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
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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.
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