CN109971766B

CN109971766B - Plant stress tolerance associated protein PwRBP1, and coding gene and application thereof

Info

Publication number: CN109971766B
Application number: CN201910244335.0A
Authority: CN
Inventors: 张凌云; 崔潇月; 张鹤华
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2021-05-25
Anticipated expiration: 2039-03-28
Also published as: CN109971766A

Abstract

The invention discloses an RNA binding protein PwRBP1 related to plant stress tolerance, and a coding gene and application thereof. The invention introduces PwRBP1 coding gene found in woody plant into Arabidopsis thaliana to obtain PwRBP1 Arabidopsis thaliana plant. Experiments prove that compared with receptor plants, the drought tolerance and salt tolerance of PwRBP1 Arabidopsis plants are obviously improved, and the PwRBP1 and the coding gene thereof have stress tolerance and are suitable for popularization and application.

Description

Plant stress tolerance associated protein PwRBP1, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an RNA binding protein PwRBP1 related to plant stress tolerance, and a coding gene and application thereof.

Background

Drought stress and salt stress are two major abiotic environmental stresses, which can cause osmotic damage, ionic damage, accumulation of active oxygen and toxic substances, etc., of plants, thereby seriously affecting various physiological and growth processes of plant seeds, such as germination, photosynthesis, plant growth and development, etc. Plants develop a series of effective action mechanisms in the long-term evolution process, wherein the effective action mechanisms comprise the regulation and the control of the expression of stress-related genes so as to deal with the external adverse environment.

RBP protein is an RNA-binding protein that has the ability to bind RNA in eukaryotes, and RNA-binding proteins play a key role in post-transcriptional level regulation, which can coordinate translation of related mrnas, much in the same way DNA-binding proteins coordinate expression of related DNA sequences. RBP proteins in plants are less studied than in other tissues due to the major lack of in vitro culture systems to study post-transcriptional level regulation. RNA binding protein can play an important role in the growth and development of organisms, coping with stresses such as low temperature, water logging, drought, high salt, high temperature and the like, and the research on the RBP gene and the plant stress resistance in model plants such as arabidopsis thaliana and rice is greatly advanced at present, but the research on the function of the RBP gene in woody plants is less reported. The present invention has been made based on this.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the stress tolerance of plants.

In order to solve the technical problems, the invention firstly provides a protein related to plant stress resistance.

The plant stress resistance related protein provided by the invention is named as PwRBP1, is derived from Picea wilsonii mask, and is a) or b) or c) or d) as follows:

a) a protein comprising the amino acid sequence shown in SEQ ID No. 2; in some embodiments, is a protein having an amino acid sequence as set forth in SEQ ID No. 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in SEQ ID NO. 2;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2;

d) and (b) a protein which has 75% or more than 75% homology with the amino acid sequence shown in SEQ ID NO.2 and has the same function.

Wherein SEQ ID NO.2 consists of 430 amino acid residues.

The label in b) may be a sequence label shown in Table 1, and the label can be connected to the amino terminal or the carboxyl terminal of the protein shown in SEQ ID NO.2 in the sequence table in order to facilitate the purification of the protein in a).

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein PwRBP1 in 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 PwRBP1 in c) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein PwRBP1 in c) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID NO.1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence for the tag shown in Table 1 above.

In order to solve the technical problems, the invention also provides a biological material related to the PwRBP1 protein.

The biological material related to the PwRBP1 protein provided by the invention is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding a PwRBP1 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 biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence of the DNA molecule is shown in SEQ ID NO.1, and in some embodiments, the DNA molecule is a cDNA molecule or a genomic DNA molecule;

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 PwRBP1 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 PwRBP1 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.1 consists of 1293 nucleotides and encodes an amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence encoding PwRBP1 of the present invention can be mutated by one of skill in the art using known experimental methods such as directed evolution and point mutation. Any artificially modified nucleotide having 75% or more identity to the nucleotide sequence of PwRBP1 isolated according to the present invention is derived from and identical to the nucleotide sequence of the present invention as long as it encodes PwRBP1 and has the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO. 2. 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.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding PwRBP1 (PwRBP1 gene expression cassette) described in A2) refers to a DNA capable of expressing PwRBP1 in a host cell, and the DNA may include not only a promoter which initiates transcription of PwRBP1 but also a terminator which terminates transcription of PwRBP 1. 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. Suitable transcription terminators include, but are not limited to: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator and the nopaline and octopine synthase terminators.

