CN117069815B - Application of GID1a protein of rabdosia lophanthide in plant increase - Google Patents
Application of GID1a protein of rabdosia lophanthide in plant increase Download PDFInfo
- Publication number
- CN117069815B CN117069815B CN202311264030.9A CN202311264030A CN117069815B CN 117069815 B CN117069815 B CN 117069815B CN 202311264030 A CN202311264030 A CN 202311264030A CN 117069815 B CN117069815 B CN 117069815B
- Authority
- CN
- China
- Prior art keywords
- gid1a
- protein
- plant
- gene
- fungus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000196324 Embryophyta Species 0.000 title claims abstract description 84
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 72
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 32
- 241001183967 Isodon Species 0.000 title claims description 13
- 241000233866 Fungi Species 0.000 claims abstract description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 32
- 239000013598 vector Substances 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 12
- 230000002018 overexpression Effects 0.000 claims description 12
- 241000589158 Agrobacterium Species 0.000 claims description 6
- 239000012620 biological material Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 4
- 241000233805 Phoenix Species 0.000 claims 2
- 239000000758 substrate Substances 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 abstract description 22
- 241000219194 Arabidopsis Species 0.000 abstract description 18
- 238000009395 breeding Methods 0.000 abstract description 8
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- 238000010353 genetic engineering Methods 0.000 abstract description 5
- 230000001276 controlling effect Effects 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 239000002773 nucleotide Substances 0.000 abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 abstract description 3
- 241000404370 Lophanthus Species 0.000 abstract description 2
- 230000008827 biological function Effects 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000007787 solid Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000013599 cloning vector Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000012215 gene cloning Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 101100282746 Oryza sativa subsp. japonica GID1 gene Proteins 0.000 description 2
- 240000007377 Petunia x hybrida Species 0.000 description 2
- 101100156295 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) VID30 gene Proteins 0.000 description 2
- 240000006394 Sorghum bicolor Species 0.000 description 2
- 235000015505 Sorghum bicolor subsp. bicolor Nutrition 0.000 description 2
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 101100438273 Arabidopsis thaliana CAN1 gene Proteins 0.000 description 1
- 241001672739 Artemia salina Species 0.000 description 1
- 241000336315 Cistanche salsa Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101150089337 GID1 gene Proteins 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 101710172184 Gibberellin receptor GID1 Proteins 0.000 description 1
- 241001517074 Iris sanguinea Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000124015 Salix viminalis Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical group C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 230000028604 virus induced gene silencing Effects 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)
- C12N15/8241—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of plant genetic engineering, and discloses application of a GID1a protein of a lineata in plant height increase. The nucleotide sequence of the GID1a protein of the lophanthus gracilis is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the gene can increase the plant height of transgenic arabidopsis after being overexpressed in the arabidopsis, thereby influencing the plant height of the arabidopsis. Through systematic research, the invention provides the biological function of the GID1a protein of the brookfield fungus in regulating and controlling plant height, which not only can provide an important theoretical basis for the regulating and controlling research of the brookfield fungus strain height, but also can be used as an excellent gene resource to be applied to the strain height breeding of other flower plants.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a GID1a protein of a lineata and application thereof in plant height increase.
Background
For garden plants, the plant height can directly influence the overall ornamental effect, and the beautiful and neat plant height is the basis for improving the ornamental value of ornamental plants. The plant height change of the prior plants is mostly regulated by the traditional breeding method or by adopting measures such as external nutrition and hormone application. Along with the development of scientific technology, the restriction of the traditional breeding technology can be broken through by the genetic engineering technology, so that the plant height is changed on the molecular level, the ornamental value is increased, and the method has become one of important means for plant height improvement.
The river and the fungus (Iris sanguinea Donn ex hornem.) are perennial herbaceous plants of the genus Iris, have peculiar flowers, various colors and strong cold resistance, have low requirements on soil, can be used for greening wetland such as lakeside, river bank, pond edge and the like, shallow water and flower bed and flower environment, can also be used for cutting flowers, has higher ornamental value, is an excellent material for greening cold landscaping, and has wide application prospect. In recent years, research on the broomcorn is mainly focused on the aspects of germplasm resource preservation, innovation, resistant physiology, crossbreeding, micropropagation technology, quality improvement and the like. When the material is used as a flower viewing and cutting material, the market demand of increasing the plant height is often met, in order to meet the requirement of garden application on the plant height of the brook, the development of genetic engineering plant height breeding is very important, and the digging and functional verification of plant height regulation genes can lay a foundation for the development of genetic engineering breeding.
