CN107828794A - 一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 - Google Patents
一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 Download PDFInfo
- Publication number
- CN107828794A CN107828794A CN201710937465.3A CN201710937465A CN107828794A CN 107828794 A CN107828794 A CN 107828794A CN 201710937465 A CN201710937465 A CN 201710937465A CN 107828794 A CN107828794 A CN 107828794A
- Authority
- CN
- China
- Prior art keywords
- osrr22
- rice
- mutant
- gene
- crispr
- 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
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 57
- 235000009566 rice Nutrition 0.000 title claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 41
- 150000003839 salts Chemical class 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000196324 Embryophyta Species 0.000 title claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 title claims abstract description 10
- 240000007594 Oryza sativa Species 0.000 title description 40
- 108091033409 CRISPR Proteins 0.000 claims abstract description 30
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 20
- 238000005516 engineering process Methods 0.000 claims abstract description 9
- 101000852217 Homo sapiens THO complex subunit 6 homolog Proteins 0.000 claims abstract description 8
- 102100036435 THO complex subunit 6 homolog Human genes 0.000 claims abstract description 8
- 241000209094 Oryza Species 0.000 claims abstract 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 12
- 108020005004 Guide RNA Proteins 0.000 claims description 11
- 239000000969 carrier Substances 0.000 claims description 11
- 238000013461 design Methods 0.000 claims description 11
- 230000029087 digestion Effects 0.000 claims description 7
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 6
- 108091034117 Oligonucleotide Proteins 0.000 claims description 6
- 238000010367 cloning Methods 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 206010064571 Gene mutation Diseases 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 230000015784 hyperosmotic salinity response Effects 0.000 abstract description 9
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 240000008467 Oryza sativa Japonica Group Species 0.000 abstract 1
- 102000012410 DNA Ligases Human genes 0.000 description 6
- 108010061982 DNA Ligases Proteins 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000036579 abiotic stress Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 108091079001 CRISPR RNA Proteins 0.000 description 2
- 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
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 1