The recombinant vector containing the PwRBP1 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, pCAMBIA1205, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. When the PwRBP1 gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, can be added in front of the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, 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 proper 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 the transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes expressing an enzyme or a luminescent compound which produces a color change in plants (GUS gene, GFP gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin, chloramphenicol marker, etc.), or anti-chemical agent marker genes (e.g., anti-herbicide gene), etc. 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.

Primer sets for amplifying the full length of the PwRBP1 gene or any fragment thereof are also within the scope of the present invention.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. In the embodiment of the present invention, the recombinant vector may be specifically a recombinant expression vector obtained by inserting the above-mentioned PwRBP1 gene (SEQ ID NO.1) between BamHI and SmaI sites of pCAMBIA1205 vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. In the present example, the Agrobacterium used is GV 3101.

In the above biological material, none of the transgenic plant cell lines comprises propagation material.

In order to solve the technical problems, the invention also provides a new application of the PwRBP1 protein or the biological material.

The invention provides an application of PwRBP1 protein or the biological material in regulating and controlling plant stress tolerance.

The invention also provides the application of the PwRBP1 protein or the biological material in cultivating transgenic plants with improved or reduced stress tolerance.

The invention also provides the application of the PwRBP1 protein or the biological material in plant breeding.

In the application, the stress tolerance is salt tolerance and/or drought tolerance.

In the above application, the plant is a monocotyledon or a dicotyledon, and the dicotyledon can be a leguminous plant and/or a cruciferous plant and/or an asteraceae plant; the leguminous plant can be soybean, Lotus corniculatus, alfalfa or wampee; the cruciferous plant may be arabidopsis thaliana or brassica napus; the Compositae plant can be sunflower; the Arabidopsis thaliana may be Arabidopsis thaliana (Columbia ecotype col-0).

In order to solve the above technical problems, the present invention finally provides a method for breeding transgenic plants with improved stress resistance; in some embodiments, the improvement is an increase; in other embodiments, the improvement is a decrease.

The method for cultivating the transgenic plant with improved stress resistance comprises the steps of improving the expression quantity and/or activity of PwRBP1 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has higher tolerance to high salt and drought than the recipient plant.

In the above method, the method for increasing the expression level and/or activity of the PwRBP1 protein in the recipient plant is to express or overexpress the PwRBP1 protein in the recipient plant.

In the above method, the expression or overexpression is carried out by introducing a gene encoding a PwRBP1 protein into a recipient plant; the nucleotide sequence of the coding gene of the PwRBP1 protein is a DNA molecule shown in SEQ ID NO. 1.

In one assay method of the present invention, the gene encoding the PwRBP1 protein (i.e., the nucleotide sequence shown in SEQ ID NO.1) was introduced into Agrobacterium GV3101 via a recombinant vector pCAMBIA1205-PwRBP1 containing an expression cassette for the gene encoding the PwRBP1 protein. The recombinant vector pCAMBIA1205-PwRBP1 is obtained by inserting PwRBP1 shown in SEQ ID NO.1 in a sequence table between BamHI and SmaI enzyme cutting sites of an expression vector pCAMBIA1205 and keeping other sequences of the pCAMBIA1205 vector unchanged. The recombinant vector pCAMBIA1205-PwRBP1 expresses PwRBP1 protein.

The transgenic plant in the method has higher stress resistance than the wild receptor plant, and the specific expression of the transgenic plant in the method is that the following modes are generated under the stress of adversity: the transgenic plant has a greater seed germination rate and/or seedling root length and/or survival rate than the recipient plant under the stress of high salt concentration or high mannitol concentration. The high-salt environment may specifically be an environment caused by 100mM, 200mM NaCl aqueous solution; the drought environment can be specifically a drought environment obtained by simulating 200mM and 300mM mannitol aqueous solution, or a drought treatment environment in which watering is stopped for 11 days.