In recent years, with intensive studies on rice and the model plant Arabidopsis thaliana, the basic pathways and molecular mechanisms of Gibberellin (GA) signal transduction have been gradually clarified, and it has been confirmed that a plurality of genes related to GA can exert an influence on the plant heights of various plants. GA signal transduction starts from the perception of active GA by gibberellin receptor GID1, and GA combines with GID1 to form GA-GID1 complex, and then combines with DELLA protein to form GA-GID1-DELLA trimeric complex, which is ubiquitinated, and DELLA protein is hydrolyzed to release inhibition to plant growth, thus generating GA effect. Research shows that GID1 gene has regulation effect on plant height, for example, ectopic expression of the artemia salina (Salix viminalis) SvGID s gene enhances gibberellin effect, resulting in plant height increase; induction of PhGID gene silencing in Petunia (Petunia x hybrid) using VIGS technology can lead to dwarfing of plants. The invention provides a GID1a protein of the brookfield fungus, and verifies the plant height regulating and controlling function in model plant arabidopsis thaliana, and aims to provide theoretical reference and important gene resources for the brookfield fungus plant height breeding work.
Disclosure of Invention
The invention aims to provide an application of the GID1a protein of the brook fungus in plant height increase, provides a new way for garden plant height increase, has important significance for exploring the function of the GID1a protein of the brook fungus and revealing the dwarf formation mechanism of the plant, and provides an important reference for molecular breeding of the arabidopsis plant.
The invention aims at providing a GID1a protein of the brook fungus.
The second object of the present invention is to provide a gene encoding GID1a protein of the group of the fungus.
The invention also aims to provide biological materials related to the GID1a protein of the rabdosia.
The fourth purpose of the invention is to obtain arabidopsis thaliana transformed plants containing the GID1a gene of the broomrape.
The invention aims at providing an application of the GID1a protein of the rabdosia lophanthide in plant height increase.
The aim of the invention is realized by the following technical scheme:
provides a GID1a protein of the brookfield fungus, which is characterized by having an amino acid sequence shown as SEQ ID NO.1 in a sequence table.
Provides a gene for encoding the GID1a protein of the brookfield fungus, which is characterized by having a nucleotide sequence shown as SEQ ID NO.2 in a sequence table.
Providing a biological material related to the GID1a protein of the brookfield, which is characterized by comprising any one of the following (A1) to (A5):
(A1) Recombinant cloning vector containing gene for encoding GID1a protein of the brook fungus;
(A2) A recombinant plant over-expression vector containing a gene for encoding the GID1a protein of the linearis;
(A3) A recombinant plant overexpression vector obtained by connecting a label to the N end or/and the C end of the gene (A2);
(A4) A bioengineering bacterium comprising the recombinant plant overexpression vector of (A2) or (A3);
(A5) A transgenic plant comprising the recombinant plant overexpression vector of (A2) or (A3).
(A2) And/or (A3) the plant over-expression vector containing the gene encoding the above-mentioned GID1a protein of the Rabdosia is a DNA capable of over-expressing the above-mentioned GID1a protein of the Rabdosia in a host cell, which may contain not only a promoter for initiating transcription of the GID1a gene but also a terminator for terminating transcription of the GID1a gene.
The plant expression vector is pCAMBIA1300-GFP.
The bioengineering bacteria is Agrobacterium GV3101 (only, china).
The application of the GID1a protein of the Xiangzhuan in plant height increase;
A method of regulating plant height increase comprising the steps of:
(B1) Introducing a recombinant plant over-expression vector containing the GID1a gene of the rabdosia into a recipient plant;
The method for introducing a plant expression vector into a recipient plant is characterized by comprising the following steps:
plant expression vector containing the GID1a gene of the broomcorn is used for transforming plant tissues through an inflorescence infection method, and the transformed plant tissues are cultivated into plants.
According to the technical scheme of the invention, the receptor plant is a monocotyledonous plant or a dicotyledonous plant, preferably a linearis or an arabidopsis thaliana.