- 108010043325 Aryl-alcohol dehydrogenase Proteins 0.000 description 1
- 108010049668 Betaine-Aldehyde Dehydrogenase Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101100096644 Escherichia coli (strain K12) srlD gene Proteins 0.000 description 1
- 102100034013 Gamma-glutamyl phosphate reductase Human genes 0.000 description 1
- 101710198928 Gamma-glutamyl phosphate reductase Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100165208 Mustela putorius furo BCO2 gene Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 101150110620 RR22 gene Proteins 0.000 description 1
- 102100035914 S-adenosylmethionine decarboxylase proenzyme Human genes 0.000 description 1
- 108050004491 S-adenosylmethionine decarboxylase proenzyme Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100076587 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MET22 gene Proteins 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 101000613883 Streptomyces lividans pH-gated potassium channel KcsA Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 101150045189 cmo gene Proteins 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction 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
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
-
- 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
- C12N15/8273—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 for drought, cold, salt resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明涉及一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列及该突变体的创制方法。属于植物生物技术领域。本发明将自选育粳稻品种WDR58通过CRISPR/Cas9技术定点编辑了水稻耐盐基因OsRR22，获得了一个具有重要应用价值的水稻耐盐基因OsRR22功能缺失突变体新种质WDR58‑cas‑1。该突变体显著提高水稻的耐盐性，可以应用于水稻的高产、耐盐育种。
Description
技术领域
本发明属于植物生物技术领域，具体涉及一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法。
背景技术
水稻(Oryza sativa L．)是全球三大主要粮食作物之一，是全球22亿人口包含我国65％以上人口的口粮。水稻产量是否供给充足直接影响全球粮食安全。水稻生产面临着各种非生物胁迫，例如：干旱、盐害、冷害、高温等。水稻非生物胁迫成为21世纪农业面临的重要问题之一。
水稻是对盐害极其敏感的作物，盐害已经成为影响水稻生长的主要非生物胁迫因素。目前，全世界6％以上的陆地表现出盐碱化；可耕地中19.5％的水田和2.1％的旱地受到不同程度的盐害。水田盐害主要是人类不当灌溉引起的次级盐害，我国水田的15％受到次级盐害的影响。在这些土地贫瘠产出量低的盐渍化地区，培育耐盐作物显得尤为重要。截至目前，已经克隆的耐盐相关的基因有：SKC1, DST, OsRR22,HAL2, P5CS, CMO, BADH,mtlD, gutD和SAMDC等。其中，OsRR22是调控水稻耐盐性的最重要基因之一。OsRR22基因编码一个696个氨基酸的B型反应调节蛋白转录因子，参与细胞分裂素信号转导和代谢，它的功能缺失显著提高了耐盐性。
CRISPR/Cas9系统是近几年发展的一种准确、便捷、高效率的生物基因组编辑方法。CRISPR/Cas9 的原理是crRNA（CRISPR RNA）通过碱基配对与tracrRNA（transactivating CRISPR RNA）结合形成 tracrRNA/crRNA双元复合体，此复合体引导内切酶Cas9 蛋白在与crRNA 配对的序列靶位点剪切双链DNA。通过人工设计改造这两种RNA，可以形成单个具有引导作用的sgRNA（single-guide RNA），足以引导Cas9 核酸酶对DNA 进行定点切割。它可以对任何靶位点后紧随NGG（PAM）的20bp序列进行定点编辑。目前，CRISPR/Cas9系统不但在酵母、果蝇、鼠、人等生物中，而且已成功在拟南芥、烟草、水稻、小麦、高粱等植物中实现了定点基因组编辑。但是，对水稻育种中有重要价值的产量、抗性、育性等关键基因的定点编辑研究鲜有报道，更缺乏对优良水稻优良亲本进行基因编辑改良的报告。
发明内容
