Application of GmXTH91 protein in regulation and control of plant stress resistance and plant height
阅读说明：本技术 GmXTH91蛋白在调控植物抗逆性和株高中的应用 (Application of GmXTH91 protein in regulation and control of plant stress resistance and plant height ) 是由 姜振峰 *** 李文滨 曹晓东 于 2019-11-28 设计创作，主要内容包括：本发明公开了GmXTH91蛋白在调控植物抗逆性和株高中的应用,GmXTH91蛋白的氨基酸序列如SEQ ID No：1所示。实验证明,在野生型拟南芥中表达GmXTH91基因可以降低拟南芥的抗旱性,抗旱性降低表现为种子萌发率降低和根长减小；在东农50中表达GmXTH91基因可以降低大豆的抗旱性,抗旱性降低表现为种子萌发率降低。GmXTH91蛋白可以调控植物的抗旱性和/或株高。本发明具有重要的应用价值。(The invention discloses an application of GmXTH91 protein in regulating and controlling plant stress resistance and plant height, wherein the amino acid sequence of the GmXTH91 protein is shown as SEQ ID No: 1 is shown. Experiments prove that the drought resistance of arabidopsis can be reduced by expressing the GmXTH91 gene in wild arabidopsis, and the reduction of the drought resistance is represented by reduction of seed germination rate and root length; the GmXTH91 gene expressed in Dongnong 50 can reduce the drought resistance of soybean, and the reduction of the drought resistance is expressed as the reduction of the seed germination rate. The GmXTH91 protein can regulate the drought resistance and/or plant height of plants. The invention has important application value.)
The application of GmXTH91 protein is S1) or S2) or S3) or S4):
s1) regulating and controlling the stress resistance of the plants;
s2) cultivating transgenic plants with changed stress resistance;
s3) regulating and controlling the plant height of the plant;
s4) cultivating the transgenic plant with the changed plant height.
2. The use of claim 1, wherein: the GmXTH91 protein is a1) or a2) or a 3):
a1) the amino acid sequence is SEQ ID No: 1;
a2) in SEQ ID No: 1, the N end or/and the C end of the protein shown in the formula 1 is connected with a label to obtain a fusion protein;
a3) the sequence shown in SEQ ID No: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and the protein related to plant stress resistance and/or plant height is obtained.
3. Use of a nucleic acid molecule encoding the GmXTH91 protein of claim 1 or 2, being S1) or S2) or S3) or S4):
s1) regulating and controlling the stress resistance of the plants;
s2) cultivating transgenic plants with changed stress resistance;
s3) regulating and controlling the plant height of the plant;
s4) cultivating the transgenic plant with the changed plant height.
4. Use according to claim 3, characterized in that: the nucleic acid molecule encoding the GmXTH91 protein is a DNA molecule shown in the following b1) or b2) or b3) or b 4):
b1) the coding region is SEQ ID No: 3, a DNA molecule shown in seq id no;
b2) the nucleotide sequence is SEQ ID No: 3, a DNA molecule shown in seq id no;
b3) the nucleotide sequence is SEQ ID No: 2;
b4) a DNA molecule having 75% or more identity with the nucleotide sequence defined by b1) or b2) or b3) and encoding the GmXTH91 protein of claim 1 or 2;
b5) a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) and which encodes a GmXTH91 protein as claimed in claim 1 or 2.
5. Use according to any one of claims 1 to 4, wherein:
the regulation and control of the plant stress resistance is to reduce the plant stress resistance;
the transgenic plant with the changed stress resistance is cultured with the reduced stress resistance;
the plant height of the plant is regulated and controlled to be increased;
the transgenic plant with the changed plant height is cultured to be the transgenic plant with the increased plant height.
6. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the GmXTH91 protein of claim 1 or 2 in a starting plant to obtain a transgenic plant; the transgenic plants have reduced stress resistance and/or increased plant height compared to the starting plants.
7. The method of claim 7, wherein: the "increasing the expression level and/or activity of the GmXTH91 protein in the starting plant" is effected by introducing a nucleic acid molecule encoding the GmXTH91 protein into the starting plant.
8. A method of plant breeding comprising the steps of: increasing the content and/or activity of the GmXTH91 protein according to claim 1 or 2 in a plant, thereby reducing stress resistance and/or increasing plant height.
9. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8, wherein: the stress resistance is drought resistance.
10. The use of claim 1, 2, 3, 4, 5 or 9 or the method of any one of claims 6 to 9, wherein: the plant is any one of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) soybean; c5) dongnong 50 soybean variety; c6) a cruciferous plant; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of GmXTH91 protein in regulation and control of stress resistance and plant height of plants.
Background
Under the stress of adversity, a series of response reactions are generated in plants, and a plurality of physiological, biochemical and developmental changes are accompanied. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance is gradually deepened to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the plant growth characteristics can be improved by utilizing biotechnology, and the adaptability of plants to stress is further improved.
Under the adverse conditions of environmental stresses such as drought, high salinity, nutrient deficiency and the like, the plant can be correspondingly adjusted on molecular, cellular and overall levels so as to reduce the damage caused by the environment to the maximum extent and survive. Many genes are induced to express by stress, and the products of the genes not only can be directly involved in the stress response of plants, but also can regulate the expression of other related genes or be involved in signal transduction pathways, so that the plants can avoid or reduce damage, and the resistance to the stress environment is enhanced.
Adversity stresses such as drought, high salinity and low temperature are barrier factors affecting plant growth and development. Therefore, understanding the plant response to stress conditions and the signal transduction mechanism, thereby improving the stress resistance of plant varieties, becomes one of the important tasks of plant genetic research and plant variety improvement.
The plant height of the crops is an important agronomic trait. The crop stalks with dwarf characters have strong lodging resistance and high efficiency of utilizing light energy by groups. By shaping the dwarf variety of an ideal plant type and carrying out reasonable close planting, the single yield level of domestic soybean is expected to be broken through. The plant height of soybean is generally determined by the variety itself, and may be influenced by factors such as cultivation conditions.
Disclosure of Invention
The invention aims to regulate and control the stress resistance (such as drought resistance) and/or plant height of plants.
The invention firstly protects the application of GmXTH91 protein, which can be S1) or S2) or S3) or S4):
s1) regulating and controlling the stress resistance of the plants;
s2) cultivating transgenic plants with changed stress resistance;
s3) regulating and controlling the plant height of the plant;
s4) cultivating the transgenic plant with the changed plant height.
In the above application, the GmXTH91 protein can be a1) or a2) or a 3):
a1) the amino acid sequence is SEQ ID No: 1;
a2) in SEQ ID No: 1, the N end or/and the C end of the protein shown in the formula 1 is connected with a label to obtain a fusion protein;
a3) the sequence shown in SEQ ID No: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and the protein related to plant stress resistance and/or plant height is obtained.
Wherein, SEQ ID No: 1 consists of 316 amino acid residues.
To facilitate purification of the protein of a1), the protein of SEQ ID No: 1 to which the tags shown in Table 1 were attached at the amino terminus or the carboxyl terminus.
TABLE 1 sequence of tags
Label (R)
Residue of
Sequence of
Poly-Arg
5-6 (typically 5)
FLAG
8
DYKDDDDK
Strep-tag II
8
WSHPQFEK
c-
10
EQKLISEEDL
The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.
The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.
The gene encoding the protein of a3) above can be obtained by converting the amino acid sequence of SEQ ID No: 3, and/or is missense mutated by one or more base pairs, and/or is obtained by linking the coding sequence of the tag shown in table 1 above at its 5 'end and/or 3' end.
The invention also protects the application of the nucleic acid molecule for coding the GmXTH91 protein, which can be S1) or S2) or S3) or S4):
s1) regulating and controlling the stress resistance of the plants;
s2) cultivating transgenic plants with changed stress resistance;
s3) regulating and controlling the plant height of the plant;
s4) cultivating the transgenic plant with the changed plant height.
In the above application, the nucleic acid molecule encoding GmXTH91 protein can be a DNA molecule represented by b1) or b2) or b3) or b4) as follows:
b1) the coding region is SEQ ID No: 3, a DNA molecule shown in seq id no;
b2) the nucleotide sequence is SEQ ID No: 3, a DNA molecule shown in seq id no;
b3) the nucleotide sequence is SEQ ID No: 2;
b4) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined by b1) or b2) or b3) and encoding said GmXTH91 protein;
b5) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) or b3) under strict conditions and codes the GmXTH91 protein.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
Wherein, SEQ ID No: 3 consists of 951 nucleotides, SEQ ID No: 3 encodes the nucleotide sequence of SEQ ID No: 1.
The nucleotide sequence of the GmXTH91 protein of the invention encoding can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of the GmXTH91 protein isolated according to the invention, as long as they encode the GmXTH91 protein, are derived from and identical to the nucleotide sequence of the invention.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID No: 1, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the GmXTH91 protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In any of the above applications, the regulating plant stress resistance may be reducing plant stress resistance.
