CN111018959A

CN111018959A - Application of BMDR protein and its encoding gene in regulating plant drought resistance

Info

Publication number: CN111018959A
Application number: CN201911423838.0A
Authority: CN
Inventors: 巩志忠; 王瑜; 綦元鹏
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-17
Anticipated expiration: 2039-12-31
Also published as: CN111018959B

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of a BMDR protein and a coding gene thereof in regulating and controlling plant drought resistance. The invention finds that the BMDR gene of the corn positively regulates the drought resistance of the plant, and can effectively improve the drought resistance of the plant by improving the expression quantity of the BMDR gene. The BMDR overexpression transgenic corn plant is constructed, the growth condition of the plant under the drought condition is obviously superior to that of a wild corn plant, and the leaf wilting degree is obviously reduced; and the transpiration rate and the stomatal conductance are both obviously lower than those of wild corn plants, so that the water loss of the plants is effectively reduced, and the drought resistance is obviously improved. The discovery of the drought-resistant function of the BMDR provides a new gene target and resource for cultivating new varieties of drought-resistant plants, and provides a theoretical basis for clarifying the molecular mechanism of small peptides in plant drought stress signal response.

Description

Application of BMDR protein and coding gene thereof in regulating and controlling plant drought resistance

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of a BMDR protein and a coding gene thereof in regulating and controlling plant drought resistance.

Background

Drought is one of the important environmental factors that restrict agricultural development. As one of the three major food crops, corn grows in tropical areas with high temperature and humidity initially, and has high water demand, so that the drought resistance is low. In addition to pest stress in biotic stress, drought stress causes the most severe losses to crops such as maize, which dominates all abiotic stresses. The cultivation of new drought-resistant varieties is an effective means for improving the yield and quality of crops under drought conditions. Hybridization and high-quality character screening have the obvious advantages of stability, safety and the like as the traditional breeding mode, but the breeding period is longer. With the development of molecular biology, the improvement of plant stress resistance by modifying genome through genetic engineering means has become one of the development directions of molecular breeding. The gene participating in the plant metabolism or stress response process is found and is subjected to overexpression or mutation to improve the stress resistance of crops, so that the gene is a common means for molecular breeding and has important significance for improving the yield and quality of crops under the adverse circumstances. With the continuous development of corn resources, more inbred lines with high transformation efficiency are applied to genetic improvement, technical guarantee is provided for developing high-quality new corn varieties, and the improvement of the yield and the quality of the corn under the adverse circumstances is facilitated.

Cell-to-cell communication plays a crucial role in many processes in the life cycle of plants. Besides phytohormones and second messengers, plant small peptides can also participate in signal transmission among plant cells, thereby influencing the growth, development and stress resistance of plants. The plant small peptide has about 10-80 amino acids, is rich in cysteine, is secreted to the outside of cells to play a role, and needs to be subjected to posttranslational modification. Classical plant small peptides include systemin in tomato that regulate repair of plant injury, CLVATA3 that affect the development of arabidopsis apical meristem, and EPFs that affect the development of arabidopsis stomata. In recent years, an article reports that CLE family genes can influence the drought resistance of arabidopsis thaliana, and the CLEs family genes encode a class of plant secretory small peptides. The function of most plant small peptides is still unknown at present.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide the application of the BMDR protein and the coding gene thereof in regulating and controlling the drought resistance of plants.

The invention obtains more than 5000 transgenic corn strains by transgenically over-expressing more than 1000 genes in the corn, performs drought treatment on the plants and observes a drought-resistant phenotype, wherein the corn plants over-expressing BMDR genes have excellent drought-resistant performance. The BMDR gene positively regulates and controls the drought resistance of corn, the expression quantity of the BMDR gene is improved through transgenic overexpression, and the drought resistance of plants can be obviously enhanced.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides the use of a BMDR protein, a gene encoding it or a biological material comprising a gene encoding it for modulating drought resistance in a plant.

In a second aspect, the present invention provides the use of a BMDR protein, a gene encoding it or a biological material comprising a gene encoding it for modulating the growth and/or survival of plants under drought conditions.

In a third aspect, the present invention provides the use of a BMDR protein, a gene encoding it or a biological material comprising a gene encoding it for modulating plant transpiration rate or stomatal conductance.

In a fourth aspect, the present invention provides the use of a BMDR protein, a gene encoding it or a biological material comprising a gene encoding it in drought resistance genetic breeding of plants.

In a fifth aspect, the invention provides the use of a BMDR protein, a gene encoding the same, or a biological material comprising the gene encoding the same for the improvement of plant germplasm resources.

Preferably, in the above application, the drought resistance of the plant is improved, or the growth and/or survival rate of the plant under drought conditions is improved, by increasing the expression level and/or activity of the BMDR protein in the plant.

