CN105969799B - 根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 - Google Patents
根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 Download PDFInfo
- Publication number
- CN105969799B CN105969799B CN201610538815.4A CN201610538815A CN105969799B CN 105969799 B CN105969799 B CN 105969799B CN 201610538815 A CN201610538815 A CN 201610538815A CN 105969799 B CN105969799 B CN 105969799B
- Authority
- CN
- China
- Prior art keywords
- water chestnut
- stem blight
- bacterium
- chinese water
- chinese
- 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.)
- Expired - Fee Related
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 87
- 244000103152 Eleocharis tuberosa Species 0.000 title claims abstract description 75
- 235000014309 Eleocharis tuberosa Nutrition 0.000 title claims abstract description 75
- 230000002068 genetic effect Effects 0.000 title claims abstract description 22
- 230000001404 mediated effect Effects 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000001131 transforming effect Effects 0.000 title claims abstract description 12
- 241000589155 Agrobacterium tumefaciens Species 0.000 title claims abstract description 11
- 241000589158 Agrobacterium Species 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 238000012216 screening Methods 0.000 claims abstract description 15
- 230000009466 transformation Effects 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000001965 potato dextrose agar Substances 0.000 claims description 12
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 claims description 9
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 claims description 9
- 229940097277 hygromycin b Drugs 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000003501 co-culture Methods 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 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
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 239000001913 cellulose Substances 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 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 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 244000153158 Ammi visnaga Species 0.000 claims description 3
- 235000010585 Ammi visnaga Nutrition 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 229960002727 cefotaxime sodium Drugs 0.000 claims description 3
- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].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 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims 1