The invention has the following beneficial effects:
(1) According to the invention, the GID1a gene is cloned from basal leaves of the lophanthus gracilis, a GV1300-GID1a-GFP plant expression vector is constructed, agrobacterium GV3101 (only, china) is transformed by a freeze thawing method, the GV1300-GID1a-GFP plant expression vector is transformed into Arabidopsis thaliana by an inflorescence infection method, and the result shows that the over-expression of the GID1a gene in the Arabidopsis thaliana leads to the increase of the plant height of the Arabidopsis thaliana.
(2) The transgenic arabidopsis plant is increased, which shows that the GID1a gene of the rabdosia has the function of regulating and controlling plant height.
(3) The GID1a gene of the brookfield fungus can be used as an excellent gene resource, lays a foundation for researching the color regulating mechanism of the swallow flower, and provides gene resources for flower color breeding of garden plants, especially iris plants.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PCR identification electrophoresis patterns of the transgenic arabidopsis thaliana of the present invention, wherein M is DL2000 Maker,1 is GV1300-GID1a-GFP recombinant plasmid, 2 is wild type arabidopsis thaliana, 3 is transgenic arabidopsis thaliana, and 4-10 are transgenic arabidopsis thaliana lines.
In FIG. 2, A is IsGID a transgenic Arabidopsis thaliana plant height phenotype statistical diagram, B is IsGID a transgenic Arabidopsis thaliana plant height phenotype comparison diagram, wherein WT is wild type Arabidopsis thaliana, empty load is transgenic carrier Arabidopsis thaliana, and OE is transgenic Arabidopsis thaliana.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but it should be understood that the scope of the invention is not limited to the specific embodiments. The experimental methods in the following implementation methods are all conventional methods, and materials, reagents and the like used in the examples are all commercially available unless otherwise specified.
The invention aims to overcome the defects of the prior art and provides an application of a GID1a protein of the rabdosia in plant height increase.
The relevant culture medium formulation method in the examples is as follows:
LB liquid medium: 5g/L yeast extract+10 g/L tryptone+10 g/L sodium chloride;
LB solid medium: 5g/L yeast extract+10 g/L tryptone+10 g/L sodium chloride+15 g/L agar;
YEP liquid medium: 10g/L yeast extract+10 g/L tryptone+5 g/L sodium chloride;
YEP solid medium: 10g/L yeast extract+10 g/L tryptone+5 g/L sodium chloride+15 g/L agar;
1/2MS solid medium: 2.47 g/L1/2MS+20g/L sucrose+7.8g/L agar.
The specific test scheme of this example is as follows:
EXAMPLE 1 cloning of the GID1a Gene of the Rabdosia
1. Total RNA extraction from plants
The total RNA of the plant is extracted by adopting OminiPlant RNAKit (Dnase I) (century, china) kit, and the operation steps are carried out according to the instruction book.
2. CDNA Synthesis
CDNA was synthesized using PRIMESCRIPT TM RT REAGENT KIT WITH GDNA ERASER (PERFECT REAL TIME) (Takara, japan) kit, and the procedure was followed according to the instructions, and cDNA was diluted 10-fold and used as a template for gene cloning.
3. Gene cloning
The Primer 5 is used for designing a cloning specific Primer of the GID1a gene of the rabdosia, and the Primer sequence is as follows:
GID1a-F1：5’-ATGGCTGGAAGCAACGAG-3’
GID1a-R1：5’-AAGTTTGAACTGTAACAACTGCTAG-3’
A50. Mu.L PCR reaction system was prepared, which included 2. Mu.L of template cDNA, 25. Mu.L of 2X PCR buffer for KOD FX, 10. Mu.L of 2mM dNTPs, 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of KOD FX and 10. Mu.L of ddH 2 O, and the above reaction solution was placed on ice, and the reaction solution was mixed uniformly and centrifuged at a low speed (2500 rpm).
The PCR reaction procedure was: 95 ℃ for 5min; cycling for 35 times at 95 ℃ for 30s,59 ℃ for 45s and 72 ℃ for 90 s; and at 72℃for 10min.