In any of the above applications, the breeding of transgenic plants with altered stress resistance may be breeding of transgenic plants with reduced stress resistance.
In any of the above applications, the regulating the plant height may be increasing the plant height.
In any of the above applications, the transgenic plant with the altered plant height may be a transgenic plant with an increased plant height.
The invention also provides a method for cultivating transgenic plants, which comprises the following steps: increasing the expression quantity and/or activity of the GmXTH91 protein in the starting plant to obtain a transgenic plant; the transgenic plants have reduced stress resistance and/or increased plant height compared to the starting plants.
In the above method, the "increasing the expression level and/or activity of the GmXTH91 protein in the starting plant" can be achieved by a method known in the art, such as transgenosis, multicopy, and changing a promoter and a regulatory factor, so as to achieve the effect of increasing the expression level and/or activity of any of the GmXTH91 proteins in the starting plant.
In the above method, the "increasing the expression level and/or activity of the GmXTH91 protein in the starting plant" may be specifically achieved by introducing a nucleic acid molecule encoding the GmXTH91 protein into the starting plant.
In the above method, the "introducing into the starting plant the nucleic acid molecule encoding the GmXTH91 protein" may be achieved by introducing into the starting plant a recombinant vector; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the GmXTH91 protein into an expression vector.
The recombinant vector can be specifically a recombinant plasmid pCAMBIA3300-GmXTH 91. The recombinant plasmid pCAMBIA3300-GmXTH91 can be specifically obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes XbaI and BamHI of a pCAMBIA3300 expression vector with a DNA fragment shown in SEQ ID No: 2 to obtain the recombinant plasmid.
The transgenic plant may be specifically C1, C7 and C9 mentioned in example 2. At this time, the starting plant is Arabidopsis thaliana, specifically, the Columbia-0 subtype of wild type Arabidopsis thaliana.
The transgenic plants can be specifically
The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of the GmXTH91 protein in the plant, thereby reducing stress resistance and/or increasing plant height.
Any of the above stress resistance may be drought resistance.
Any of the above-described reduced stress resistance may be manifested as a reduced rate of seed germination and/or a reduced root length (starting plant is Arabidopsis thaliana).
Any of the above mentioned reduced stress resistance may be manifested as a reduced rate of seed germination (starting plant is soybean).
Any of the plants described above may be any of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) leguminous plants; c4) soybean; c5) dongnong 50 soybean variety; c6) a cruciferous plant; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.
Experiments prove that the drought resistance of arabidopsis can be reduced by expressing the GmXTH91 gene in wild arabidopsis, and the reduction of the drought resistance is represented by reduction of seed germination rate and root length; the GmXTH91 gene expressed in Dongnong 50 can reduce the drought resistance of soybean, and the reduction of the drought resistance is expressed as the reduction of the seed germination rate. The GmXTH91 protein can regulate the stress resistance (such as drought resistance) and/or plant height of plants. The invention has important application value.
Drawings
FIG. 1 is the effect of BGmXTH91 gene on Arabidopsis thaliana drought resistance in step five of example 2.
FIG. 2 shows the effect of the six GmXTH91 genes on the plant height of Arabidopsis thaliana in example 2.
FIG. 3 shows the real-time fluorescent quantitative detection T3The expression level of GmXTH91 gene in different soybean strains transformed with GmXTH91 gene by generation-homozygous transformation.
FIG. 4 is T3Protein level detection results of different strains of soybean with generation-homozygous GmXTH91 gene.
FIG. 5 shows the germination status of the seeds cultured for 24 hours according to the sixth step in example 3.
FIG. 6 shows the germination status of the seeds cultured for 48h according to step six in example 3.
FIG. 7 shows the germination status of the seeds cultured for 72 hours according to step six in example 3.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A geneticin between column responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004,36: 167-.
The pGM-T cloning vector, the pCAMBIA3300 expression vector, the Agrobacterium tumefaciens EHA105, the soybean variety Charleston, Dongnong 594 and Dongnong 50 are all provided by the Soybean science research institute of northeast agriculture university. Hereinafter, the soybean variety Charleston is abbreviated as Charleston, Dongnong is abbreviated as DN594, and Dongnong is abbreviated as DN 50.
The plant RNA extraction kit and the reverse transcription kit are both products of Tiangen Biotechnology (Beijing) Co. DNA Marker is a product of Shunhua Biotechnology, Inc. in Shanghai.