Increasing the expression level of said BMDR protein in said plant can be achieved by overexpressing a gene encoding said BMDR protein in said plant.

In the present invention, the BMDR protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) an amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No. 1; preferably, the homology is at least 90%; more preferably 95%.

The amino acid sequence shown as SEQ ID No.1 is the amino acid sequence of the BMDR protein of corn, and a person skilled in the art can obtain the BMDR protein mutant with the same function as the amino acid sequence shown as SEQ ID No.1 by substituting, deleting and/or adding one or more amino acids on the premise of not influencing the activity according to the amino acid sequence shown as SEQ ID No.1, conservative substitution of the amino acids and other conventional technical means in the field.

In the present invention, the encoding gene of the BMDR protein has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) the coding nucleotide sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2;

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown as SEQ ID NO.2 under strict conditions.

The nucleotide sequence shown as SEQ ID NO.2 is the sequence of the BMDR gene in the B73 type corn. The number in the maize genomic database is Zm00001d030748(V3 version gene number AC210816.3_ FG 006). The maize BMDR gene consists of 1464 bases and is in frame from the 601 st base to the 864 th base of the 5' end, wherein no intron is present. Because of natural variation of genotypes of different inbred lines of corn, BMDR genes of different inbred lines are in the protection scope of the patent. All nucleotide sequences encoding the BMDR protein are within the scope of the present invention in view of codon degeneracy.

In the invention, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

The invention provides a cloning vector or various expression vectors containing the encoding gene of the BMDR protein. The invention also provides a host cell containing the vector, a transformed plant cell or a transgenic plant containing the encoding gene of the BMDR protein.

Specifically, the expression vector can be a pBCXUN vector (the pBCXUN vector takes a commercial vector pCAMBIA1300 as a framework, the hygromycin resistance gene hpt in the pBCXUN vector is replaced by a herbicide resistance gene barM, and meanwhile, the promoter of the maize ubiquitin gene Ubi is cloned to the vector in an enzyme digestion connection mode to drive the transcription of a downstream overexpression gene).

In a sixth aspect, the invention provides a method for breeding drought-resistant corn, which comprises the following steps: improving the expression quantity and/or activity of BMDR protein in corn by transgenic, hybridization, backcross, self-crossing or asexual propagation; the BMDR protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

Preferably, the transgene is a transgenic maize line obtained by introducing into maize an overexpression vector comprising the gene encoding the BMDR protein.

As an embodiment of the invention, the breeding method of the drought-resistant corn comprises the following steps:

(1) extracting total RNA of corn, carrying out reverse transcription to obtain cDNA, amplifying BMDR gene by taking the cDNA as a template and taking a sequence shown as SEQ ID NO.3-4 as a primer, and connecting an amplification product to a plant expression vector pBCXUN to obtain a recombinant expression vector;

(2) transforming agrobacterium with the recombinant expression vector obtained in the step (1) to obtain recombinant agrobacterium;

(3) infecting the young corn embryo with the recombinant agrobacterium obtained in the step (2) to obtain a transformed seedling, and screening positive plants by herbicide screening and PCR identification;

by utilizing the method, the transgenic corn plant with over-expressed BMDR is obtained, and the drought resistance of the plant is obviously improved compared with that of wild corn.

In the present invention, the plant is a monocotyledon or a dicotyledon. Such plants include, but are not limited to, corn, rice, wheat, cotton, or soybean. Preferably, the monocotyledonous plant is a gramineous plant. More preferably corn.

The invention has the beneficial effects that: the invention finds that the BMDR gene of the corn positively regulates the drought resistance of the plant, and can effectively improve the drought resistance of the plant by improving the expression quantity of the BMDR gene. The BMDR overexpression transgenic corn plant is constructed, the growth condition of the plant under the drought condition is obviously superior to that of a wild corn plant, and the leaf wilting degree is obviously reduced; and the transpiration rate and the stomatal conductance are both obviously lower than those of wild corn plants (respectively reduced by 16.1 and 15.4%), the water loss of the plants is effectively reduced, and the drought resistance of the plants is obviously improved. The discovery of the drought-resistant function of the BMDR provides a new gene target and resource for cultivating new varieties of drought-resistant plants, and provides a theoretical basis for clarifying the molecular mechanism of small peptides in plant drought stress signal response. Compared with the traditional breeding mode, the breeding method of the drought-resistant plant provided by the invention has the advantages of short breeding time, strong purposiveness and the like, obviously shortens the breeding period and improves the breeding efficiency.

Drawings

FIG. 1 is a diagram showing the detection of the expression level of BMDR gene in a BMDR overexpressing maize strain in example 2 of the present invention; wherein WT represents a wild-type maize plant and BMDR OE represents a BMDR gene over-expressed maize line.

FIG. 2 shows the growth of a BMDR over-expressed corn strain after drought treatment in example 3 of the present invention; wherein WT represents a wild-type maize plant, and BMDR OE represents a BMDR gene over-expressed maize strain; the left and right pictures are respectively front view and top view pictures, wherein the two pots near the left are wild type corn plants, and the two pots near the right are BMDR gene overexpression corn strains.

FIG. 3 shows the transpiration rate and stomatal conductance measurements of a BMDR overexpressing maize strain in example 4 of the present invention; wherein A is transpiration rate, and B is porosity conductance; WT stands for wild-type maize plant and BMDR OE stands for BMDR gene over-expressed maize line.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The wild-type maize used in the following examples was maize inbred line B73; the agrobacterium strain is EHA 105; the main reagents used included: restriction enzymes, DNA polymerases, T4 ligases, etc. from biological companies such as NEB and Toyobo; reverse transcription kit from Thermo corporation; RNA extraction kit from magenta; quantitative PCR reagents of Taraka corporation; the plasmid extraction kit and the DNA recovery kit are purchased from Tiangen corporation; reagents such as MS culture medium, agar powder, agarose, ampicillin, kanamycin, gentamicin sulfate, rifampicin and other antibiotics are purchased from Sigma company; the various other chemical reagents used in the examples were all imported or domestic analytical reagents; primer synthesis and sequencing was performed by handsome, meiji corporation.

Example 1 construction and identification of BMDR Gene overexpression vector

In this example, an overexpression vector of BMDR gene was constructed by the following specific method:

extracting total RNA of corn (Zea mays L.), carrying out reverse transcription to obtain cDNA, amplifying BMDR gene by using the cDNA as a template and F and R as primers, wherein the primers are provided with enzyme cutting sites and are connected to an over-expression vector after enzyme cutting.

(1) The total RNA of the corn is extracted by using an RNA extraction kit of magenta company, and the specific steps refer to the kit instruction.

(2) The RNA was reverse transcribed to give cDNA using a reverse transcription kit from Thermo, and the detailed procedures were as described in the kit's instructions.

(3) And (3) amplifying BMDR gene cDNA by taking the cDNA as a template and F and R as primers, running electrophoresis on an amplification product, cutting gel and recovering, wherein the recovery method refers to a reagent kit of Tiangen corporation.

The sequences of primers used for amplifying the BMDR gene are as follows:

SEQ ID NO. 3: an upstream primer F: GCTCTAGAATGAACATCAACGCGAAC, respectively;

SEQ ID NO. 4: a downstream primer R: CCATCGATCATCTTGGCGAAGGAGACGG are provided.

(4) The recovered BMDR gene cDNA and pBCXUN vector (the pBCXUN vector uses commercial vector pCAMBIA1300 as a framework, and the hygromycin resistance gene hpt in the pBCXUN vector is replaced by herbicide resistance gene bare M; simultaneously, the promoter of the maize ubiquitin gene Ubi is cloned on the vector by enzyme digestion connection to drive the transcription of downstream overexpression gene) are subjected to double enzyme digestion by Xba I and Cla I, and the enzyme digestion product is subjected to electrophoresis and gel cutting for recovery; the recovered product was ligated with T4 ligase to join the BMDR gene to the pBCXUN vector, driving the expression of the BMDR gene with Ubi promoter.

(5) Taking 5 mu L of the product of the enzyme digestion-connection system in the step (4), transforming the competence of escherichia coli, screening on an LB (Langmuir-Blodgett) plate containing 50 mu g/mL kanamycin, identifying a monoclonal by colony PCR (polymerase chain reaction), and selecting a positive clone for sequencing; the obtained recombinant expression vector with correct sequencing is named pBCXUN-BMDR.

The primer sequences for colony PCR identification and sequencing are as follows:

SEQ ID NO.5：UbiP-seq:TTTTAGCCCTGCCTTCATACGC；

SEQ ID NO.6：NosR-seq:AGACCGGCAACAGGATTCAATC。

example 2 construction and detection of maize overexpressing the BMDR Gene

The pBCXUN-BMDR overexpression plasmid constructed in example 1 was transformed into competent Agrobacterium EHA105 strain by heat shock method, and positive clones were identified by colony PCR. Single colonies of Agrobacterium identified as correct were inoculated into 2-3mL liquid cultures containing 100. mu.g/mL kanamycin and 50. mu.g/mL rifampicinIn the medium, shaking culture is carried out overnight at 28 ℃, the cells are transferred to a liquid medium containing a large amount of antibiotics for shaking culture in the next day, the cells are collected after being transferred for several times, and the cells are resuspended to OD₆₀₀Between 0.8 and 1.0. And infecting the young B73 corn embryo picked out under aseptic condition with the obtained recombinant agrobacterium suspension, and inducing the young corn embryo to callus and grow into seedlings. Screening positive transgenic plants by adopting herbicide screening and PCR identification. The positive transgenic plant is self-bred to obtain T3 generation for subsequent experiment.

The RNA of 4 transgenic T3 generation inbred lines for transforming pBCXUN-BMDR overexpression plasmids is extracted, cDNA is reversely transcribed, the quantitative PCR detection of the transgenic overexpression condition proves that 4 lines are BMDR gene overexpression lines, the quantitative PCR detection result of one transgenic inbred line is shown in figure 1, and the BMDR gene expression amount in the transgenic plants is about 20 times of that of wild type control plants which are not transgenic and is far higher than that of the control plants.

Example 3 maize drought treatment phenotype detection of BMDR Gene overexpression

A drought treatment experiment is carried out on the BMDR gene overexpression corn strain T3 generation constructed in the example 2, and the specific method is as follows:

adding 140g of soil into each small pot, adding water into a tray, putting 4 seeds into each small pot, covering 50ml of soil, pouring the residual water in the tray after full water absorption, culturing in a greenhouse at 25 ℃, removing one seedling with irregular growth after about three days after seedling emergence, adding 1L of water into the tray, pouring the water after full water absorption, starting drought treatment (continuously culturing in the greenhouse at 25 ℃, no water is supplied during the culturing process), treating the seedlings for about one week, and observing the phenotypes of wild plants, transgenic plants, such as growth, leaf wilting degree and the like under the drought treatment condition. Wild type and transgenic plants, 3 pots each, were used as biological replicates. The results are shown in fig. 2, the growth conditions of the maize plants over-expressing BMDR are obviously better than the control, and the leaf wilting degree is obviously lower than the control, which indicates that the drought resistance of the transgenic plants is obviously improved compared with the control.

Example 4 measurement of transpiration Rate and stomatal conductance of maize overexpressing the BMDR Gene

The transpiration rate and stomatal conductance of the BMDR gene over-expression corn strain constructed in the example 2 are measured by the following specific method:

and respectively planting the single wild type control and over-expression BMDR corn plants in a big barrel, growing to 7-8 leaf stages, and measuring the indexes such as leaf transpiration rate, stomatal conductance and the like. The measuring instrument is LI-6400XT of LI-COR company, and the using method refers to the using instruction of the product. The results are shown in fig. 3, compared with the wild type control, the transpiration rate and the stomatal conductance of the over-expressed BMDR corn plant are respectively reduced by 16.1 and 15.4%, and it can be seen that the transpiration rate and the stomatal conductance of the over-expressed BMDR corn plant are significantly lower than those of the control, which indicates that the water loss of the over-expressed BMDR corn plant in a transpiration manner is obviously slower than that of the control, and this may be one of the reasons for drought resistance of the over-expressed plant.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. Application of BMDR protein, its encoding gene or biological material containing its encoding gene in regulating drought resistance of plants.

2. Application of BMDR protein, its encoding gene or biological material containing its encoding gene in regulating the growth and/or survival rate of plants under drought conditions.

3. The application of BMDR protein, its encoding gene or biological material containing its encoding gene in regulating plant transpiration rate or stomatal conductance.

4. Application of BMDR protein, its encoding gene or biological material containing its encoding gene in genetic breeding of plant drought resistance.

5. Application of BMDR protein, its encoding gene or biological material containing its encoding gene in the improvement of plant germplasm resources.

The use according to any one of claims 1 to 5, characterized in that, by increasing the expression and/or activity of the BMDR protein in the plant, the drought resistance of the plant is improved, or the Growth and/or survival of plants under drought conditions.

7. The application according to any one of claims 1 to 6, wherein the BMDR protein has any of the following amino acid sequences:

(1) amino acid sequence as shown in SEQ ID NO.1;

(2) the amino acid sequence of the protein with the same function obtained by the replacement, insertion or deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO.1;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably 95%.

8. The use according to any one of claims 1 to 7, wherein the biological material is an expression cassette, a vector, a host cell or a recombinant bacteria.

9. A breeding method for drought-resistant corn, characterized in that, by the method of transgenic, hybridization, backcrossing, self-crossing or vegetative propagation, the expression amount and/or activity of BMDR protein in corn is improved;

The BMDR protein has any of the following amino acid sequences:

(1) amino acid sequence as shown in SEQ ID NO.1;

10 . The method according to claim 9 , wherein the transgenic method comprises introducing an overexpression vector comprising the encoding gene of the BMDR protein into maize to obtain a transgenic maize line. 11 .