Amplifying by PCR to obtain an amplified product; detecting the amplification result by 1% agarose gel electrophoresis, and recycling the target band by gel; the recovered product was then ligated to cloning vector pEASY-Blunt Zero Cloning Vector (full gold, china), transformed into E.coli (ESCHERICHIA COLI) DH 5. Alpha. (full gold, china) and spread on LB solid containing 100mg/L Amp resistance, cultured upside down in an incubator at 37℃for 10-12h, the monoclonal strain was picked up for bacterial liquid PCR identification, positive clones were expanded for 10mL in LB liquid medium containing 100mg/L Amp resistance, and then sent to Bio-company for sequencing.
The nucleotide sequence of the obtained GID1a gene of the Xiang is shown as follows:
the ORF region of the GID1a gene of the Xun contains 1032 bases and codes 343 amino acids, and the amino acid sequence is shown as follows:
EXAMPLE 2 construction of the plant overexpression vector of the GID1a Gene of the Rabdosia
(1) Designing a carrier homology arm primer with Sal I and BamH I enzyme cutting sites respectively by using a homologous recombination method, carrying out PCR amplification by using a cloning vector plasmid of a connected xihong GID1a gene as a template, wherein a PCR reaction system and a reaction program are the same as those of gene cloning, and recovering a target fragment by glue after the PCR is finished, wherein the sequences of the carrier homology arm primers (the underlined parts are Sal I and BamH I enzyme cutting sites) are as follows:
GID1a-F2：5’-TTGATACATATGCCCGTCGACATGGCTGGAAGCAACGAG-3’
GID1a-R2：5’-CCCTTGCTCACCATGGATCCGCAATGGGAGCTCACAAA-3’
(2) The expression vector GV1300-GFP plasmid is digested by Sal I and BamH I restriction enzymes, the vector fragment is recovered, the linearization vector is connected with the GID1a gene fragment added with a vector homology arm, the competent DH5 alpha of the escherichia coli is transformed and coated on LB solid containing 100mg/L Amp resistance, the mixture is inversely cultured for 10-12 hours in a 37 ℃ incubator, a monoclonal strain is selected for bacterial liquid PCR identification, positive clone is amplified and cultured for 10mL in LB liquid medium containing 100mg/L Amp resistance, and then the positive clone is sent to a biological company for sequencing. The vector construction was carried out using ClonExpress IIOne Step Cloning Kit (Nor praise, china) homologous recombination kit according to the instructions.
Example 3 application of GID1a Gene of Xuangzhan in plant height control
1. Cultivation of Arabidopsis thaliana
In an ultra-clean workbench, a proper amount of empty carrier arabidopsis thaliana and wild arabidopsis thaliana seeds (Columbia type Col-0) are placed in a 1.5mL centrifuge tube, sterilized with 75% alcohol for 1min, sterilized with sterile water for 3 times, sterilized with 0.8% sodium hypochlorite solution (Siberian) for 10min, and sterilized with sterile water for 5 times. The sterilized wild Arabidopsis seeds are inoculated on a 1/2MS solid medium, placed in a refrigerator at 4 ℃ for vernalization for 2-3d under dark conditions, and then placed in a plant culture chamber for culture. Transplanting the germinated Arabidopsis seedlings into a flowerpot filled with a culture medium (peat soil, vermiculite and perlite are mixed according to a ratio of 5:3:2, and are subjected to high-temperature and high-pressure sterilization), placing the flowerpot in a plant culture room for continuous culture for 3 weeks, and preparing genetic transformation when Arabidopsis is in a process of taking out the flower and the more buds are generated. The environmental conditions of the plant culture room are as follows: 16h of light/8 h of darkness, 20-22 ℃.
2. Preparation of infectious microbe liquid
Agrobacterium competent GV3101 (only, china) was transformed, the transformation procedure was as described above, and then spread on YEP solid medium containing 50mg/L Kana and 25mg/L Rif, and cultured upside down at 28℃for 36h; selecting a monoclonal to perform bacterial liquid PCR verification, and performing amplification culture on a positive bacterial colony containing a target gene in a YEP (or LB) liquid culture medium containing Kana for 10mL under the culture condition of 28 ℃ and 180rpm shaking culture for 12-16h; sucking 1mL of the cultured bacterial liquid, adding the bacterial liquid into 50mL of fresh YEP (or LB) liquid culture medium containing corresponding antibiotics, and continuously shaking and culturing until OD 600 is about 0.6-0.8; the cultured bacterial liquid is centrifuged at 5000rpm for 5min, the supernatant is discarded in an ultra-clean workbench to collect bacterial cells, and an equal volume (50 mL) of infection liquid (pH 5.8) containing 5% sucrose and 3% silwet-77 is used for resuspension of bacterial cells, so that arabidopsis thaliana is infected.