本发明要解决的技术问题是：提出一种提高了耐盐性并保持了其自身优良综合农艺性状的水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法。
本发明为解决上述技术问题提出的技术方案（一）是：一种水稻耐盐基因OsRR22突变体，其核苷酸序列如 SEQ ID NO:1 所示。
本发明为解决上述技术问题提出的技术方案（二）是：一种水稻耐盐基因OsRR22突变体编码的多肽，其氨基酸序列如SEQ ID NO:2所示。
本发明为解决上述技术问题提出的技术方案（三）是：一种水稻耐盐基因OsRR22突变体的创制方法，包括以下步骤：
S1、引导RNA靶点序列设计与选择：根据控制水稻耐盐基因OsRR22的基因组序列和CRISPR-Cas9技术的设计靶位点的原则，设计、选择并合成1个OsRR22引导RNA靶点序列；
S2、CRISPR/Cas9-gRNA载体的构建：将步骤S1中合成的引导RNA靶点序列退火形成双链，与BsaI酶切后的pYLgRNA-OsU3/LacZ连接得到gRNA表达盒，然后通过Golden gatecloning方法将gRNA表达盒装载到CRISPR/Cas9载体上，得到CRISPR/Cas9-gRNA载体；
S3、农杆菌介导水稻愈伤遗传转化：将步骤S2构建好的CRISPR/Cas9-gRNA载体转化到水稻品种WDR58中，获得了不含有CRISPR元件T-DNA成分的纯合OsRR22基因突变体。
进一步的，在所述步骤S1中，靶位点设计在OsRR22基因的第一个外显子上；
所述步骤S1中，所述的OsRR22引导RNA靶点序列的寡核苷酸序列为：
RR22-F:5’-ggcAGAGGGATCAATTCCCCGT-3’
RR22-R:5’-aaacACGGGGAATTGATCCCTCT-3’。
进一步的，在步骤S2中，所述Golden gate cloning方法中用到的引物为：
U3-F: 5’-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3’
U3-R: 5’-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3’。
进一步的，所述水稻品种WDR58为秀水123///秀水123//秀水123/75-1-127，即将75-1-127与秀水123杂交，再将其后代与秀水123回交两次再自交五代所得的稳定品系。
本发明为解决上述技术问题提出的技术方案（四）是：一种水稻耐盐基因Osrr22突变体植株，上述的靶点CRISPR/Cas9-sgRNA载体转化水稻愈伤组织，并转化得到的转基因植株。
本发明的有益效果是：
本发明较现有水稻育种技术具有以下优点：
1、本发明通过设计特定的位点，利用CRISPR/Cas9技术定点编辑耐盐基因OsRR22，改良现有优良水稻品系的单个基因位点，获得了一个具有应用价值的水稻耐盐基因OsRR22突变体。这个突变体显著提高了耐盐性并保持了其自身优良综合农艺性状，可以直接应用于水稻的耐盐育种。
2、本发明创制的突变体获得方法的成功实施，为将来不同水稻品种进行不同基因（fgr、wx等基因）突变体的创制提供了重要参考价值，为水稻育种提供了一种新的技术手段。
附图说明
下面结合附图对本发明的水稻耐盐基因OsRR22突变体、其编码的氨基酸序列及该突变体的创制方法作进一步说明。
图1是OsRR22基因靶点序列引物设计示意图；
图2是OsRR22突变体的测序分析图；
图3是OsRR22纯合突变体的耐盐性表型鉴定图。
具体实施方式
实施例
本实施例涉及到水稻OsRR22基因敲除突变体的获得，即创制方法。
1、引导RNA靶点序列设计与选择
根据控制水稻耐盐基因OsRR22的基因组序列和CRISPR-Cas9技术的设计靶位点的原则，本发明把靶位点设计在OsRR22基因的第一个外显子上，设计、选择并合成1个OsRR22引导RNA靶点序列，见图1和表1。
表1 gRNA靶点的寡核苷酸序列
| gRNA靶点序列 | 寡核苷酸序列（5’-3’） |
| RR22-F | ggcAGAGGGATCAATTCCCCGT |
| RR22-R | aaacACGGGGAATTGATCCCTCT |
2、CRISPR/Cas9-gRNA载体的构建
参考Ma等人的方法（Ma et al., 2015, Molecular Plant, DOI:10.1016/j.molp.2015.04.007）, 取等量靶点gRNA寡核苷酸链的上下游引物（表1）混合(终浓度1 µmol /L)，95℃处理30 s，然后移至室温冷却完成退火形成双链接头；取pYLgRNA-OsU3/LacZ等质粒各1 μg，在25 μl反应用10 U Bsa I酶（NEB公司）切20min；然后将酶切后的pYLgRNA-OsU3/LacZ质粒与对应的双链接头利用T4 DNA ligase 22℃连接30min后得到gRNA表达盒，具体连接体系为：
| 成分 | 加入量 | 终浓度(量) |
| 10×T4 DNA ligase Buffer | 1 μl | 1× |
| pYLgRNA-OsU3/LacZ质粒 | 0.5 μl | 10~20 ng |
| 接头 | 0.5μl | 0.05 μM |
| T4 DNA ligase(Takara) | 0.05 μl | ~18 U |
| ddH2O | 补足到 10 μl |
然后用表2中所列引物对gRNA表达盒进行扩增，采用Golden Gate cloning (Engleret al., 2008; 2009)方法组装Cas9载体和gRNA表达盒片段。
表2扩增靶点gRNA表达盒的引物
| 名称 | 寡核苷酸序列（5’-3’） |
| U3-F | TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG |
| U3-R | AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC |
扩增体系为1ul质粒模板，2.5 uL 10×Buffer，0.5 uL KOD plus聚合酶，1ul 25mM的MgSO4，2.5 uL 2mM的dNTPs，10 uM的上下游引物各0.5 uL，补充dd H2O至25 uL ；PCR扩增程序为：95℃ 2min，98 ℃ 10s，60℃ 15s ，68 ℃ 20 s 20个循环。
PCR产物纯化后用20 U Bsa I酶在37℃酶切30 min，然后75℃处理5 min；将酶切片段纯化后与Bsa I酶切回收的pYLCRISPR/Cas9Pubi-H载体片段利用T4 DNA ligase(NEB) 20℃连接2 h，连接体系为：
| ddH2O | Up to 10 μl |
| 10 × T4 DNA ligase Buffer | 1 μl |
| Bsa I酶切后pYLCRISPR/Cas9Pubi-H载体 | 4 μl |
| 靶点gRNA表达盒扩增产物 | 3 μl |
| T4 DNA ligase | 1 μl |
最后连接产物转化DH5α感受态细胞并挑取阳性克隆测序验证得到靶点CRISPR/Cas9-sgRNA载体。
3、农杆菌介导水稻愈伤遗传转化
将步骤2构建好的CRISPR/Cas9-gRNA载体通过农杆菌介导的方法侵染水稻材料WDR58的愈伤组织，具体方法参考于恒秀等（2005）进行，获得转基因水稻植株。
4、转基因水稻的DNA提取及突变体的测序分析
采用CTAB法提取步骤3中获得的水稻植株的基因组DNA，对潮霉素基因检测为阳性的植株利用引物Cas9-cexu F和Cas9-cexu R对OsRR22突变位点进行PCR扩增。其中鉴定引物为：
Cas9-cexu F：5’- AGAGGAAGGGATTGATGGG-3’
Cas9-cexu R：5’- CATGTCCCTGTTCTCCCTGA-3’
PCR扩增体系为： 20ng水稻基因组DNA模板，10 uL Taq PCR Mastermix（天根生化科技(北京)有限公司），10 uM的前后引物各1 uL，补充dd H2O至20 uL。反应程序为： 95℃预变性5 min，然后按95℃ 30 s，58℃下 30 s，72℃下 45 s进行30个循环，最后72℃下延伸 5min。
对PCR扩增产物进行的测序结果分析表明，OsRR22突变频率91.6%，其中25%为纯合突变，见图2。
5、无转基因成分OsRR22突变体的获得及耐盐性表型调查
将纯合突变体T1代转基因植株播种成苗，苗期检测潮霉素基因的有无，保留不含有潮霉素基因而含有OsRR22基因纯合突变位点的个体。水稻苗三叶一心期，放入0.75%氯化钠浓度的培养液中下处理7天后，进行耐盐性筛选（图3）。获得耐盐性比对照提高的株系，命名为WDR58-cas-1。
其中，耐盐突变体WDR58-cas-1，耐盐基因OsRR22的氨基酸序列如SEQ ID NO:2所示。
耐盐突变体WDR58-cas-1，耐盐基因OsRR22的核苷酸序列见SEQ ID NO:1 所示。
本发明的不局限于上述实施例，本发明的上述各个实施例的技术方案彼此可以交叉组合形成新的技术方案，另外凡采用等同替换形成的技术方案，均落在本发明要求的保护范围内。
<110>上海市农业生物基因中心
<120>一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法
<160> 2
<210> 1
<211> 2092
<212> DNA
<213>人工序列
<400> 1
atgcttctgg gtgctttgag gatggaggag aggaagggat tgatggggag ggagagggat 60
caattcccac gtcggcatgc gggtcctcgc cgtcgacgat gacccggtgt gcctcaaggt 120
tcttgagacc ctcctccggc gctgccaata ccatgtaaca tcaaccaacc aggctattac 180
tgcgttgaag ctgctcaggg agaacaggga catgtttgat cttgtcatca gtgatgtcca 240
catgcccgac atggacggat ttaagctcct tgagcttgtg gggcttgaaa tggatctccc 300
agtcatcatg ttatcagtaa atggagagac aaagactgtg atgaagggga taactcatgg 360
tgcctgtgac tatcttctaa aaccggtccg aatcgaagaa ctaaggaaca tatggcagca 420
tgttgttagg aggaagttcg gtaatcgtga gcgaaacaat cttgatttct ccaaagaatg 480
caataagccg caaagcgcgg atactgatca tggaccatac caacctacct gtggttcttc 540
tgatcaaaat gggaggtcca gcaggaaaag gaaagaacta cacggcgagg acgacgatga 600
aggcgatgat aatgattatc aagaaaatga tgagccctca gctgcaaaga agcccagagt 660
tgtatggtca gttgagctgc accgaaaatt tgttgccgct gtcaaccagc ttggaattga 720
caaagctgta ccaaaaagaa ttcttgagct tatgaatgtg gagaaactca ccagggaaaa 780
tgttgcaagt catctacaga agtacaggct ttacctcaag agactaggtg ctgtagcatc 840
acaacaagcc agcattgttg ctgcctttgg aggcagagat ccctccttct tgcatattgg 900
agcatttgaa ggactccaga gctatcaacc ttttgcacct tctgctgctc ttccatcttt 960
caatccacat ggcctgctaa cccgaactag cgccgccgcg gctttcggac ttcaggagct 1020