- 230000001717 pathogenic effect Effects 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 6
- 230000007547 defect Effects 0.000 abstract description 3
- 238000011835 investigation Methods 0.000 abstract description 3
- 230000008506 pathogenesis Effects 0.000 abstract description 3
- 235000003283 Pachira macrocarpa Nutrition 0.000 description 23
- 235000014364 Trapa natans Nutrition 0.000 description 23
- 235000009165 saligot Nutrition 0.000 description 23
- 241001083492 Trapa Species 0.000 description 17
- 240000001085 Trapa natans Species 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 241000233866 Fungi Species 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 235000011305 Capsella bursa pastoris Nutrition 0.000 description 3
- 240000008867 Capsella bursa-pastoris Species 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000461762 Cylindrosporium Species 0.000 description 2
- 241000234646 Cyperaceae Species 0.000 description 2
- 241000202829 Eleocharis Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000003950 pathogenic mechanism Effects 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JFALSRSLKYAFGM-UHFFFAOYSA-N uranium(0) Chemical compound [U] JFALSRSLKYAFGM-UHFFFAOYSA-N 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 244000205754 Colocasia esculenta Species 0.000 description 1
- 235000006481 Colocasia esculenta Nutrition 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 235000019082 Osmanthus Nutrition 0.000 description 1
- 241000333181 Osmanthus Species 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 241000893045 Pseudozyma Species 0.000 description 1
- 241000287420 Pyrus x nivalis Species 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 241000226724 Sporisorium scitamineum Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 241000509513 Ustilago hordei Species 0.000 description 1
- 244000301083 Ustilago maydis Species 0.000 description 1
- 241000228452 Venturia inaequalis Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000001058 brown pigment Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000035568 catharsis Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 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
- 239000000523 sample Substances 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于以荸荠秆枯病菌分生孢子为转化受体材料，将含有双元载体的根癌农杆菌与荸荠秆枯病菌分生孢子混合后共培养，用抗生素筛选，获得抗性转化子，从而建立了荸荠秆枯病菌高效的遗传转化体系。该法操作简单、转化效率高且稳定性好，以此构建荸荠秆枯病菌突变体库，由农杆菌介导介导荸荠秆枯病菌的遗传转化获得的转化子可为筛选弱致病性和致病性缺陷的荸荠秆枯病菌菌株提供了丰富的筛选菌源，同时为克隆荸荠秆枯病菌致病性相关基因，深入研究荸荠秆枯病菌致病机理奠定基础。
Description
技术领域
本发明属于微生物基因工程技术领域，尤其涉及一种根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法。
背景技术
荸荠(Eleocharis dulcis)属莎草科(Cyperaceae)荸荠属(Eleocharis)，是多年生草本植物，又名地栗、乌芋、马莽、红慈姑、马蹄等。原产于我国南方和印度，在我国有逾2000年的栽培历史。目前，荸荠广泛栽培于我国长江流域及其以南各省，广西则以合浦、桂林、贺州等地区广泛种植；在美国、朝鲜、越南、澳大利亚、日本等国亦有栽培。据不完全统计，1998年我国荸荠栽培面积约为3.5万hm2，年产75万t左右；2011年广西荸荠种植面积达2万hm2，总产量达60万t，面积和产量占全国的一半以上，荸荠产业收入达18亿元以上，主要供国内市场鲜销，仅部分加工成罐头或冷藏，经我国香港或台湾转销东南亚和欧美各国，并且出口量逐年增加，如美国1983～1987年4年间进口数量几乎增加了2.5倍。
荸荠球茎营养丰富，汁多味甜，自古以来就有“地下雪梨”的美誉，北方人更将其视为“江南人参”。荸荠富含多种营养物质，如蛋白质、脂肪、碳水化合物、粗纤维、胡萝卜素、维生素B1、B2、C、尼克酸和各种矿物质。荸荠既可生吃，亦可熟食，还可制成各种罐头、饮料。荸荠的地上茎秆和地下球茎均可入药，具有很好的医疗保健效果，如抗肿瘤作用、抗菌作用、治疗呼吸道疾病、利肠通便和利尿排淋作用、治疗糖尿病、清肺化痰。荸荠球茎表皮中含有丰富的棕色素，提取后可用做食品添加剂。另外，荸荠植株可以吸收湿地中放射性物质铀，对湿地铀污染有一定的净化作用。荸荠因其独特的营养价值和药用功效，现今已成为大众喜爱的季节性蔬菜。
但是，荸荠在栽培过程中常受病害侵扰，其中以秆枯病(俗称“荸荠瘟”)发生最为严重。该病主要为害叶鞘、叶状茎、花器等部位，严重影响荸荠的产量和品质。该病病原为荸荠柱盘孢菌Cylindrosporium eleocharidis Lentz，属半知菌类黑盘孢目柱盘孢属。近几年来，随着荸养种植面积的扩大和连作，荸荠秆枯病也呈逐年上升趋势。发病年一般病秆率20％～50％，重病田块可达100％；球茎损失在45％以上，以至有的田块颗粒无收。尽管政府部门已对秆枯病予以高度重视，广大的科技人员也付出大量的心血研究控制措施，但由于致病机理尚不清楚，远不能达到有效地遏制该病蔓延和传播的要求。为了更好地防治该病，对该病菌的致病分子机理进行深入研究具有重要意义。分离和鉴定荸荠秆枯病菌致病相关基因是关键所在，而建立真菌遗传转化技术创造致病性缺陷突变体则是分离和鉴定致病性相关基因的一种有效途径。
目前，丝状真菌遗传转化技术已成为植物病原真菌分子遗传和基因功能研究的重要手段。由农杆菌介导的真菌遗传转化技术(ATMT)已经成功应用于U.maydis、Pseudozymaantarctica、U.hordei、S.scitamineum等黑粉菌及其它丝状真菌中。农杆菌介导的真菌转化相比其它几种真菌转化方法(PEG-MT、REMI、基因枪等)具有以下的优点：1)转化受体可以是多种类型，既可以是原生质体，又可以是菌丝、分生孢子，甚至蘑菇的菌丝体组织。因此，相比PEG-MT和REMI，利用ATMT转化可以省略原生质体制备的繁琐步骤，省力又省时。2)成本低，转化不需要复杂的仪器，任何实验室只要具有微生物培养的基本实验条件，都可以进行这种转化。3)转化效率较高，据报道同时用PEG和ATMT对Venturia inaequalis转化后发现，ATMT的转化效率要比PEG-MT的高出10倍。4)随机插入的T-DNA多为单拷贝。5)获得的转化子稳定，由ATMT得到的转化子，在没有选择压力的培养基上培养几代后也不会丧失抗生素抗性。迄今，未见荸荠秆枯病菌遗传转化体系相关报道，因此，建立荸荠秆枯病菌的遗传转化体系，应用于荸荠杆枯病菌致病相关基因的研究将有助于解析荸荠杆枯病菌的致病机制，对推动荸荠秆枯病菌分子生物学研究意义重大。
发明内容
本发明要解决的技术问题是提供一种操作简单、转化效率高、稳定性好的根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，该法获得的转化子可为筛选弱致病性和致病性缺陷性突变体提供丰富的筛选菌源。
为解决上述技术问题，本发明采用以下技术方案：根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，以荸荠秆枯病菌分生孢子为转化受体材料，将含有双元载体的根癌农杆菌与荸荠秆枯病菌分生孢子混合后共培养，用抗生素筛选，获得抗性转化子。
荸荠秆枯病菌分生孢子按以下操作制备：在马铃薯-葡萄糖-琼脂培(PDA)养基上将荸荠秆枯病菌培养15-20天后，用灭菌的去离子水将荸荠秆枯病菌的分生孢子刮下，然后用无菌擦镜纸过滤去除菌丝，得到的荸荠秆枯病菌分生孢子滤液用去离子水调整到浓度为105-108孢子/毫升，获得荸荠秆枯病菌分生孢子悬浮液备用。
含有双元载体的根癌农杆菌按以下操作准备：利用携带绿色荧光蛋白基因eGFP和潮霉素抗性筛选基因hpth的质粒pEV为转化载体，通过电击转化将质粒pEV载体转入农杆菌感受态细胞中；挑含有双元载体的根癌农杆菌菌体至5mL的MM液体培养液中，28℃，200rpm，培养过夜，用IM液体培养液将菌液稀释OD600至0.15左右，28℃下预诱导培养6h，使菌液OD600调至为0.5备用。