3. Infection of Arabidopsis thaliana
Taking a wild arabidopsis plant to be transformed, taking bud dew as a standard, and removing the opened flowers. The arabidopsis thaliana to be converted is soaked in an infection liquid for 1min, is covered with a light-tight black plastic bag for 24h, is uncovered, is cultured in a plant culture room, and simultaneously is provided with an empty carrier arabidopsis thaliana which is not infected by agrobacterium and a wild arabidopsis thaliana as negative control. Extracting DNA of transgenic Arabidopsis plants, and carrying out PCR identification by using GID1a gene homology arm primers to obtain transgenic positive Arabidopsis plants.
4. Identification of transgenic Positive Arabidopsis plants
The obtained 7 transgenic Arabidopsis plants were subjected to DNA extraction and PCR identification using homology arm primers (GID 1a-F2 and GID1 a-R2), wherein the transgenic vector Arabidopsis and wild type Arabidopsis were negative controls, GV1300-GID1a-GFP recombinant plasmids were positive controls, and as a result, 7 transgenic Arabidopsis lines contained the desired band, which indicated that the GID1a gene had been successfully inserted into 7 Arabidopsis genomes containing the desired fragment (FIG. 1).
5. Plant height phenotype observation of transgenic positive Arabidopsis plants
By comparing the plant heights of the transgenic empty vector Arabidopsis thaliana, the wild type Arabidopsis thaliana and the transgenic Arabidopsis thaliana, the plant heights of the wild type Arabidopsis thaliana and the transgenic empty vector Arabidopsis thaliana are not significantly different, compared with a control, the plant heights of the over-expressed IsGID a gene Arabidopsis thaliana are significantly increased, and the plant heights of 3 transgenic lines are respectively increased by 16.01 percent, 16.43 percent and 17.67 percent compared with the transgenic empty vector Arabidopsis thaliana, which indicates that the over-expression of the IsGID a gene in the Arabidopsis thaliana can promote the growth of the plant heights.
Claims (7)
1. The gene sequence of the GID1a protein of the rabdosia lophanthide is shown as SEQ ID NO. 1.
2. The gene for encoding the GID1a protein of the brook fungus as defined in claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.
3. Biological material related to the GID1a protein of the group of the stick fungus as claimed in claim 1, characterized by comprising any one of the following (A1) to (A2):
(A1) A recombinant plant over-expression vector containing a gene for encoding the GID1a protein of the linearis;
(A2) A bioengineering bacterium comprising the recombinant plant overexpression vector of (A1).
4. A biological material related to the GID1a protein of the group of the phoenix fungus according to claim 3, wherein:
the plant expression vector is pCAMBIA1300-GFP.
5. A biological material related to the GID1a protein of the group of the phoenix fungus according to claim 3, wherein:
the bioengineering bacteria is agrobacterium GV3101.
6. The use of the biological material related to the GID1a protein of the flexible substrate web of the river fungus as defined in any one of claims 3-5 for controlling the increase of plant height, wherein the recombinant plant over-expression vector containing the gene for encoding the GID1a protein of the river fungus is introduced into the Arabidopsis thaliana.