tgctgccccc tccagcacaa ttcagacttc tacaggaaat gtcacagttg gccattgctt 1080
ggaagaaaac cagcaggcaa atctagcaca aggcttgacc gcggcgatcg ggcaacctca 1140
gcttcaacag aactggattc atcaagaagg taatggtctg tctgatgttt tttctgggag 1200
ttctctgacc aacactttgt ccagcacact ccaaagagtt ccaagcagtt cattgccacc 1260
acaagaactc ttggagtgca aacaagccaa agttagcatg ccgccatcga tacggatacc 1320
gccttctagt tcagcacttc ttgagaggac tcttggggtt tccaccaatt tgggagattc 1380
tagtatatcc cagcagggtg ctcttccaat agatggtgga ttttctgctg acaggttacc 1440
attgcacagt tcatttgatg gcgctgttgc aacaaagcta gatactagtt tggcagcttc 1500
acagagagag attggccagc aggggaaatt ttcagttagc atgcttgtct ccccttctga 1560
caatcttgca ttagccaaaa atgccaaaac tggagctagt tcttctggca gtactataat 1620
tctccctctt gatactgcaa gacattcaga ctacttgcag ttcggaggtg caagcaattc 1680
tttgcagaaa atggatggac agaaacaaga tcatatacag agctcaaaca ttatatggag 1740
ttcaatgcca agcactcaac tgccaagtga tacccaaatt cataatactc aaaaccaaag 1800
attggacagc ggaagtttta accataatat tggtgcccat ttggctgacc aaacaaatgc 1860
aagtgcgtca atacttccgc aaatgaagtt tgacacaaga atatcagaag agaaaatgaa 1920
gcagaagaat acatatgact tgggtagttc aaagctgcag ggtggattta attctagtgg 1980
ctgcaatttt gatggccttc tcaattccat aatcaaagtg gagaaggatg atctcccatt 2040
catggacaat gaattgggct gtgacctttt tccacttggt gcctgcatat ga 2092
<210> 2
<211> 33
<212> PRT
<213>人工序列
<400> 2
MLLGALRMEERKGLMGRERDQFPRRHAGPRRRR*
Claims (8)
1.一种水稻耐盐基因OsRR22突变体，其特征在于：其核苷酸序列如 SEQ ID NO:1 所示。
2.一种如权利要求1所述水稻耐盐基因OsRR22突变体编码的多肽，其特征在于：其氨基酸序列如SEQ ID NO:2所示。
3.一种如权利要求1所述水稻耐盐基因OsRR22突变体的创制方法，包括以下特征步骤：
S1、引导RNA靶点序列设计与选择：根据控制水稻耐盐基因OsRR22的基因组序列和CRISPR-Cas9技术的设计靶位点的原则，设计、选择并合成1个OsRR22引导RNA靶点序列；
S2、CRISPR/Cas9-gRNA载体的构建：将步骤S1中合成的引导RNA靶点序列退火形成双链，与BsaI酶切后的pYLgRNA-OsU3/LacZ连接，然后通过Golden gate cloning方法将gRNA表达盒装载到CRISPR/Cas9载体上，得到CRISPR/Cas9-gRNA载体；
S3、农杆菌介导水稻愈伤遗传转化：将步骤S2构建好的CRISPR/Cas9-gRNA载体转化到水稻品种WDR58中，获得了不含有CRISPR元件T-DNA成分的纯合OsRR22基因突变体。
4.根据权利要求3所述水稻耐盐基因OsRR22突变体的创制方法，其特征在于：在所述步骤S1中，靶位点设计在OsRR22基因的第一个外显子上；
所述步骤S1中，所述的OsRR22引导RNA靶点序列的寡核苷酸序列为：
RR22-F:5’-ggcAGAGGGATCAATTCCCCGT-3’
RR22-R:5’-aaacACGGGGAATTGATCCCTCT-3’。
5.根据权利要求3或4所述水稻耐盐基因OsRR22突变体的创制方法，其特征在于：在步骤S2中，所述Golden gate cloning方法中用到的引物为：
U3-F: 5’-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3’
U3-R: 5’-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3’。
6.根据权利要求3或4所述水稻耐盐基因OsRR22突变体的创制方法，其特征在于：所述水稻品种WDR58为秀水123///秀水123//秀水123/75-1-127，即将75-1-127与秀水123杂交，再将其后代与秀水123回交两次再自交五代所得的稳定品系。
7.根据权利要求5所述水稻耐盐基因OsRR22突变体的创制方法，其特征在于：所述水稻品种WDR58为秀水123///秀水123//秀水123/75-1-127，即将75-1-127与秀水123杂交，再将其后代与秀水123回交两次再自交五代所得的稳定品系。
8.一种水稻耐盐基因Osrr22突变体植株，其特征在于：是通过将权利要求3所述的靶点CRISPR/Cas9-sgRNA载体转化水稻愈伤组织，并转化得到的转基因植株。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710937465.3A CN107828794A (zh) | 2017-09-30 | 2017-09-30 | 一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710937465.3A CN107828794A (zh) | 2017-09-30 | 2017-09-30 | 一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107828794A true CN107828794A (zh) | 2018-03-23 |
Family
ID=61647645
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710937465.3A Pending CN107828794A (zh) | 2017-09-30 | 2017-09-30 | 一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107828794A (zh) |
Cited By (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| CN109207509A (zh) * | 2018-08-29 | 2019-01-15 | 湖南农业大学 | 一种定向、高效培育耐盐水稻品种的育种方法 |
| CN109825638A (zh) * | 2019-04-11 | 2019-05-31 | 上海市农业生物基因中心 | 一种水稻耐盐基因OsRR22引导引物、应用和靶点载体及靶点载体制备方法 |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| CN109988774A (zh) * | 2019-04-24 | 2019-07-09 | 广东省生物工程研究所（广州甘蔗糖业研究所） | 调控水稻矮化的b型响应调节基因orr2及其应用 |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| CN110885837A (zh) * | 2019-12-03 | 2020-03-17 | 湖南杂交水稻研究中心 | 水稻OsRR22-1突变型基因及其鉴定方法、鉴定用KASP分型引物及应用 |
| CN110885838A (zh) * | 2019-12-03 | 2020-03-17 | 湖南杂交水稻研究中心 | 水稻OsRR22-7突变型基因及其鉴定方法、鉴定用KASP分型引物及应用 |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| CN111909956A (zh) * | 2020-06-30 | 2020-11-10 | 电子科技大学 | 阻断或减弱水稻OsNAC092基因表达以提高水稻抗旱性的方法 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112226427A (zh) * | 2020-09-25 | 2021-01-15 | 华中农业大学 | 桃氰丙氨酸合成酶基因在提高植物耐盐性方面的应用 |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN114717352A (zh) * | 2022-01-28 | 2022-07-08 | 上海市农业生物基因中心 | 水稻耐高温调控基因Hsp70的分子标记及其应用 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| JP2023505138A (ja) * | 2019-12-03 | 2023-02-08 | 湖南雑交水稲研究中心 | 物理的及び化学的突然変異誘発植物のｍ１世代突然変異を同定する方法、と突然変異体を取得する方法、及びイネの突然変異同定用のタイピングプライマー、突然変異遺伝子及び使用 |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US12390514B2 (en) | 2017-03-09 | 2025-08-19 | President And Fellows Of Harvard College | Cancer vaccine |
| US12406749B2 (en) | 2017-12-15 | 2025-09-02 | The Broad Institute, Inc. | Systems and methods for predicting repair outcomes in genetic engineering |
| US12435330B2 (en) | 2019-10-10 | 2025-10-07 | The Broad Institute, Inc. | Methods and compositions for prime editing RNA |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981842A (en) * | 1995-10-12 | 1999-11-09 | Cornell Research Foundation, Inc. | Production of water stress or salt stress tolerant transgenic cereal plants |
| CN105063061A (zh) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | 一种水稻千粒重基因tgw6突变体及其制备方法与应用 |
| CN105063026A (zh) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | 一种水稻千粒重基因tgw6引导rna靶点序列及应用 |
-
2017