MM液体培养液为基本培养基，其配方为：1.45g KH2PO4，2.05g K2HPO4，0.5gNH4NO3，0.01g CaCl2，0.3g(NH4)SO4，2g Glucose，0.01g FeSO4，5ml Z-buffer，加双蒸水定容至1000mL，pH6.7-7.0；诱导培养基(induce media，IM)配方为：1g Glucose，7.808g MES，1.45g KH2PO4，0.5g NH4NO3，0.6g MgSO4·7H2O，0.01g CaCl2，0.3g NaCl，0.5g(NH4)SO4，HCl调pH值到5.6，双蒸水定容至1000mL。
共培养按以下操作进行：分别取荸荠秆枯病菌分生孢子悬浮液和农杆菌培养液各100μL混合后涂板于含AS的覆盖有纤维素滤膜的IM固体培养基上，24℃下共培养48h-72h；IM固体培养基由每升诱导培养基(IM)加25克琼脂构成。
抗生素筛选按以下操作进行：转移纤维素滤膜至含有潮霉素B和头孢噻肟钠的PDA，28℃下培养8-12d；将长出的单菌落转化子用牙签挑到含有潮霉素B的PDA上进行二次筛选，第二次筛选获得的抗潮霉素B的荸荠秆枯病菌即为转化子。
为研究荸荠杆枯病菌的致病机制，发明人建立了一种根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于以荸荠秆枯病菌分生孢子为转化受体材料，将含有双元载体的根癌农杆菌与荸荠秆枯病菌分生孢子混合后共培养，用抗生素筛选，获得抗性转化子，从而建立了荸荠秆枯病菌高效的遗传转化体系。该法操作简单、转化效率高且稳定性好，以此构建荸荠秆枯病菌突变体库，由农杆菌介导介导荸荠秆枯病菌的遗传转化获得的转化子可为筛选弱致病性和致病性缺陷的荸荠秆枯病菌菌株提供了丰富的筛选菌源，同时为克隆荸荠秆枯病菌致病性相关基因，深入研究荸荠秆枯病菌致病机理奠定基础。
附图说明
图1是荸荠秆枯病菌菌落和分生孢子，图中：A荸荠秆枯病菌菌落，B荸荠秆枯病菌分生孢子。
图2是双元载体结构示意图。
图3是荸荠秆枯病菌荧光鉴定结果他，图中：A是在绿色荧光滤镜下的荸荠秆枯病菌菌落，B是在明场下的荸荠秆枯病菌菌落。
图4是荸荠秆枯病菌转化子PCR鉴定图，图中：M是marker 2k，1-13是荸荠秆枯病菌转化子，14是荸荠秆枯病菌野生型，15是无菌水阴性对照，16是质粒pEV。
图5是荸荠秆枯病菌转化子southern blot图，图中：Marker：marker 1kb，1-11：荸荠秆枯病菌转化子
具体实施方式
实施例1荸荠秆枯病菌T-DNA插入转化子的获得
1>荸荠秆枯病菌分生孢子的制备(一切操作在无菌条件下进行)
在马铃薯-葡萄糖-琼脂培养基(PDA)上将荸荠秆枯病菌(图1A)培养15-20天后，用灭菌的去离子水将荸荠秆枯病菌的分生孢子刮下，然后用无菌擦镜纸过滤去除菌丝，得到的荸荠秆枯病菌分生孢子滤液用去离子水调整到浓度为105-108孢子/毫升，获得荸荠秆枯病菌分生孢子(图1B)悬浮液备用。
2>含载体农杆菌的准备
利用携带绿色荧光蛋白基因eGFP和潮霉素抗性筛选基因hpth(图2)的质粒pEV为转化载体，通过电击转化将质粒pEV载体转入农杆菌感受态细胞中；挑含有双元载体的根癌农杆菌菌体至5mL的MM液体培养液中(状观霉素100μg/mL和利福平50μg/mL)，28℃，200rpm，培养过夜，用IM液体培养液将菌液稀释OD600至0.15左右，28℃下预诱导培养6h，28℃，200rpm，使菌液OD600调至为0.5左右备用。
其中，MM液体培养液为基本培养基(minimal media，MM)，其配方为：1.45g KH2PO4，2.05g K2HPO4，0.5g NH4NO3，0.01g CaCl2，0.3g(NH4)SO4，2g Glucose，0.01g FeSO4，5ml Z-buffer(每种成分各0.01％CuSO4*5H2O，ZnSO4*7H2O，MnSO4*H2O，H3BO3和NaMbO4*2H2O)，加双蒸水定容至1000mL，pH6.7-7.0；
诱导培养基(induce media，IM)配方为：1g Glucose，7.808g MES，1.45g KH2PO4，0.5g NH4NO3，0.6g MgSO4·7H2O，0.01g CaCl2，0.3g NaCl，0.5g(NH4)SO4，HCl调pH值到5.6，双蒸水定容至1000mL。
3>转化
分别取荸荠秆枯病菌分生孢子悬浮液和农杆菌培养液各100μL混合后涂板于含AS(200μ mol/L)的覆盖有纤维素滤膜的IM固体培养基(由每升诱导培养基(IM)加25克琼脂构成)上，24℃下共培养48h-72h(黑暗条件下)；转移纤维素滤膜至含有潮霉素B(200μg/mL)和头孢噻肟钠(300μg/mL)的PDA，28℃下培养8-12d；将长出的单菌落转化子用牙签挑到含有潮霉素B(100μg/mL，)的PDA上进行二次筛选，第二次筛选获得的抗潮霉素B的荸荠秆枯病菌即为转化子。
实施例2荸荠秆枯病菌转化子的鉴定
1>荧光显微镜鉴定
将荸荠秆枯病菌转化子菌落置于荧光显微镜下观察荧光。荸荠秆枯病菌野生型没有检测到荧光，经农杆菌转化后的转化子在荧光显微镜紫外激发光下发绿光，证明农杆菌的绿色荧光蛋白GFP已经成功转到荸荠秆枯病菌中并得以表达(图3)。
2)PCR验证
以双元载体pEV上的潮霉素基因(hpth(SEQ.ID.No.1))为模板设计引物hpth-F：GCGTGGAGAAGTTCCTCATCGAGA(SEQ.ID.No.2)/hpth-R：GAGCCTTGGGCTTCCAGCAA(SEQ.ID.No.3)对转化子进行PCR扩增，以初步验证是否得到真正有插入的突变体。
PCR反应液组成(25μL体系)：25.0μL反应体系中，加入2.5μL 10×PCR Buffer、2.0μL 2.5μmol/L dNTP、1.0μL 5μ mol/L正向引物hpth-F、1.0μL 5μ mol/L反向引物hpth-R，1.0U Taq DNA聚合酶和1.0μL DNA，用ddH20补至25.0μL。PCR扩增反应程序：95℃预变性2min，然后94℃变性40sec，55℃退火40sec，72℃延伸1min，进行30个循环；最后72℃延伸10min。
结果表明，所有测试的转化子及质粒pEV均能扩增出预期的约758bp大小的片段，而未经转化的野生型菌株和无菌水对照则扩增不到任何条带，说明转化子含有T-DNA插入片段(图4)。
3)southern blot验证
为进一步验证荸荠秆枯病菌转化子T-DNA插入和拷贝数，对荸荠秆枯病菌转化子进行southern blot。对荸荠秆枯病菌转化子DNA采用EcoR I内切酶进行酶切，然后进行0.8％琼脂糖凝胶电泳。将电泳凝胶转入尼龙膜(Roche，Cat.No.11 417 240 001)，以引物hpth-F/hpth-R扩增得潮霉素基因hpth片段为探针，PCR反映体系同2)，随后的分子杂交采用PCR DIG Probe Synthesis Kit(Roche，Cat.No.11 636 090 910)，显色反应采用DIGNucleic Acid Detection Kit(Roche，Cat.No.11 175 041 910)进行，具体步骤参考产品使用说明书。
结果表明，所有测试的11个荸荠秆枯病菌转化子均能检测到杂交信号，且杂交信号表现丰富带型；除了2个转化子为双拷贝外，其他转化子均为单拷贝，说明T-DNA在转化子中为随机插入，且80％以上为单拷贝(图5)。
Claims (5)
1.一种根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于以荸荠秆枯病菌分生孢子为转化受体材料，将含有双元载体的根癌农杆菌与荸荠秆枯病菌分生孢子混合后共培养，用抗生素筛选，获得抗性转化子；所述含有双元载体的根癌农杆菌按以下操作准备：利用携带绿色荧光蛋白基因eGFP和潮霉素抗性筛选基因hpth的质粒pEV为转化载体，通过电击转化将质粒pEV载体转入农杆菌感受态细胞中；挑含有双元载体的根癌农杆菌菌体至5mL的MM液体培养液中，28℃，200rpm，培养过夜，用IM液体培养液将菌液稀释OD600至0.15左右，28℃下预诱导培养6h，使菌液OD600调至为0.5备用。
2.根据权利要求1所述的根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于所述荸荠秆枯病菌分生孢子按以下操作制备：在马铃薯-葡萄糖-琼脂培养基(PDA)上将荸荠秆枯病菌培养15-20天后，用灭菌的去离子水将荸荠秆枯病菌的分生孢子刮下，然后用无菌擦镜纸过滤去除菌丝，得到的荸荠秆枯病菌分生孢子滤液用去离子水调整到浓度为105-108孢子/毫升，获得荸荠秆枯病菌分生孢子悬浮液备用。
3.根据权利要求2所述的根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于所述MM液体培养液为基本培养基，其配方为：1.45g KH2PO4，2.05g K2HPO4，0.5g NH4NO3，0.01g CaCl2，0.3g(NH4)SO4，2g Glucose，0.01g FeSO4，5ml Z-buffer，加双蒸水定容至1000mL，pH6.7-7.0；所述诱导培养基(induce media,IM)配方为：1g Glucose，7.808g MES，1.45g KH2PO4，0.5g NH4NO3，0.6g MgSO4·7H2O，0.01g CaCl2，0.3g NaCl，0.5g(NH4)SO4，HCl调pH值到5.6，双蒸水定容至1000mL。
4.根据权利要求1所述的根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于所述共培养按以下操作进行：分别取所述荸荠秆枯病菌分生孢子悬浮液和农杆菌培养液各100μL混合后涂板于含AS的覆盖有纤维素滤膜的IM固体培养基上，24℃下共培养48h-72h；所述IM固体培养基由每升诱导培养基(IM)加25克琼脂构成。
5.根据权利要求4所述的根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法，其特征在于所述抗生素筛选按以下操作进行：转移纤维素滤膜至含有潮霉素B和头孢噻肟钠的PDA，28℃下培养8-12d；将长出的单菌落转化子用牙签挑到含有潮霉素B的PDA上进行二次筛选，第二次筛选获得的抗潮霉素B的荸荠秆枯病菌即为转化子。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610538815.4A CN105969799B (zh) | 2016-07-08 | 2016-07-08 | 根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610538815.4A CN105969799B (zh) | 2016-07-08 | 2016-07-08 | 根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105969799A CN105969799A (zh) | 2016-09-28 |
| CN105969799B true CN105969799B (zh) | 2019-06-21 |
Family