7. The use according to claim 6, characterized in that the plant tissue is transformed by using agrobacterium mediation and the transformed plant tissue is cultivated into a plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311264030.9A CN117069815B (en) | 2023-09-28 | 2023-09-28 | Application of GID1a protein of rabdosia lophanthide in plant increase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311264030.9A CN117069815B (en) | 2023-09-28 | 2023-09-28 | Application of GID1a protein of rabdosia lophanthide in plant increase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117069815A CN117069815A (en) | 2023-11-17 |
| CN117069815B true CN117069815B (en) | 2024-06-25 |
Family
ID=88702657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311264030.9A Active CN117069815B (en) | 2023-09-28 | 2023-09-28 | Application of GID1a protein of rabdosia lophanthide in plant increase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117069815B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101248178A (en) * | 2005-04-14 | 2008-08-20 | 国立大学法人名古屋大学 | Gene capable of controlling differentiation/growth of plant, and use of the same |
| CN107475263A (en) * | 2017-09-14 | 2017-12-15 | 东北林业大学 | Participation plant forms build up the white birch SPL2 genes and its albumen with flower development |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3044152A1 (en) * | 2018-05-29 | 2019-11-29 | Agrisoma Biosciences Inc. | Brassica carinata producing seed with reduced glucosinolate content |
| CN109651493B (en) * | 2018-12-04 | 2022-06-17 | 重庆市农业科学院 | Gibberellin receptor protein, transgenic tobacco thereof and application of gibberellin receptor protein in tobacco |
| CN109819891A (en) * | 2019-02-26 | 2019-05-31 | 黑龙江省科学院自然与生态研究所 | Small stream sweet-smelling grass tissue-cultured seedling60Co- gamma Rays method for mutation breeding |
| CN116655758B (en) * | 2023-04-25 | 2024-06-07 | 东北林业大学 | IlMYB5 protein, and coding gene and application thereof |
-
2023
- 2023-09-28 CN CN202311264030.9A patent/CN117069815B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101248178A (en) * | 2005-04-14 | 2008-08-20 | 国立大学法人名古屋大学 | Gene capable of controlling differentiation/growth of plant, and use of the same |
| CN107475263A (en) * | 2017-09-14 | 2017-12-15 | 东北林业大学 | Participation plant forms build up the white birch SPL2 genes and its albumen with flower development |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117069815A (en) | 2023-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112626080B (en) | R gene for controlling soybean-rhizobium matching property, protein and application thereof | |
| CN110643618A (en) | Jatropha curcas MYB transcription factor JcMYB16 gene and application thereof in improving drought resistance of plants | |
| CN112322628A (en) | Transcription factor GhWRKY1-like gene for regulating and controlling verticillium wilt and drought resistance of cotton and application thereof | |
| CN102181456B (en) | Cotton flowering hormone GhFT and vector, construct, cell and polypeptide thereof | |
| CN110358772B (en) | OsEBP89 gene for improving abiotic stress resistance of rice, and preparation method and application thereof | |
| CN113621625B (en) | Application of sesame SiERF103 gene in enhancing plant resistance | |
| CN102796760A (en) | Application of zinc finger protein gene in adjusting paddy rice root growth | |
| CN109096380B (en) | Application of OsBICs gene in regulation and control of plant height and flowering time | |
| CN117069815B (en) | Application of GID1a protein of rabdosia lophanthide in plant increase | |
| CN116497042A (en) | ANR gene clone of swallow flower and application thereof | |
| CN109207487B (en) | Rape stain-resistant gene BnalPP1, and preparation method and application thereof | |
| CN107177565A (en) | Anti- tomato stem wilt gene mLCB2b and its application | |
| CN114085854A (en) | Rice drought-resistant and salt-tolerant gene OsSKL2 and application thereof | |
| WO2013010368A1 (en) | Use of rice aerenchyma formation key gene oslsd2 | |
| CN113444740B (en) | Recombinant vector capable of improving soybean aphid resistance gene and application thereof | |
| CN113151293B (en) | Stress-resistant gene line AcDwEm and application thereof in improving salt resistance, drought resistance and high temperature resistance of crops | |
| CN104498489A (en) | Coriander flower symmetry gene CsCYC2, and plant expression vector and building method thereof | |
| CN117209581B (en) | Application of GAI protein of herba lophatheri in plant dwarfing | |
| CN116535478B (en) | Swallow flower MYB4 protein and application thereof in color regulation and control | |
| CN116375835B (en) | Application of Yan flower MYB4b protein in regulation and control of plant leaf morphology | |
| CN113150088B (en) | Efficient stress-resistant module SyDcw capable of intelligently responding to stress signals and application of efficient stress-resistant module SyDcw in crop breeding | |
| CN110183524B (en) | Gene GmKRP2a for promoting soybean main root elongation, protein and application thereof | |
| CN111850031B (en) | Application of protein GmFULc in regulation and control of plant yield | |
| CN116218903A (en) | Application of cotton light signal regulating factor GhSPA1 and coding gene thereof in cotton | |
| CN117447568A (en) | Application of EsDREB2B gene and protein of Sophora angustifolia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |