CN118048366A - 一种大豆根系镉抗性相关基因GmEIL3及应用 - Google Patents
一种大豆根系镉抗性相关基因GmEIL3及应用 Download PDFInfo
- Publication number
- CN118048366A CN118048366A CN202410350877.7A CN202410350877A CN118048366A CN 118048366 A CN118048366 A CN 118048366A CN 202410350877 A CN202410350877 A CN 202410350877A CN 118048366 A CN118048366 A CN 118048366A
- Authority
- CN
- China
- Prior art keywords
- gmeil
- gene
- soybean
- root system
- cadmium resistance
- 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
- 244000068988 Glycine max Species 0.000 title claims abstract description 64
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 51
- 229910052793 cadmium Inorganic materials 0.000 title claims abstract description 47
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 241000196324 Embryophyta Species 0.000 claims abstract description 21
- 238000003209 gene knockout Methods 0.000 claims abstract description 16
- 239000013604 expression vector Substances 0.000 claims abstract description 11
- 239000013598 vector Substances 0.000 claims abstract description 9
- 108091026890 Coding region Proteins 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000000463 material Substances 0.000 claims description 10
- 238000010362 genome editing Methods 0.000 claims description 9
- 230000002018 overexpression Effects 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 8
- 241000589156 Agrobacterium rhizogenes Species 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 230000018109 developmental process Effects 0.000 abstract description 5
- 230000009261 transgenic effect Effects 0.000 abstract description 5
- 230000010496 root system development Effects 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001404 mediated effect Effects 0.000 abstract description 3
- 230000001131 transforming effect Effects 0.000 abstract description 3
- 238000003208 gene overexpression Methods 0.000 abstract 1
- 230000035882 stress Effects 0.000 description 12
- 101100279542 Arabidopsis thaliana EIN3 gene Proteins 0.000 description 11
- 101100279534 Arabidopsis thaliana EIL1 gene Proteins 0.000 description 9
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 9
- 239000005977 Ethylene Substances 0.000 description 9
- 101100279532 Oryza sativa subsp. japonica EIL1A gene Proteins 0.000 description 9
- 101100279533 Oryza sativa subsp. japonica EIL1B gene Proteins 0.000 description 9