ID=56951398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610538815.4A Expired - Fee Related CN105969799B (zh) | 2016-07-08 | 2016-07-08 | 根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105969799B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110741925A (zh) * | 2019-11-01 | 2020-02-04 | 广西壮族自治区农业科学院 | 芋杂交育种方法 |
-
2016
- 2016-07-08 CN CN201610538815.4A patent/CN105969799B/zh not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 农杆菌介导的黄瓜炭疽菌遗传转化;方丽 等;《浙江大学学报(农业与生命科学版)》;20061231;第32卷(第4期);第1.1-2.2节 * |
| 荸荠秆枯病菌生物学特性研究;潘丽 等;《长江蔬菜》;20111231(第16期);第1.1节 * |
| 采用农杆菌介导转化法的泡盛曲霉表达载体的构建;陈波 等;《食品科学》;20091231;第30卷(第1期);第182页左栏第2段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105969799A (zh) | 2016-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Colletotrichum anthracnose of Amaryllidaceae | |
| CN110894470B (zh) | 一种适合工厂化栽培的香菇菌种农香5号及其分子鉴定方法 | |
| CN106350453A (zh) | 紫花苜蓿镰刀菌根腐病病原菌分离纯化与致病性鉴定方法 | |
| CN105969799B (zh) | 根癌农杆菌介导的荸荠秆枯病菌的遗传转化方法 | |
| CN103243030A (zh) | 一株用于防治柑橘木虱的刀孢轮枝菌菌株 | |
| CN107236751B (zh) | 一种利用枯草芽孢杆菌表达系统快速、高效筛选抗菌基因的方法 | |
| CN110292011A (zh) | 一种适合盐碱地养殖对虾种虾培育方法 | |
| CN103468579A (zh) | 一种对柑橘木虱具致病力的蜡蚧菌属真菌新种 | |
| CN114292759B (zh) | 一株具有防治烟草连作障碍作用的尖孢镰刀菌 | |
| CN105274131A (zh) | 一种peg介导胶孢炭疽病菌原生质体遗传转化的方法 | |
| Vallejo-Gutiérrez et al. | Response of Capsicum pubescens genotypes to damage caused by the fungal wilt complex | |
| Zhang et al. | First report of Stagonosporopsis caricae causing chayote leaf spot in Guizhou province, China | |
| CN110862930B (zh) | 一种白色茶树菇及其分子标记鉴定方法 | |
| Olson et al. | Association of Diaporthe longicolla with black zone lines on mature soybean plants | |
| CN105779315A (zh) | 一种农杆菌介导的芦笋茎枯病菌遗传转化子的制备方法 | |
| CN110257545B (zh) | 一种用于鉴定杂交构树的分子标记及其应用 | |
| CN114933980A (zh) | 一种防治黄精根腐病的浅紫链霉菌hjb-xtbg45及其应用 | |
| Chen et al. | Diplocarpon mespilicola sp. nov. associated with Entomosporium leaf spot on hawthorn in China | |
| CN104031850B (zh) | 一种蛹虫草Rhf1基因失活的工程菌以及蛹虫草Rhf1基因的应用 | |
| CN102660469A (zh) | 一种表达植物抗性诱导因子的毕赤酵母基因工程菌株的构建方法 | |
| CN103290000B (zh) | 生防绿木霉的scar标记及其应用和定量检测方法 | |
| Li et al. | Molecular Identification and Analysis on Differential Pathogenicity of Cylindrocarpon Species Associated With Ginseng Root Rust Rot in Northeastern China | |
| CN101892319A (zh) | 德国镜鲤抗逆基因标记及其核心选育群体构建方法 | |
| CN107058148A (zh) | 一种植物乳杆菌菌株及其应用 | |
| Lee et al. | Leaf spot of rhubarb caused by Didymella rhei in Korea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190621 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |