化子一般在10天左右再生。
按上述方法培养真姬菇转化受体的单核菌丝获得节孢子,用0.05%吐温-20洗脱节孢子,过滤后计数。图2为光学显微镜观察的血球计数板上的节孢子。按上述方法对获得的节孢子用包含pTSdi-GUS的EHA105农杆菌进行侵染,经过共培养和筛选获得对真菌杀菌剂萎锈灵有抗性的阳性转化子。图3为培养10天后在筛选平板上再生的阳性转化子。
四、阳性转化子的分子检测:
从转化受体的菌丝和转化子的菌丝中提取基因组DNA,针对转化质粒的DNA序列设计引物pesdiF(5'-TCACGTCCTCAATAAGCGCTC-3')
和pesdi3R(5'-TGAGTGAGGTATACAAGGTAG-3'),对转化子和转化受体的基因组DNA进行PCR扩增。PCR结果显示,从转化子和转化质粒pSdi-GUS中都能扩增出617bp的预期DNA片段,但转化受体中没有扩增产物(图4)。说明转化质粒上的外源DNA片段已经整合到了真姬菇转化子的基因组中。
五、阳性转化子的GUS染色鉴定:
挑取阳性转化子的少量菌丝体浸入含有GUS染色液的1.5ml离心管中,在37℃下孵育24小时后,转化子的菌丝体被染成蓝色(图5),说明转入的外源GUS基因不仅整合到了真姬菇转化子的基因组中,而且可以在转化子的细胞中正常表达。
SEQUENCE LISTING
<110> 上海市农业科学院
<120> 一种真姬菇的遗传转化方法
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 12295
<212> DNA
<213> 人工序列
<400> 1
gatctgaggg taaatttcta gtttttctcc ttcattttct tggttaggac ccttttctct 60
ttttattttt ttgagctttg atctttcttt aaactgatct attttttaat tgattggtta 120
tggtgtaaat attacatagc tttaactgat aatctgatta ctttatttcg tgtgtctatg 180
atgatgatga tagttacaga accgacgact cgtccgtcct gtagaaaccc caacccgtga 240
aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga 300
tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt 360
taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg 420
cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt 480
cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta 540
tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac 600
cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga 660
cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca 720
tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac 780
gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga 840
tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag 900
cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga 960
actcgaagtc acagccaaaa gccagacaga gtctgatatc tacccgcttc gcgtcggcat 1020
ccggtcagtg gcagtgaagg gccaacagtt cctgattaac cacaaaccgt tctactttac 1080
tggctttggt cgtcatgaag atgcggactt acgtggcaaa ggattcgata acgtgctgat 1140
ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta 1200
cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga 1260
aactgctgct gtcggctttc agctgtcttt aggcattggt ttcgaagcgg gcaacaagcc 1320
gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc 1380
gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc 1440
caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc 1500
aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc 1560
tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg 1620
gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc 1680
ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc 1740
cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga 1800
tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt 1860
cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt 1920
cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat 1980
gaacttcggt gaaaaaccgc agcagggagg caaacaagct agccaccacc accaccacca 2040
cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg ttcaaacatt tggcaataaa 2100
gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga 2160
attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt 2220
ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg 2280
caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattaaact 2340
atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa 2400
taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc 2460
caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt 2520
gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc 2580
taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt 2640
agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc 2700
gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac 2760
caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc 2820
accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt 2880
gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc 2940
gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc 3000
accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt 3060
tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag 3120
tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac 3180
caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg 3240
taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc 3300
ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa 3360
gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag 3420
agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa 3480
ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg 3540
ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt 3600
aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac 3660
gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac 3720
gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt 3780
cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct 3840
aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg 3900
cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa 3960
ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc 4020
cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc 4080
ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct 4140
ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg 4200
cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga 4260
ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag 4320
aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag 4380
gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg 4440
ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc 4500
gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag 4560
tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga 4620
cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt 4680
tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc 4740
gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt 4800
tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat 4860
cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg 4920
cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc 4980
tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt 5040
cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag 5100
aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg 5160
cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg 5220
gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg 5280
gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca 5340
tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct 5400
tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg 5460
agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg 5520
ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac 5580
gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg 5640
gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg 5700
acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc 5760
gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc 5820
tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca 5880
cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa 5940
agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg 6000
atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg 6060
cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc 6120
tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa 6180
tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg 6240
accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac 6300
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 6360
agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 6420
cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 6480
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 6540
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 6600
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 6660
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 6720
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 6780
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 6840
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 6900
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 6960
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 7020
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 7080
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 7140
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 7200
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7260
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7320
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7380
aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa atataatatt 7440
ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt 7500
cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg 7560
ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt 7620
tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta 7680
aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat 7740
ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc 7800
tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat 7860
acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc 7920
catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct 7980
ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat 8040
aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca 8100
ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt 8160
cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc 8220
ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga actccaattc 8280
actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc 8340
aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc 8400
agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc 8460
aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc 8520
cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt 8580
ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat 8640
tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg 8700
tactgaatta acgccgaatt aattcggggg atctggattt tagtactgga ttttggtttt 8760
aggaattaga aattttattg atagaagtat tttacaaata caaatacata ctaagggttt 8820
cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt atctgggaac 8880
tactcacaca ttattatgga gaaactcgag tcacgtcctc aataagcgct cgcgacaacg 8940
cggattcaca tcctgccccc acatcactct ccaagaatct cttgttcaaa ggtatcccgt 9000
atttgtaagt caagccaacg tttattccat gatacatggc tggccacatt catggtattc 9060
tctccgtaaa tagccattta ccacccatga cacgacatat tcggactcct gccgcttgtg 9120
aaccgttcaa ctatgcaaat gctaatgcat ggcttctgag actgccaacg ctgtttggag 9180
aaaccttctc aaaaacggct gctgaagacc taagccagcg ctgcaccgac attttctcaa 9240
cactcaatag aagttgcaac taggtcaaca aagttggcat tcgatcgcct gtgactacaa 9300
attgattcgg ggggttcgac gatggacacc atgactgagc tttggggtcc gttacgacga 9360
gaacatccga agtcccttcc tcaactttca agggaatttc gaggcctcag agcgccgttg 9420
aactcaggtt tccgagtgat acctcaattg ctttccccct tttctgatca gagtactcga 9480
ggtacagctt gggtacggca catattctac cttgtatacc tcactcataa cgttccgagg 9540
agagtggcat cacacagcgc gctggcctaa aagtggtgtc aagttggaag ggaagcgagt 9600
cggggtcatt ggaactgggg ccagcgccga gttcagatca tccaagaggc aggcaaattg 9660
gcacaagagc tcgtggtatt acagcgtaca ccgaatctga cattccaccc gcgacaaccc 9720
acgcaagcca aagtgtcaga agttgtcgaa atgcagtgga atcatgctca cgaagaggcc 9780
ccgagtagtg accctctcaa gtaaacaagc atcgtcgatt tcttaacact tctagttgct 9840
ccgtccacgt gtatatatac cttcgactga agagcatgat tggcgcaaag attacgttct 9900
atcgctcttc tctccttatt ttgataatcg aaagccgcac tactcagcgc agcagttcta 9960
catgtttttc taaaaatcta ccgggtcacc gctgacgcgc tgacggccgg taagcttcaa 10020
gctaaatttc aaaagttcca agtaacctgc agtcccaatc gggcaattcc gaagtgcgcc 10080
ggcatttacc gaagagtaac atgtgccctc tgattggttc ggatcgcggg ggctcctttc 10140
ccgctccgaa ttcacgcgac gggatcccgc cgcatctctc ctcttcaccg tcgtcgttga 10200
cgtccccgaa acacagaaat caccgaatca tgcaggcgct cacctccagg tcgttggctc 10260
gctcatcccg ctcgattcgt gctttctcca cctcatctgg aaggtggcag gctgagcccc 10320
tccagaagcc cgttctccag aaagaattca agatctatcg ttgggtgagc tgagacgctt 10380
gtatatccag atgtgttgct cacatcgcag cccagaatcc ggatgaacca gccaagaagc 10440
ctcatctcca atcgtacacc attgacttga accagacggg ccccatggta cgtacaattc 10500
caaggcgatt gtctctatgc tcacgggctc gtagattctg gacgctctta tcaagatcaa 10560
gaacgaaatc gatcctacgc tcacattccg tcgttcgtgt agagaaggga tttgcggctc 10620
gtgcgcgatg aatattgacg gacagaacac gttggcttgc ctgtgccgaa tcgaccgtaa 10680
cgctagcaag gacagcaaga tctacccttt gccgcacagt atgacatgtc tttcggctcc 10740
taatagcatt ggctgacggt cggttacagt gtacatcgtg aaagacctcg tacccgacct 10800
cacgcttttc tacaagcagt acaagtccat caagccatac ctgcagaacg acaatattcc 10860
cgagagggag cacctccagt cgccagagga ccgcaagaag ttggacggga tgtatgaatg 10920
catcctgtgc gcgtgctgca gcacatcgtg ccccagctat tggtggaacc aagatgagta 10980
tttgggaccg gctgctttga tggccgcgta taggtggatt gcggattcac gagtacgttg 11040
tatcctcgtc gctccattcg ctgaataagt tgacgtttcg taggatacat atggcgcaca 11100
acgcaaggag cacttccaga atgagatgag tttgttccgc tgcctcacca ttttcaattg 11160
tacgtcgctt tcgccttgat atggattcgc tattaaacat atcccttctc aaggctctcg 11220
cacttgtcca aagggcctca accctgcgaa agccatcgca gagatcaagc tcgcgcttgc 11280
tactgagtaa accctagtca acagtcacgg atcaaaagca tcaagtcaga ggcagatatc 11340
tttcctgtag cagttcgcag ttctttccac ttcatcgtat ggtgtccatt gcaaacatct 11400
aatcatattc attctatcac atccacttgt tcctgagcca ctcttaaggt aggcatatcc 11460
ttggtctcac ttgcggtgct gagctcggta cccggggatc ctctagagtc gacctgcagg 11520
catgcaagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 11580
acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 11640
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ctagagcagc 11700
ttgagcttgg atcagattgt cgtttcccgc cttcagttta gcttcatgga gtcaaagatt 11760
caaatagagg acctaacaga actcgccgta aagactggcg aacagttcat acagagtctc 11820
ttacgactca atgacaagaa gaaaatcttc gtcaacatgg tggagcacga cacacttgtc 11880
tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga gacttttcaa 11940
caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg tcactttatt 12000
gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga taaaggaaag 12060
gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg 12120
agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat 12180
atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga cccttcctct 12240
atataaggaa gttcatttca tttggagaga acacggggga ctcttgacca tggta 12295
<210> 2
<211> 21
<212> DNA
<213> 人工序列
<400> 2
tcacgtcctc aataagcgct c 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<400> 3
tgagtgaggt atacaaggta g 21
Claims (5)
1.一种真姬菇的遗传转化方法,包括:
(1)挑取含有转化质粒pTSdi-GUS的EHA105农杆菌单克隆,接菌到含有利发霉素和卡那霉素的LB液体培养基中,经摇床培养得到农杆菌菌液用于侵染;
(2)将上述农杆菌菌液与重悬于IM液体培养基的真姬菇节孢子混合进行侵染,随后涂布到含琼脂的IM液体培养基上,遮光培养;最后用吐温-20洗下IM固体培养基上的节孢子,涂布在含有萎锈灵和头孢噻肟霉素的YMG平板上进行筛选,得到真姬菇转化子。
2.根据权利要求1所述的方法,其特征在于:所述步骤(1)中的LB液体培养基的组成为:10g/L胰蛋白胨,5g/L酵母提取物,5g/L NaCl,pH 7.0。
3.根据权利要求1所述的方法,其特征在于:所述步骤(2)中的IM培养基的组成为:10mMK2HPO4,10mM KH2PO4,2.5mM NaCl,2mM MgSO4·7H2O,0.7mM CaCl2,9μM FeSO4·7H2O,4mM(NH4)2SO4,10mM葡萄糖,0.5%甘油,0.2mM乙酰丁香酮,40mM 2-(N-吗啉基)乙磺酸,pH 5.6。
4.根据权利要求1所述的方法,其特征在于:所述步骤(2)中的YMG平板的组成为:1%葡萄糖,1%麦芽提取物,0.4%酵母提取物,2%琼脂,pH7.0。
5.根据权利要求1所述的方法,其特征在于:所述步骤(2)中的萎锈灵浓度为2μg/mL,头孢噻肟霉素浓度为300μg/mL。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911233767.8A CN110923258A (zh) | 2019-12-05 | 2019-12-05 | 一种真姬菇的遗传转化方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911233767.8A CN110923258A (zh) | 2019-12-05 | 2019-12-05 | 一种真姬菇的遗传转化方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110923258A true CN110923258A (zh) | 2020-03-27 |
Family
ID=69857008
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911233767.8A Pending CN110923258A (zh) | 2019-12-05 | 2019-12-05 | 一种真姬菇的遗传转化方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110923258A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111269930A (zh) * | 2020-02-14 | 2020-06-12 | 江南大学 | 一种检测丝状真菌转化体系遗传稳定性的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011078394A (ja) * | 2009-10-06 | 2011-04-21 | Kimio Munemura | 食用きのこの発酵培地及び製造方法 |
CN106350461A (zh) * | 2016-07-21 | 2017-01-25 | 中国农业科学院生物技术研究所 | 根瘤农杆菌介导的特异腐质霉的遗传转化方法及其表达载体 |
CN108950041A (zh) * | 2018-06-29 | 2018-12-07 | 上海市农业科学院 | 一种杏鲍菇遗传转化筛选标记 |
CN109055420A (zh) * | 2018-06-29 | 2018-12-21 | 上海市农业科学院 | 一种杏鲍菇萎锈灵抗性筛选转化载体及其构建方法和应用 |
-
2019
- 2019-12-05 CN CN201911233767.8A patent/CN110923258A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011078394A (ja) * | 2009-10-06 | 2011-04-21 | Kimio Munemura | 食用きのこの発酵培地及び製造方法 |
CN106350461A (zh) * | 2016-07-21 | 2017-01-25 | 中国农业科学院生物技术研究所 | 根瘤农杆菌介导的特异腐质霉的遗传转化方法及其表达载体 |
CN108950041A (zh) * | 2018-06-29 | 2018-12-07 | 上海市农业科学院 | 一种杏鲍菇遗传转化筛选标记 |
CN109055420A (zh) * | 2018-06-29 | 2018-12-21 | 上海市农业科学院 | 一种杏鲍菇萎锈灵抗性筛选转化载体及其构建方法和应用 |
Non-Patent Citations (2)
Title |
---|
JIN JING ZHANG: "An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus", 《MICROBIOLOGICAL RESEARCH》 * |
姜宁等: "农杆菌介导香菇菌丝遗传转化体系的研究", 《菌物学报》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111269930A (zh) * | 2020-02-14 | 2020-06-12 | 江南大学 | 一种检测丝状真菌转化体系遗传稳定性的方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111909953B (zh) | 用于桑黄遗传表达的重组载体及构建方法和遗传转化方法 | |
CN105838733A (zh) | Cas9 介导的香石竹基因编辑载体和应用 | |
CN109456973A (zh) | SpCas9n&PmCDA1&UGI碱基编辑***在植物基因编辑中的应用 | |
CN109679949B (zh) | 调控miR156及其靶基因IPA1同时提高水稻抗病与产量的育种方法 | |
CN113583885B (zh) | 酵母菌株及其在石油烃降解中的应用 | |
CN106916828A (zh) | 一种调控杨树叶片发育的生长调节因子基因及其应用 | |
CN110923258A (zh) | 一种真姬菇的遗传转化方法 | |
CN108165579B (zh) | 一种优化的鉴定月季RhPDS基因的VIGS沉默体系的方法 | |
CN106350526A (zh) | 大豆圣豆9号MYB转录因子家族基因GmMYB84及其应用 | |
CN113215136B (zh) | 一种壳聚糖酶突变体CsnT及其应用 | |
CA2521752A1 (en) | Plant cells and plants with increased tolerance to environmental stress | |
CN114774427B (zh) | 一种提高金银花中木犀草素含量的重组基因及其应用 | |
CN101818151B (zh) | 一种大豆种子特异性启动子及其应用 | |
CN110184290B (zh) | 一种用于生产大分子量透明质酸的遗传重组质粒及工程菌与应用 | |
CN113122556B (zh) | 振荡型基因表达***、构建方法及其在鼠李糖脂发酵中的应用 | |
CN112708633B (zh) | 含有玉米种子荧光报告基团的CRISPR-Cas9基因编辑***及应用 | |
CN110923235B (zh) | 一种控制玉米籽粒灌浆的非编码基因及其应用 | |
CN112538493B (zh) | 全基因组沉默子筛选***及其应用 | |
CN112011563A (zh) | 一种基于ai-2群体感应自诱导蛋白表达载体及应用 | |
CN110872584B (zh) | 大麦α-淀粉酶及其编码基因与应用 | |
CN106755059B (zh) | 一种用于基因工程的骨干质粒载体及应用 | |
CN114561388B (zh) | 一种辣椒外源aba诱导型启动子、表达载体及其应用 | |
CN108893487A (zh) | 一种含有C-Myc蛋白融合标签的植物表达质粒载体及其载体的构建方法 | |
CN114457082B (zh) | 一种辣椒NaCl诱导启动子、重组载体及其应用 | |
CN111893139A (zh) | 一种基于CRISPR-Cas9***进行芽孢杆菌基因组编辑的方法及其应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200327 |