Application of corn 3-phosphoglycerol dehydrogenase ZmGPDH4 and coding gene thereof in regulation and control of plant stress tolerance
阅读说明：本技术 玉米3-磷酸甘油脱氢酶ZmGPDH4及其编码基因在调控植物耐逆性中的应用 (Application of corn 3-phosphoglycerol dehydrogenase ZmGPDH4 and coding gene thereof in regulation and control of plant stress tolerance ) 是由 徐晶宇 刘梦 李佐同 赵莹 贺琳 魏金鹏 赵长江 于 2019-11-13 设计创作，主要内容包括：本发明公开了玉米3-磷酸甘油脱氢酶ZmGPDH4及其编码基因在调控植物耐逆性中的应用。本发明以玉米GPDH基因家族成员ZmGPDH4为研究对象,将其转入拟南芥突变体获得T3代纯合转化子。选取其中COM-1和COM-2两个转化子进行抗病功能鉴定。以拟南芥突变体为对照,研究ZmGPDH4转基因拟南芥在病菌胁迫下的3-磷酸甘油、甘油含量及生理表型的变化。结果表明：在病菌Pst DC3000胁迫处理条件下,ZmGPDH4转基因拟南芥中3-磷酸甘油、甘油含量显著高于拟南芥突变体,且ZmGPDH4转基因拟南芥的感病情况显著优于对照。表明ZmGPDH4能显著提高转基因植物的抗病能力,ZmGPDH4作为耐逆基因可以被应用于培育玉米耐逆品种。(The invention discloses application of corn 3-phosphoglycerol dehydrogenase ZmGPDH4 and a coding gene thereof in regulating and controlling plant stress tolerance. The invention takes a corn GPDH gene family member ZmGPDH4 as a research object, and transfers the corn GPDH gene family member ZmGPDH4 into an arabidopsis thaliana mutant to obtain a T3 generation homozygous transformant. Two transformants of COM-1 and COM-2 are selected for disease resistance identification. The arabidopsis mutant is used as a control to research the change of 3-phosphoglycerol, glycerol content and physiological phenotype of ZmGPDH4 transgenic arabidopsis under the stress of pathogenic bacteria. The results show that: under the stress treatment condition of a pathogen Pst DC3000, the contents of 3-phosphoglycerol and glycerol in the ZmGPDH4 transgenic Arabidopsis are obviously higher than those of Arabidopsis mutants, and the infection condition of the ZmGPDH4 transgenic Arabidopsis is obviously better than that of a control. The ZmGPDH4 is shown to be capable of obviously improving the disease resistance of transgenic plants, and ZmGPDH4 as a stress tolerance gene can be applied to the cultivation of corn stress tolerance varieties.)
The application of ZmGPDH4 protein in regulating and controlling plant stress tolerance;
or, the ZmGPDH4 protein is used for regulating and controlling the content of 3-phosphoglycerol/glycerol in plants.
2. Use according to claim 1, characterized in that:
the ZmGPDH4 protein is a protein shown in a) or b) or c) or d) as follows:
a) the amino acid sequence is a protein shown in a sequence 2;
b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;
c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;
d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.
3. The application of biological materials related to ZmGPDH4 protein in regulating and controlling plant stress tolerance;
or, the use of a biomaterial related to the ZmGPDH4 protein for modulating the glycerol-3-phosphate/glycerol content in plants;
or, the use of a biological material related to the ZmGPDH4 protein for the cultivation of transgenic plants with increased stress tolerance.
4. Use according to claim 3, characterized in that:
the biomaterial is any one of the following A1) to A12):
A1) a nucleic acid molecule encoding a ZmGPDH4 protein;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising a4) said recombinant vector;
A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);
A10) a transgenic plant cell line comprising the expression cassette of a 2);
A11) a transgenic plant cell line comprising the recombinant vector of a 3);
A12) a transgenic plant cell line comprising the recombinant vector of a 4).
5. Use according to claim 4, characterized in that: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):
1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a sequence 1;
2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes ZmGPDH4 protein;
3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes for ZmGPDH4 protein.
6. Use according to any one of claims 1 to 5, characterized in that: the stress tolerance is disease resistance.
7. A method for producing a transgenic plant having improved stress tolerance, comprising the step of increasing the expression level and/or activity of ZmGPDH4 protein in a recipient plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance than the recipient plant.
8. The method of claim 7, wherein: the stress tolerance is disease resistance;
the transgenic plant has higher stress tolerance than the recipient plant is found in any one of the following (1) to (3):
(1) the content of 3-phosphoglycerol in the transgenic plant is higher than that in the receptor plant;
(2) the transgenic plant has a higher glycerol content than the recipient plant;
(3) the leaf infection area of the transgenic plant is smaller than that of the receptor plant.
9. The method according to claim 7 or 8, characterized in that: the method for improving the expression amount and/or activity of the ZmGPDH4 protein in the receptor plant comprises the steps of over-expressing the ZmGPDH4 protein in the receptor plant;
or, the overexpression method is to introduce the encoding gene of ZmGPDH4 protein into the receptor plant;
or, the nucleotide sequence of the encoding gene of the ZmGPDH4 protein is a DNA molecule shown in sequence 1.
10. The use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9, wherein: the plant is a monocotyledon or a dicotyledon.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of corn 3-phosphoglycerol dehydrogenase ZmGPDH4 and a coding gene thereof in regulation and control of plant stress tolerance.
Background
Improving the stress tolerance of crops has become a hotspot and a difficulty of technical research in the field of agriculture and animal husbandry at present, and is also a major problem to be solved urgently at present. In recent years, with the development of functional genomics and molecular biology, the development of stress tolerance key genes and the cultivation of new crop varieties with good stress tolerance characters by using genetic engineering technology have become one of means for effectively improving the stress tolerance of crops.
Glycerol-3-phosphate (Glycerol-3-phosphate/G-3-P) is an important intermediate product in the oil and fat metabolism process and is involved in various physiological and biochemical processes in plants. Glycerol-3-phosphate can be dephosphorylated to glycerol by glycerol-3-phosphate phosphorylase (GPP) and can also be used as a synthetic precursor for glycerols, including triacylglycerols, glycerophospholipids and glyceroglycolipids. Glycerol-3-phosphate Dehydrogenase (GPDH) catalyzes the reversible redox reaction between Glycerol-3-phosphate and dihydroxyacetone phosphate, and such enzymes are widely present in various organisms and have tissue-and cell-specific properties. In addition to the model plant Arabidopsis thaliana and some algae, few studies have been reported on the GPDH family in other higher plants, especially in maize.
Disclosure of Invention
The technical problem to be solved by the invention is how to regulate and control the stress tolerance of plants.
In order to solve the technical problems, the invention firstly provides a new application of the ZmGPDH4 protein.
The invention provides an application of ZmGPDH4 protein in regulation and control of plant stress tolerance.
The invention also provides application of the ZmGPDH4 protein in regulating and controlling the content of vegetable glycerol and 3-phosphoglycerol.
In the above application, the ZmGPDH4 protein is a protein shown in a) or b) or c) or d) as follows:
a) the amino acid sequence is a protein shown in a
b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the
c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the
d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the
In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the
TABLE 1 sequence of tags
Label (R)
Residue of
Sequence of
Poly-Arg
5-6 (typically 5)
RRRRR
Poly-His
2-10 (generally 6)
FLAG
8
DYKDDDDK
Strep-
8
WSHPQFEK
c-
10
EQKLISEEDL
The protein of c) above, 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 in the c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.
The gene encoding the protein of c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.
In the above d), "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence represented by the
In order to solve the technical problems, the invention also provides a new application of the biological material related to the ZmGPDH4 protein.
The invention provides application of biological materials related to ZmGPDH4 protein in regulation and control of plant stress tolerance.
The invention also provides application of the biological material related to the ZmGPDH4 protein in regulating and controlling the content of vegetable glycerol and 3-phosphoglycerol.
The invention also provides application of the biological material related to the ZmGPDH4 protein in culturing transgenic plants with improved stress tolerance.
The biomaterial is any one of the following A1) to A12):
A1) a nucleic acid molecule encoding a ZmGPDH4 protein;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising a4) said recombinant vector;
A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);
A10) a transgenic plant cell line comprising the expression cassette of a 2);
A11) a transgenic plant cell line comprising the recombinant vector of a 3);
A12) a transgenic plant cell line comprising the recombinant vector of a 4).
In the above application, the nucleic acid molecule of A1) is a gene as shown in 1) or 2) or 3) below:
1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a
2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes ZmGPDH4 protein;
3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes for ZmGPDH4 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.
The nucleotide sequence encoding ZmGPDH4 protein of the present invention 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 to have 75% or more identity to the nucleotide sequence encoding the ZmGPDH4 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the ZmGPDH4 protein and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
In the above application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and
In the above applications, the expression cassette containing a nucleic acid molecule encoding a ZmGPDH4 protein according to a2) refers to a DNA capable of expressing a ZmGPDH4 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of ZmGPDH4 but also a terminator that terminates transcription of ZmGPDH 4. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters.