- 2017-09-30 CN CN201710937465.3A patent/CN107828794A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981842A (en) * | 1995-10-12 | 1999-11-09 | Cornell Research Foundation, Inc. | Production of water stress or salt stress tolerant transgenic cereal plants |
| CN105063061A (zh) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | 一种水稻千粒重基因tgw6突变体及其制备方法与应用 |
| CN105063026A (zh) * | 2015-07-28 | 2015-11-18 | 华南农业大学 | 一种水稻千粒重基因tgw6引导rna靶点序列及应用 |
Non-Patent Citations (6)
| Title |
|---|
| FUJUN WANG: "Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922", 《PLOS ONE》 * |
| HIROKI TAKAGI: "MutMap Accelerates Breeding of a Salt-Tolerant Rice Cultivar", 《NATURE BIOTECHNOLOGY》 * |
| ORIGIN: "PREDICTED:Oryza sativa Japonica Group two-component response regulator ORR22(LOC4340320),mRNA", 《NCBI》 * |
| 王加峰: "基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建", 《作物学报》 * |
| 王彩芬: "水稻耐盐基因SKC1特异性CAPS标记的开发与验证", 《分子植物育种》 * |
| 陈薇: "《生物技术发展年鉴（2013）》", 31 December 2014, 军事医学科学出版社 * |
Cited By (67)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US12215365B2 (en) | 2013-12-12 | 2025-02-04 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US12398406B2 (en) | 2014-07-30 | 2025-08-26 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US12344869B2 (en) | 2015-10-23 | 2025-07-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US12084663B2 (en) | 2016-08-24 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US12390514B2 (en) | 2017-03-09 | 2025-08-19 | President And Fellows Of Harvard College | Cancer vaccine |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US12435331B2 (en) | 2017-03-10 | 2025-10-07 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US12359218B2 (en) | 2017-07-28 | 2025-07-15 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US12406749B2 (en) | 2017-12-15 | 2025-09-02 | The Broad Institute, Inc. | Systems and methods for predicting repair outcomes in genetic engineering |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| CN109207509A (zh) * | 2018-08-29 | 2019-01-15 | 湖南农业大学 | 一种定向、高效培育耐盐水稻品种的育种方法 |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US12281303B2 (en) | 2019-03-19 | 2025-04-22 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN109825638A (zh) * | 2019-04-11 | 2019-05-31 | 上海市农业生物基因中心 | 一种水稻耐盐基因OsRR22引导引物、应用和靶点载体及靶点载体制备方法 |
| CN109988774B (zh) * | 2019-04-24 | 2020-12-04 | 广东省生物工程研究所（广州甘蔗糖业研究所） | 调控水稻矮化的b型响应调节基因orr2及其应用 |
| CN109988774A (zh) * | 2019-04-24 | 2019-07-09 | 广东省生物工程研究所（广州甘蔗糖业研究所） | 调控水稻矮化的b型响应调节基因orr2及其应用 |
| US12435330B2 (en) | 2019-10-10 | 2025-10-07 | The Broad Institute, Inc. | Methods and compositions for prime editing RNA |
| JP7503134B2 (ja) | 2019-12-03 | 2024-06-19 | 湖南雑交水稲研究中心 | 物理的及び化学的突然変異誘発植物のｍ１世代突然変異を同定する方法、と突然変異体を取得する方法、及びイネの突然変異同定用のタイピングプライマー、突然変異遺伝子及び使用 |
| CN110885837A (zh) * | 2019-12-03 | 2020-03-17 | 湖南杂交水稻研究中心 | 水稻OsRR22-1突变型基因及其鉴定方法、鉴定用KASP分型引物及应用 |
| CN110885838A (zh) * | 2019-12-03 | 2020-03-17 | 湖南杂交水稻研究中心 | 水稻OsRR22-7突变型基因及其鉴定方法、鉴定用KASP分型引物及应用 |
| CN110885838B (zh) * | 2019-12-03 | 2021-08-17 | 湖南杂交水稻研究中心 | 水稻OsRR22-7突变型基因及其鉴定方法、鉴定用KASP分型引物及应用 |
| JP2023505138A (ja) * | 2019-12-03 | 2023-02-08 | 湖南雑交水稲研究中心 | 物理的及び化学的突然変異誘発植物のｍ１世代突然変異を同定する方法、と突然変異体を取得する方法、及びイネの突然変異同定用のタイピングプライマー、突然変異遺伝子及び使用 |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN111909956A (zh) * | 2020-06-30 | 2020-11-10 | 电子科技大学 | 阻断或减弱水稻OsNAC092基因表达以提高水稻抗旱性的方法 |
| CN112226427A (zh) * | 2020-09-25 | 2021-01-15 | 华中农业大学 | 桃氰丙氨酸合成酶基因在提高植物耐盐性方面的应用 |
| CN112226427B (zh) * | 2020-09-25 | 2022-06-10 | 华中农业大学 | 桃氰丙氨酸合成酶基因在提高桃耐盐性方面的应用 |
| CN114717352B (zh) * | 2022-01-28 | 2023-11-17 | 上海市农业生物基因中心 | 水稻耐高温调控基因Hsp70的分子标记及其应用 |
| CN114717352A (zh) * | 2022-01-28 | 2022-07-08 | 上海市农业生物基因中心 | 水稻耐高温调控基因Hsp70的分子标记及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107828794A (zh) | 一种水稻耐盐基因OsRR22突变体、其编码的氨基酸序列、植株及该突变体的创制方法 | |
| CN107988229B (zh) | 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 | |
| CN110317828B (zh) | 修饰水稻OsSWEET基因启动子培育广谱抗白叶枯病水稻的方法 | |
| CN109234310B (zh) | 快速获得无转基因基因编辑植株的重组载体及使用方法 | |
| CN109825638A (zh) | 一种水稻耐盐基因OsRR22引导引物、应用和靶点载体及靶点载体制备方法 | |
| CN120098948A (zh) | 水稻OsRMT1基因在提高水稻耐盐能力中的应用 | |
| CN114907465B (zh) | 水稻孕穗期耐冷性相关的OsLEA9蛋白及其相关生物材料与应用 | |
| CN115820672B (zh) | 一个玉米氮利用相关基因ZmNCRG2及其应用 | |
| CN117646029A (zh) | TaSLC25A4-7A基因在小麦育种及提高抗逆能力中的应用 | |
| CN113943740B (zh) | 一种可调控烟叶钾含量的NtCHA1基因及其应用 | |
| CN115806605A (zh) | 一种玉米氮高效利用基因及其分子标记和应用 | |
| WO2018196744A1 (zh) | 转基因大豆事件gc1-1外源插入片段旁侧序列及其应用 | |
| CN113429467B (zh) | Npf7.6蛋白在调控豆科植物根瘤耐氮性中的应用 | |
| CN112048489A (zh) | 水稻中两个二磷酸腺苷葡萄糖焦磷酸化酶基因及其编码蛋白的工程应用 | |
| CN112094865A (zh) | 一种培育耐低钾水稻的方法 | |
| CN119193619B (zh) | 水稻基因OsPOK1用于调控水稻粒型的用途及其突变体 | |
| CN120060349B (zh) | 二穗短柄草BdSTAR1蛋白及其编码基因在调控植物种子粒长中的应用 | |
| CN119162204B (zh) | 水稻基因OsABCA3用于调控水稻粒型的用途及其突变体 | |
| CN110616220A (zh) | 一种提高小麦籽粒硬度的方法 | |
| CN117025627B (zh) | 烟草氯离子通道蛋白NtCLC13及其编码基因和应用 | |
| CN119776381B (zh) | 一种提高水稻白叶枯抗性的Xa5优良等位基因的创制方法及应用 | |
| CN114790230B (zh) | 蛋白质TaARE1在调控植物耐低氮中的应用 | |
| CN118240864B (zh) | 水稻OsFD1基因启动子区的STR或靶向删除该STR的方法在水稻分子育种中的应用 | |
| CN118834912B (zh) | 水稻耐冷基因ct4.1及其应用 | |
| EP4628591A1 (en) | Method for producing caaibz1 gene-edited pepper to improve drought tolerance |
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 |
Application publication date: 20180323 |
|
| RJ01 | Rejection of invention patent application after publication |