- 108091023040 Transcription factor Proteins 0.000 description 6
- 102000040945 Transcription factor Human genes 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 229960004261 cefotaxime Drugs 0.000 description 4
- 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 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000008635 plant growth Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 241000589158 Agrobacterium Species 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 230000031018 biological processes and functions Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000008121 plant development Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000012883 rooting culture medium Substances 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000012877 elongation medium Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000027874 photomorphogenesis Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000021749 root development Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Botany (AREA)
- Immunology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Mycology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
Abstract
本发明属于分子生物学和基因工程技术领域，具体涉及一种大豆根系镉抗性相关基因GmEIL3及应用，调节大豆根系镉抗性的GmEIL3全长编码区序列如序列表SEQ ID NO.1所示；氨基酸序列如序列表SEQ ID NO.2所示。本发明提供大豆根系镉抗性相关基因并用于构建基因的表达载体和敲除载体，构建的植物表达载体经发根农杆菌介导法转化大豆幼嫩子叶，获得的基因过表达转基因毛状根在镉胁迫下发育受到抑制，获得的基因敲除转基因大豆在镉胁迫下根系发育良好，表明该基因负调控大豆根系的耐镉性。本发明对提高大豆根系耐镉的能力提供了理论依据与技术手段，具有很大的应用价值。
Description
技术领域
本发明属于分子生物学和基因工程技术领域，具体涉及一种大豆根系镉抗性相关基因GmEIL3及应用。
背景技术
镉（Cd）作为一种主要重金属污染物，对植物生长具有毒害作用。Cd污染严重抑制根系发育，降低作物产量。大豆（Glycine max）是重要的油料作物，在我国农业生产中占有重要地位。根系是植物生长发育的重要组织，在植物的生长过程中不仅可以提供水分、无机盐等营养物质，还具有合成植物激素和维持根际微生物等功能。当植物受到干旱、低温和盐胁迫等非生物胁迫时，植物通过改变侧根形状和数量适应胁迫，维持植物的正常生长。因此，根系的发育程度是影响作物产量的重要农艺性状之一，研究Cd胁迫下大豆根系发育相关基因的功能及作用机制可以为大豆高产分子育种提供新途径和新种质。
EIN3（ethylene insensitive 3）是一类植物特有的转录因子，位于细胞核中，其编码基因属于一个小的转录因子家族，在乙烯反应中发挥正向调节作用。EIN3和 EIN3-like（EIL）处于乙烯信号通路中的枢纽位置，决定着乙烯信号能否顺利传入细胞核，通过乙烯信号途径影响植物的生长发育。目前为止，人们发现大部分乙烯相关的生物学过程都是通过核心转录因子EIN3/EIL1完成的，EIN3/EIL1有大量结合靶点，涉及到诸如乙烯反应、根的发育、光形态建成、细胞分裂素协同作用、水杨酸协同作用、盐胁迫、镉胁迫、病原微生物进化等生物学过程。由此可见，EIN3/ EIL1在乙烯信号通路中发挥重要作用，还参与到植物生长发育、激素协同作用、环境胁迫其他生物学过程中，这些现象表明转录因子EIN3/EIL1是植物体内整个转录调控网络中的重要节点，具有整合信号、调控基因表达网络的作用。
EIN3/EIL1转录因子首先在拟南芥乙烯不敏感突变体中分离得到，迄今为止，研究人员也已在烟草、番茄、水稻、牡丹等植物中分离得到该基因。目前，对于EIN3/EIL1转录因子结构、功能、作用机制的研究已取得突破性进展。然而，植物中EIN3/EIL1家族成员较多，EIN3/EIL1序列多样性与其功能多样性之间的关系仍不明确。EIN3/EIL1对植物生长发育、胁迫响应及信号转导等的调节机制有待深入探索。
发明内容
为了解决上述技术问题，本发明提供一种大豆根系镉抗性相关基因GmEIL3及应用，从大豆中分离得到GmEIL3基因，构建该基因的植物表达载体及基因敲除载体，通过农杆菌介导法，将该基因转化大豆子叶中，以实现GmEIL3基因的功能分析。
本发明是这样实现的，提供一种大豆根系镉抗性相关基因GmEIL3，其编码区序列如SEQ ID NO.1所示。
提供一种大豆根系镉抗性相关基因GmEIL3编码的蛋白质，氨基酸序列如SEQ IDNO.2所示。
提供一种用于扩增大豆根系镉抗性相关基因GmEIL3的引物，包含：
OE-F:5'-ACTCTTGACCATGGTAGATCTATGATGATGATGCTTGAAGATAT-3'；
OE-R:5'-GGGGAAATTCGAGCTGGTCACCTCACTGATACCAAATAGAGATAT-3'；
其中，OE-F/OE-R的5'端的前15个碱基是构建过表达载体所需，不属于GmEIL3的基因序列，紧接着的6个碱基是酶切位点，OE-R去掉了终止密码子。
提供一种用于敲除GmEIL3基因的靶点序列，包含：
Target:5'-ACTGTGATCCCCCTCAGAGGCGG-3'；
根据靶点序列设计基因编辑引物，包含：
CR-F:5'-cagtGGTCTCatgcaACTGTGATCCCCCTCAGAGG-3'
CR-R:5'-cagtGGTCTCaaaac cctctgagggggatcacagt-3'。
提供一种用于鉴定大豆GmEIL3基因编辑材料的鉴定引物，包含：
J216162-F7:5'-AGAGGAGGATGTGGAGGGAC-3'；
J216162-R7:5'-CTGCAGTCAGAACACCCACT-3'。
提供一种含有大豆根系镉抗性相关基因GmEIL3的过表达载体，为pCAMBIA3301-CaMV35S-GFP-GmEIL3。
提供一种含有大豆根系镉抗性相关基因GmEIL3的过表达载体的应用，用于降低植物根系镉抗性。
提供一种大豆根系镉抗性相关基因GmEIL3的基因敲除载体，为pzmpl-bar-GmEIL3。
提供一种大豆根系镉抗性相关基因GmEIL3的基因敲除载体的应用，用于增强植物根系镉抗性。
提供一种大豆根系镉抗性相关基因GmEIL3在调节大豆根系镉抗性中的应用。
与现有技术相比，本发明的有益效果为：
本发明利用现有植物基因工程技术，克隆了大豆根系镉抗性相关基因GmEIL3，并通过农杆菌介导的方法将该基因转入大豆外植体，经过比较分析证明，镉胁迫下GmEIL3过表达材料根系发育指标显著下降，GmEIL3敲除材料在镉胁迫下根系发育良好，表明该基因负调控大豆根系的耐镉性，对提高大豆根系耐镉的能力提供了理论依据与技术手段，具有很大的应用价值。
附图说明
图1为GmEIL3基因全长1866 bp编码区序列的扩增结果。其中M为DL 2000；
图2为pCAMBIA3301-CaMV35S-GFP-GmEIL3重组载体测序比对结果；
图3为转化pCAMBIA3301-CaMV35S-GFP-GmEIL3的发根农杆菌菌落PCR结果。其中M为DL 2000；
图4为大豆GmEIL3基因敲除材料鉴定结果。其中M为DL 2000；
图5（a）为镉胁迫下GmEIL3过表达毛状根表型鉴定；
图5（b）为镉胁迫下GmEIL3过表达毛状根根系发育指标统计结果；
图6（a）为镉胁迫下GmEIL3基因敲除大豆根系表型鉴定；
图6（b）为镉胁迫下GmEIL3基因敲除大豆根系发育指标统计结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，下面结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明。
实施例1、大豆GmEIL3基因的克隆
（1）以大豆‘东农50’为试材。
（2）利用RNAprep pure植物总RNA提取试剂盒的方法提取总RNA，反转录合成cDNA第一链。
（3）基因的克隆：以反转录的cDNA第一链为模板，利用引物OE-F/OE-R进行PCR扩增，回收PCR产物，获得1866 bp的目的片段，结果见图1。
实施例2、植物表达载体构建
将纯化后的胶回收产物GmEIL3全长序列，应用In-Fusion HD Cloning kit连入pCAMBIA3301-CaMV35S-GFP线性化大片段中，反应体系为：2 μL 5×In-Fusion HD EnzymePremix，2 μL线性大片段，6 μL目的基因片段，50℃反应20 min。将连接产物转至大肠杆菌感受态DH5α中，经菌落PCR、摇菌、提质粒获得带有目的基因的植物表达载体pCAMBIA3301-CaMV35S-GFP-GmEIL3，测序，结果见图2。之后转化发根农杆菌R599感受态细胞，进行发根农杆菌菌落PCR验证，见图3，得到的含有重组质粒的发根农杆菌用于大豆子叶的转化。
实施例3、GmEIL3基因敲除载体构建
基因编辑载体的构建采用武汉伯远生物科技有限公司基因编辑试剂盒完成（货号：#REC44-I）。具体操作为：以cDNA为模板，使用基因编辑引物CR-F/CR-R扩增靶点序列。按照试剂盒说明进行酶切连接。将连接产物转化大肠杆菌感受态细胞，使用通用引物进行菌落PCR鉴定，提质粒获得敲除载体pzmpl-bar-GmEIL3。之后转化农杆菌感受态细胞，用于大豆种子侵染。
实施例4、转基因大豆材料的获得及验证
（1）重组质粒转化大豆子叶
选取含有pCAMBIA3301-CaMV35S-GFP-GmEIL3的发根农杆菌R599阳性克隆，接种于不加任何抗性的MS液体培养基（含100 mg/mL AS、1 mg/mL BA和1 mg/mL NAA）吸打混匀，将侵染液浓度范围控制在OD600=0.02~0.3之间，将大豆幼嫩子叶表面制造伤口，破坏其维管组织，与侵染液共培养约10 min，将外植体伤口朝下移植至含有MS固体培养基（含100 mg/mLAS）中，使用封口膜封口，放置于26℃暗培养3 d。将外植体洗菌后伤口朝上移至生根培养基中，置于26℃光照培养2~4周诱导毛状根。
提取含有pzmpl-bar-GmEIL3敲除载体的农杆菌菌液，加入到划伤处理的大豆萌发种子中，浸染10 min。再将外植体转移至共培养培养基（1/2MS固体，30 g/L蔗糖，2 mg/LBA，0.1 mg/L NAA，100 μM乙酰丁香酮，pH 5.2），25℃避光培养3~5 d。挑选共培养后的外植体，切去胚轴末端，插入恢复培养基（MS固体，30 g/L蔗糖，2 mg/L BA，0.1 mg/L NAA，400mg/L头孢噻肟，pH 5.8），恢复培养7~10 d。将长有丛生芽的外植体接种至筛选培养基（MS固体，30 g/L蔗糖，2 mg/L BA，0.1 mg/L NAA，400 mg/L头孢噻肟，5 mg/L草铵膦，pH 5.8）暗培养21 d。将经过筛选后长势良好的丛生芽，转移至伸长培养基（MS固体，30 g/L蔗糖，0.1mg/L NAA，400 mg/L头孢噻肟，pH 5.8）暗培养21 d。待幼芽生长至约5 cm时，转移至生根培养基（MS固体，30 g/L蔗糖，0.05 mg/L NAA，400 mg/L头孢噻肟，5 mg/L草铵膦，pH 5.8）暗培养21 d，洗干净幼苗根上附着的培养基，移载至装盛有营养土的育苗盘中，27℃暗培养，炼苗3～4周。
（2）GmEIL3过表达毛状根鉴定
荧光检测及表型检测：pCAMBIA3301-CaMV35S-GFP-GmEIL3过表达载体含有GFP标签，用于检测毛状根中特定基因的转化效果，仅侵染pCAMBIA3301-CaMV35S-GFP的大豆毛状根为对照，应用激光共聚焦显微镜检测大豆毛状根中GmEIL3-GFP融合蛋白的表达情况。在注射pCAMBIA3301-CaMV35S-GFP、pCAMBIA3301-CaMV35S-GFP-GmEIL3的大豆毛状根中均检测到荧光信号，表明GmEIL3-GFP融合蛋白在毛状根中成功表达。
（3）GmEIL3基因敲除材料鉴定
采用Bar试纸法直接对转基因植物内有无bar/pat蛋白进行鉴定，有条带的为转基因阳性植株。将经过Bar试纸检测的阳性幼苗移出培养基栽种到温室中直至收种。以整株T2代GmEIL3基因敲除大豆植株为实验材料，提取大豆叶片全基因组DNA，使用J216162-F7/J216162-R7引物进行PCR扩增，鉴定稳定遗传的GmEIL3基因敲除大豆株系。参考图4，PCR结果鉴定到262 bp特异条带，说明该T2代材料为GmEIL3基因敲除材料阳性植株。
实施例5、GmEIL3基因对大豆根系镉抗性的影响
培养GmEIL3过表达毛状根并转移至含有75 μmol/L CdCl2·2.5H2O的MS培养罐中，观察大豆根系形态并统计根系发育指标。参考图5（a）、图5（b）、图6（a）和图6（b），结果表明，与对照组相比，过表达GmEIL3的毛状根生长受到抑制，GmEIL3基因敲除大豆根系发育良好。镉胁迫下，GmEIL3过表达毛状根的主根长和侧根数量显著低于对照组，GmEIL3基因敲除大豆的主根长与侧根数量高于对照组。综合上述实验结果，证明了GmEIL3基因负调控大豆根系对镉的抗性。
Claims (10)
1.一种大豆根系镉抗性相关基因GmEIL3，其特征在于，基因GmEIL3的编码区序列如SEQID NO.1所示。
2.一种大豆根系镉抗性相关基因GmEIL3编码的蛋白质，其特征在于，氨基酸序列如SEQID NO.2所示。
3.一种用于扩增大豆根系镉抗性相关基因GmEIL3的引物，其特征在于，包含：
OE-F:5'-ACTCTTGACCATGGTAGATCTATGATGATGATGCTTGAAGATAT-3'；
OE-R:5'-GGGGAAATTCGAGCTGGTCACCTCACTGATACCAAATAGAGATAT-3'；
其中，OE-F/OE-R的5'端的前15个碱基是构建过表达载体所需，不属于GmEIL3的基因序列，紧接着的6个碱基是酶切位点，OE-R去掉了终止密码子。
4.一种用于敲除GmEIL3基因的靶点序列，其特征在于，包含：
Target:5'-ACTGTGATCCCCCTCAGAGGCGG-3'；
根据靶点序列设计基因编辑引物，包含：
CR-F:5'-cagtGGTCTCatgcaACTGTGATCCCCCTCAGAGG-3'
CR-R:5'-cagtGGTCTCaaaac cctctgagggggatcacagt-3'。
5.一种用于鉴定大豆GmEIL3基因编辑材料的鉴定引物，其特征在于，包含：
J216162-F7:5'-AGAGGAGGATGTGGAGGGAC-3'；
J216162-R7:5'-CTGCAGTCAGAACACCCACT-3'。
6.一种含有大豆根系镉抗性相关基因GmEIL3的过表达载体，其特征在于，为pCAMBIA3301-CaMV35S-GFP-GmEIL3。
7.一种含有大豆根系镉抗性相关基因GmEIL3的过表达载体的应用，其特征在于，用于降低植物根系镉抗性。
8.一种大豆根系镉抗性相关基因GmEIL3的基因敲除载体，其特征在于，为pzmpl-bar-GmEIL3。
9.一种大豆根系镉抗性相关基因GmEIL3的基因敲除载体的应用，其特征在于，用于增强植物根系镉抗性。
10.一种大豆根系镉抗性相关基因GmEIL3在调节大豆根系镉抗性中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410350877.7A CN118048366A (zh) | 2024-03-26 | 2024-03-26 | 一种大豆根系镉抗性相关基因GmEIL3及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410350877.7A CN118048366A (zh) | 2024-03-26 | 2024-03-26 | 一种大豆根系镉抗性相关基因GmEIL3及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118048366A true CN118048366A (zh) | 2024-05-17 |
Family
ID=91053721
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410350877.7A Pending CN118048366A (zh) | 2024-03-26 | 2024-03-26 | 一种大豆根系镉抗性相关基因GmEIL3及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118048366A (zh) |
-
2024
- 2024-03-26 CN CN202410350877.7A patent/CN118048366A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103183732B (zh) | 一种棉花Gh FPF1蛋白及其编码基因和应用 | |
| CN113337520B (zh) | 陆地棉GhA0749和GhD0744转录因子及其调控开花方面的应用 | |
| Hosokawa et al. | Genetic transformation of gentian using wild-type Agrobacterium rhizogenes | |
| CN110004154B (zh) | 茶树CsJAZ1基因的应用 | |
| CN114591966A (zh) | 拟南芥转录因子srg1基因在调控植物生长发育中的应用 | |
| WO2022213453A1 (zh) | 一种调控植物抗铝性的铝离子受体alr1基因或蛋白的应用 | |
| CN104531723B (zh) | 一种植物维管束发育基因sm‑Nvas及其应用 | |
| CN118048366A (zh) | 一种大豆根系镉抗性相关基因GmEIL3及应用 | |
| Zhao et al. | Transformation of modified cowpea trypsin inhibitor gene and anti-bacterial peptide gene in Brassica pekinensis protoplasts mediated by Agrobacterium tumefaciens | |
| CN117305266B (zh) | 一种与水稻抗逆相关的基因OsBDG1及其编码蛋白的应用 | |
| CN113912687B (zh) | 一种改变植物茎叶及花形态特性的方法及应用 | |
| CN108841831A (zh) | 成花素基因GmFT2a的应用 | |
| CN118374513B (zh) | 水孔蛋白基因OsPIP2-1在调控直播稻种子萌发活力和幼苗建成中的应用 | |
| CN113862282B (zh) | 大豆pcl同源基因编辑位点及其应用 | |
| CN118406698B (zh) | ZmWIND1基因在提高玉米遗传转化效率中的应用 | |
| CN113403326B (zh) | 茶树CsERF3基因及其应用 | |
| KR100496028B1 (ko) | 제초제 저항성 고추 식물체의 제조방법 | |
| CN106244568B (zh) | 一种钙依赖型蛋白激酶cpk32及其编码基因和应用 | |
| CN117587032A (zh) | 84K杨PagMYB4基因及其用途 | |
| CN114591965A (zh) | 一个拟南芥转录因子srg2的应用 | |
| CN118291487A (zh) | OsbHLH167基因在调控水稻深根比中的应用 | |
| CN117947045A (zh) | 一种烟草低温响应基因NtSAP9及其应用 | |
| CN117305344A (zh) | 基因BnaSPL7在独脚金内酯生物合成以及培育转基因植物中的应用 | |
| CN116284290A (zh) | 燕子花花期调控基因IlWRKY22及其应用 | |
| CN117721121A (zh) | MtSPG9基因、蛋白及应用 |
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 |