The recombinant vector containing the ZmGPDH4 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.
In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.
In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.
In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.
In the above application, the stress tolerance is disease resistance.
In order to solve the above technical problems, the present invention finally provides a method for breeding a transgenic plant having improved stress tolerance.
The method for cultivating the transgenic plant with improved stress tolerance comprises the steps of improving the expression quantity and/or activity of ZmGPDH4 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has higher stress tolerance than the recipient plant.
Further, the stress tolerance is disease resistance.
The transgenic plant has higher stress tolerance than the recipient plant is found in any one of the following (1) to (3):
(1) the content of 3-phosphoglycerol in the transgenic plant is higher than that in the receptor plant;
(2) the transgenic plant has a higher glycerol content than the recipient plant;
(3) the infected area of the transgenic plant is smaller than that of the receptor plant.
Still further, the method for increasing the expression amount and/or activity of the ZmGPDH4 protein in the recipient plant is to express the ZmGPDH4 protein in the recipient plant; the expression method in the receptor plant is to introduce the encoding gene of ZmGPDH4 protein into the receptor plant; the nucleotide sequence of the encoding gene of the ZmGPDH4 protein is a DNA molecule shown in a
In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the ZmGPDH4 gene into a recipient plant, but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.
In the above method or application, the plant is a monocotyledon or a dicotyledon. The dicotyledonous plant can be specifically arabidopsis thaliana; the Arabidopsis thaliana can be specifically T-DNA insertion mutant chl-KO (SALK _062006) of Arabidopsis thaliana AtGPDHp1(AT5G 40610).
The invention takes a corn GPDH gene family member ZmGPDH4(GRMZM6G161711_ T01) as a research object, and transfers the research object into arabidopsis thaliana mutant chl-KO to obtain a T3 generation homozygous transformant. Two transformants of COM-1 and COM-2 are selected for disease resistance identification. The 3-phosphoglycerol, the glycerol content and the physiological phenotype change of the ZmGPDH4 transgenic Arabidopsis under the stress of the pathogen Pst DC3000 are researched by taking the arabidopsis mutant chl-KO as a control. The result shows that the content of glycerol-3-phosphate and glycerol of ZmGPDH4 transgenic Arabidopsis is obviously higher than that of the Arabidopsis mutant chl-KO under the DC3000 treatment condition, and the susceptibility of the transgenic line is obviously lower than that of the control. These results indicate that ZmGPDH4 can significantly improve the disease resistance of transgenic plants, and ZmGPDH4 as stress tolerance gene can be applied to the cultivation of maize stress tolerance varieties.
Drawings
FIG. 1 shows the expression pattern analysis of ZmGPDH4 gene.
FIG. 2 shows the glycerol-3-phosphate content of the third day treated Arabidopsis thaliana mutant (chl-KO) and transgenic Arabidopsis thaliana (COM-1 and COM-2) with Pst DC 3000.
FIG. 3 shows glycerol content of Arabidopsis thaliana mutant (chl-KO) and transgenic Arabidopsis thaliana (COM-1 and COM-2) on the third day of Pst DC3000 treatment.
FIG. 4 is a statistic of bacterial growth of Arabidopsis thaliana mutant (chl-KO) and transgenic Arabidopsis thaliana (COM-1 and COM-2) under PstDC3000 treatment.
FIG. 5 shows a comparison of the staining of Arabidopsis mutants (chl-KO) and transgenic Arabidopsis (COM-1 and COM-2) under Pst DC3000 treatment.
FIG. 6 shows the phenotypic differences between Arabidopsis mutants (chl-KO) and transgenic Arabidopsis (COM-1 and COM-2) under Pst DC3000 treatment.
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.
Statistical analysis of data in the following examples: statistical analysis was performed using SPSS 16.0(SPSS Inc, Chicago, IL, USA) software. One-way ANOVA compared whether the differences between the different strains (CK, COM-1 and COM-2) were significant under the same treatment conditions (P <0.05, one-way ANOVA).
The pBI121-GFP vector in the examples described below is a product of Shanghai Xinyu Biotechnology Ltd, catalog No. or Commodity No. XY 2117.
The EHA105 Agrobacterium in the examples described below is a product of Shanghai Diego biol, Inc. under catalog number or cat number AC 1010S.
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