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by introducing the PwRBP1 gene into a recipient plant but also its progeny. 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, the recipient plant is a monocotyledon or dicotyledon, and the dicotyledon can be a plant of the family Leguminosae and/or a plant of the family Brassicaceae and/or a plant of the family Compositae; the leguminous plant can be soybean, Lotus corniculatus, alfalfa or wampee; the cruciferous plant may be arabidopsis thaliana or brassica napus; the Compositae plant can be sunflower; the Arabidopsis thaliana may be Arabidopsis thaliana (Col-0 ecotype Columbia).

The invention firstly discovers a new gene PwRBP1, then introduces the new gene PwRBP1 into Arabidopsis thaliana to obtain PwRBP1 Arabidopsis thaliana, and discovers that the tolerance capability of the PwRBP1 Arabidopsis thaliana to high salt and drought is higher than that of the original receptor plant. The concrete expression is as follows: under the stress of NaCl with the concentration of 100mM, the seed germination rate and the root length of the transgenic plant are obviously higher than those of the original wild plant, wherein the germination rate is improved by 45.82%, and the root length is improved by 43.32% compared with that of the wild control group. Under the stress of irrigating NaCl solution with 200mM concentration, the survival rate of the transgenic seedlings after 11 days of treatment is improved by 53.39 percent compared with that of the wild plants; the germination rate and the root length of the PwRBP1 Arabidopsis thaliana transferred seeds obtained by the invention are obviously higher than those of wild plants under the stress of 200mM concentration mannitol culture medium, wherein the germination rate is 1.41 times of that of the wild plants, and the root length is 18.13% higher than that of wild control groups. The survival rate of the transgenic seedlings is improved by 1.55 times compared with the wild plants after the transgenic seedlings are rehydrated for 3 days after the watering is stopped in the culture medium for 11 days. The results show that the PwRBP1 or the protein coded by the PwRBP1 has the function of improving the drought tolerance and the salt tolerance of plants.

Drawings

FIG. 1 shows the expression of PwRBP1 gene in tissues of Picea wilsonii.

FIG. 2 shows the result of molecular assay of PwRBP1 transgenic Arabidopsis thaliana.

FIG. 3 is an observation that PwRBP 1-transgenic Arabidopsis and wild-type Arabidopsis seeds germinated for 8 days under 100mM NaCl treatment.

FIG. 4 shows the germination rate of seeds of PwRBP 1-transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana treated with 100mM NaCl.

FIG. 5 is an observation of seeds of PwRBP 1-transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana germinated for 8 days under 200mM mannitol treatment.

FIG. 6 shows the germination rate of seeds of PwRBP 1-transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana treated with 200mM mannitol.

FIG. 7 is the observation of the root length of seeds of PwRBP1 transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana after germination, which were grown for 8 days on plates containing NaCl and mannitol at different concentrations.

FIG. 8 is a numerical statistic of root length of PwRBP1 transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana seeds after germination, after 8 days of growth, transformed into plates containing different concentrations of NaCl and mannitol.

FIG. 9 is a graph of observations of PwRBP1 transgenic Arabidopsis and wild type Arabidopsis seedlings after 11 days of 200mM NaCl treatment.

FIG. 10 is an observation of PwRBP1 transgenic Arabidopsis and wild type Arabidopsis seedlings after 11 days of drought treatment and 3 days of rehydration.

FIG. 11 shows the survival rate of PwRBP 1-transgenic Arabidopsis and wild-type Arabidopsis seedlings after salt treatment and drought treatment.

Detailed Description

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.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The pCAMBIA1205 vector in the following examples is available from the applicant (Beijing university of forestry), and the biomaterial is used only for repeating the relevant experiments of the present invention, and is not used for other purposes.

Example 1 acquisition of PwRBP1 and Gene encoding the same

One, PwRBP1 and obtaining of its coding gene

The Picea wilsonii cDNA library constructed by Gateway method and completed by Yinxie fundi (Shanghai) company is used as a template, 5'-CTGATGAATATCGACTTTGGAAAGTC-3' and 5'-ATGATGCAACCCACAGCAGG-3' are adopted for PCR amplification, and a positive cDNA sequence is obtained and sequenced.

The sequencing result shows that: the cDNA nucleotide sequence is shown as SEQ ID NO.1 in the sequence table and consists of 1293 nucleotides, the gene is named as PwRBP1, the protein coded by PwRBP1 is named as PwRBP1, and the amino acid sequence is SEQ ID NO.2 in the sequence table and consists of 431 amino acids.

The cDNA can be obtained by artificial synthesis.

Secondly, fluorescent quantitative PCR analysis of tissue expression of PwRBP1 gene

Extracting the RNA of the pollen, the root, the stem, the needle leaf and the seed tissue of the picea wilsonii, and carrying out reverse transcription to synthesize cDNA; and respectively taking the cDNA of each tissue as a template and 5'-TATGGGTTTGTGAGGTTTGGCG-3' and 5'-T ATGAGATCCGCCATTGCCTG-3' as detection primers, carrying out PCR amplification, and detecting the expression condition of the PwRBP1 gene in different tissues of picea wilsonii. Meanwhile, the EF 1-alpha gene is used as an internal reference, and the 5' primer for amplifying the EF 1-alpha gene is as follows: 5'-AACTGGAGAAGGAACCCAAG-3', the 3 ' primer is: 5'-AACGACCCAATGGAGGATAC-3' are provided. RT-qPCR reaction conditions: pre-denaturation at 95 ℃ for 15 min; 33 cycles of 95 ℃ 20sec, 56 ℃ 28sec, 72 ℃ 50 sec; extension was then carried out at 72 ℃ for 5 min.

The expression of PwRBP1 gene in each tissue is shown in FIG. 1. The result shows that PwRBP1 is expressed in all tissues, wherein the expression level is highest in roots and lower in needles.

Example 2 application of PwRBP1 Gene in improving stress tolerance in plants

First, construction of PwRBP1 Arabidopsis thaliana

1. Acquisition of PwRBP1 Gene

The following amplification primer pairs were prepared:

primer 1: 5'-ATGATGCAACCCACAGCAGG-3', respectively;

primer 2: 5'-CTGATGAATATCGACTTTGGAAAGTC-3' are provided.

And (2) carrying out PCR amplification by using cDNA of the picea wilsonii as a template and using the

primers

1 and 2 to obtain a PCR product with the size of 1293bp, and carrying out sequencing after connecting the PCR product with a pEASY-T1 vector.

The sequencing result shows that: the nucleotide sequence of the PCR product is SEQ ID NO.1 in the sequence table, and the PCR product can encode protein PwRBP1 shown in SEQ ID NO.2 in the sequence table.

2. Obtaining of recombinant expression vectors

The following primer pairs were prepared:

primer 1: 5'-CGCGGATCCATGATGCAACCCACAGCAGG-3', respectively;

primer 2: 5'-TCCCCCGGGCTGATGAATATCGACTTTGGAAAGTC-3' are provided.

PCR amplification was carried out using the plasmid pEASY-T1 ligated with the PwRBP1 sequence as a template, and the PCR product was digested with BamHI and SmaI restriction enzymes to obtain a digested product. The digested product was ligated with the same digested pCAMBIA1205 vector, and the ligation product was transformed into E.coli DH 5. alpha. competent cells and plated on LB plates containing 35ug/ml chloramphenicol for overnight culture. Picking white single colony to culture in LB liquid culture medium containing 35ug/ml chloramphenicol overnight and carrying out colony PCR identification; meanwhile, plasmid DNA is extracted by an alkaline method for sequence determination.

The sequencing result shows that the plasmid is a recombinant vector obtained by inserting PwRBP1 shown in SEQ ID NO.1 in the sequence table into BamHI and SmaI enzyme cutting sites of an expression vector pCAMBIA1205, and the plasmid is named as pCAMBIA1205-PwRBP 1.

3. PwRBP1 transgenic Arabidopsis thaliana

1) PwRBP1 transgenic Arabidopsis thaliana

The recombinant vector pCAMBIA1205-PwRBP1 prepared in the step 2 is transformed into competent cells of agrobacterium GV3101 (purchased from Shanghai Weidi Biotech Co., Ltd.) to obtain recombinant bacteria GV3101/pCAMBIA1205-PwRBP1 (extracted plasmid is sent for sequencing, and is the recombinant vector pCAMBIA1205-PwRBP 1).

The recombinant strain GV3101/pCAMBIA1205-PwRBP1 was inoculated in a single clone in YEB liquid medium containing 35mg/L chloramphenicol, and cultured with shaking at 28 ℃ for two days. The culture broth was centrifuged at 3000rpm/min for 5 minutes and the resulting Agrobacterium pellet was suspended in an infection solution containing 5% sucrose and 0.03% Silwet L-77.

Columbia ecotype wild type Arabidopsis thaliana (Col-0) (purchased from ABRC) was transformed by the catkin dip method, seeds inoculated to the contemporary transgenic Arabidopsis thaliana plants were harvested (T1 generation), and germinated seeds were screened in MS medium containing 40. mu.g/ml Hygromycin (Hygromycin B) and 40. mu.g/ml carbenicillin (Carbenicillin). Seedlings from T1 generations germinated on the above medium were transferred to culture soil, seeds were harvested (T2 generations), and homozygous PwRBP1 Arabidopsis plants (T4 generations) were obtained by the same screening process. Finally, seeds of PwRBP 1-transferred Arabidopsis plants (T4 generation) are directly sown in culture soil, and the grown PwRBP1 Arabidopsis plants (T4 generation) grow under long-day conditions for about two weeks and flower.

2) Molecular assay of PwRBP1 transgenic Arabidopsis thaliana

Extracting total protein of a PwRBP1 transgenic arabidopsis plant (T4 generation), detecting by taking the total protein as a template, performing SDS-PAGE gel running, membrane transferring and sealing, and then taking a GFP label as an antibody, and taking wild type arabidopsis as a control, wherein the detection result is shown in figure 2, the PwRBP1 transgenic arabidopsis plant (T4 generation) has an obvious strip after being combined by a GFP antibody, while the wild type arabidopsis does not detect the strip, and the expression level of PwRBP1 in the T4 generation strain is proved to be obviously improved.

The same method is adopted to transfer the empty vector pCAMBIA1205 into wild type arabidopsis thaliana to obtain a transferred empty vector arabidopsis thaliana, and sowing and passage are carried out to obtain an empty vector overexpression arabidopsis thaliana plant (T4 generation).

Second, function study of PwRBP1 Arabidopsis thaliana

1. Seed germination test

1) Study of salt tolerance

A seed germination experiment is carried out on an MS culture medium containing 100mM NaCl, in the experiment, a PwRBP1 Arabidopsis thaliana plant (generation T4), an empty vector Arabidopsis thaliana plant (generation T4) and a wild type Arabidopsis thaliana (WT) seed are transferred for sowing, the culture conditions are that the light is 16 hours, the dark is 8 hours, and the light intensity is 300-^-2s^-1Under illumination, the room temperature is 22-24 ℃, and the relative humidity is 70-90%; the room temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. 100 seeds per line, the experiment was repeated 3 times and the results averaged.

The results of the 8 th day experiment for counting the germination rate of each seed are shown in FIGS. 3 and 4 (WT represents wild type Arabidopsis thaliana, and T represents PwRBP1 Arabidopsis thaliana plant T4 generation). Under 100mM NaCl treatment, seeds of a wild type control strain and a Pw RBP 1-transferred strain can germinate on day 4, but the germination rate of the Pw RBP 1-transferred strain on day 1 is 45.82% higher than that of the wild type, and the result shows that under 100mM NaCl stress treatment, the germination rate of the Pw RBP 1-transferred Arabidopsis seeds is obviously higher than that of the wild type seeds, and the seeds have better salt tolerance.

There was no significant difference in the results of wild type arabidopsis (WT) and empty vector overexpression arabidopsis plants (T4 generation).

2) Study of drought tolerance

The seed germination experiment is carried out on MS culture medium containing 200mM mannitol, in the experiment, PwRBP1 Arabidopsis thaliana plant (T4 generation), empty vector Arabidopsis thaliana plant (T4 generation) and wild type Arabidopsis thaliana (WT) seed are transferred for sowing, the culture condition is 16 hours under light, 8 hours under dark, and the light intensity is 300-^-2s^-1Under illumination, the room temperature is 22-24 ℃, and the relative humidity is 70-90%; the room temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. 100 seeds per line, the experiment was repeated 3 times and the results averaged.

The results of the 8 th day experiment for counting the germination rate of each seed are shown in FIGS. 5 and 6 (WT means wild type Arabidopsis thaliana, and T means T4 generation of PwRBP1 Arabidopsis thaliana plant). Under 200mM mannitol stress treatment, seeds of a wild type and PwRBP1 transferred strain do not germinate at 1 st day of the stress treatment, but can germinate at 5 th day, and the germination rate of the seeds of the Pw RBP1 transferred strain at 2 nd day of the stress treatment is 1.41 times higher than that of the wild type, so that the result shows that under 200mM mannitol stress treatment, the germination rate of the seeds of the PwRBP1 transferred Arabidopsis is obviously higher than that of the wild type, and the seeds have better drought stress resistance.

2. Root length measurement salt and drought tolerance test

In the experiment, PwRBP1 Arabidopsis plants (T4 generation) are transferred and unloadedThe arabidopsis thaliana plant (T4 generation) and the wild type arabidopsis thaliana (WT) seed are sown under the conditions of 16 hours of illumination, 8 hours of darkness and 300-400 mu mol m light intensity^-2s^-1Under illumination, the room temperature is 22-24 ℃, and the relative humidity is 70-90%; the room temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. After the seeds germinate, the germinated arabidopsis seedlings are moved to a MS culture medium vertically and flatly laid on 100mM NaCl or 200mM mannitol by using tweezers, each strain contains 100 seeds, the experiment is repeated for 3 times, and the result is averaged.

The root length of each line was counted at day 8 of experiment, and the results are shown in FIGS. 7 and 8 (WT means wild type Arabidopsis thaliana, and T means PwRBP1 Arabidopsis thaliana plant T4 generation). Under the stress treatment of 100mM NaCl, the root length of the PwRBP 1-transferred Arabidopsis seedling is increased by 43.32 percent compared with that of a wild plant; under 200mM mannitol stress treatment, the root length of the PwRBP1 transferred Arabidopsis thaliana seedling is increased by 18.13 percent compared with that of a wild plant. The result shows that the root length of the PwRBP 1-transferred Arabidopsis seedlings is obviously longer than that of the wild type under the stress treatment of 100mM NaCl and 200mM mannitol, and the seedlings have better salt tolerance and drought tolerance.

The results show that PwRBP1 or the protein encoded by the PwRBP1 has drought tolerance and salt tolerance.

3. Stress tolerance test of seedlings

1) Study of salt tolerance

In the experiment, PwRBP1 Arabidopsis thaliana plant (generation T4), empty vector Arabidopsis thaliana plant (generation T4) and wild type Arabidopsis thaliana (WT) seed are sown under the conditions of 16 hours of illumination, 8 hours of darkness and 300 μmol m light intensity^-2s^-1Under illumination, the room temperature is 22-24 ℃, and the relative humidity is 70-90%; the room temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. After the seeds germinate, the germinated arabidopsis seedlings are transferred to a culture medium (nutrient soil: vermiculite: 1), 200mM NaCl solution is poured into the culture medium after 12 days of growth, the solution is poured every two days, and the survival rate is counted after 11 days. 100 seeds per line, 3 replicates, and the resultsAnd (6) taking an average value.

The experimental results are shown in fig. 9 and 11 (WT represents wild type arabidopsis, T represents T4 generation of transgenic PwRBP1 arabidopsis plant), and the survival rate of seedlings of transgenic PwRBP1 arabidopsis plant (T4 generation) after being treated by 200mM NaCl is improved by 53.39% compared with that of seedlings of wild type arabidopsis, and the transgenic plants have stronger salt tolerance.

2) Study of drought tolerance

In the experiment, PwRBP1 Arabidopsis thaliana plant (generation T4), empty vector Arabidopsis thaliana plant (generation T4) and wild type Arabidopsis thaliana (WT) seed are sown under the conditions of 16 hours of illumination, 8 hours of darkness and 300 μmol m light intensity^-2s^-1Under illumination, the room temperature is 22-24 ℃, and the relative humidity is 70-90%; the room temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. After the seeds germinate, the germinated arabidopsis seedlings are transferred to a culture medium (nutrient soil: vermiculite: 1), after 12 days of growth in the culture medium, watering is stopped for 11 days, and the survival rate is counted after 3 days of rehydration. 100 seeds per line, the experiment was repeated 3 times and the results averaged.

The experimental results are shown in fig. 10 and 11 (WT represents wild type arabidopsis, T represents T4 generation of the transgenic PwRBP1 arabidopsis plant), the survival rate of seedlings of the transgenic PwRBP1 arabidopsis plant (T4 generation) after drought treatment for 11 days is 1.55 times that of seedlings of wild type arabidopsis after 3 days of rehydration, and is obviously higher than that of wild type seedlings, which indicates that the transgenic PwRBP1 arabidopsis plant has better drought tolerance.

Sequence listing

<110> Beijing university of forestry

<120> plant stress tolerance associated protein PwRBP1, and coding gene and application thereof

<130> 20190124

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1293

<212> DNA

<213> Artificial sequence

<400> 1

atgatgcaac ccacagcagg cgtcggccct ccttttgcaa accctaatca aaaccagcag 60

cggcagcaat ggcttcaaca gcagcagcag atggcgatgg cgatgcagca gcaacagcag 120

ccgcctcagc aacaggcgaa ccaggccatg gcgatgcagc aacaacaagc cccaatgatg 180

gctcagcagt actatgcaca gcagcctcaa tatcagcagc agcagctacc aatggtgatg 240

cagcaacatc agccgcagtc gagtgacgag gttaagactc tctgggtggg tgatttgcag 300

ttctggatgg acgagggcta tttgcacacc tgtttttccc acactggaga gcttgtttct 360

gccaagataa tccgtaataa gtatactgga cagtcagagg gttatggctt tatggagttc 420

ataacacgta cagctgctga gaagattatg caaacttata atgggacgct aatgcccaac 480

actgaacaag ttttcagaat gaattgggca acttttagca tgggagaaag gcgtctagat 540

ggaggcccag atttttctat ttttgtggga gatttggatt cagatgtctc agatttggtc 600

ttgcaggaga ctttccaaag tcgacattca tcagtgaaag ctgctaaggt tgtcatggat 660

gcaaacacag ggcgctcaaa aggttatggg tttgtgaggt ttggcgagga gagtgagagg 720

gcccgagcca tgacagaaat gaatggtgta tattgttcta ctagacctat gcgaatcagt 780

gcagccaccc caaggaagtc tgcaggggtt cagcaccagt attcaggaag agcaggcaat 840

ggcggatctc atgcccaagg attcccgtca gacaatgatt taaacaatac aactatattt 900

gtaggccggc tagacccaaa tgcgacagat gaagatctga gacaagtctt tggccagtat 960

ggagagcttg tgtctgtaaa aatacctgtt ggtaaaggtt gtggatttgt ccagtttggt 1020

aacagggctt ctgctgagga agccttgcaa aggcttcatg gtactgttat tcgtcagcaa 1080

actgtacgtc tttcttgggg tcgaagccct gcaaacaagc agcaacccca gccccagggg 1140

caacagcctc aatctgatcc aaatcaatgg aatggtgctt actatgggca aggatatgaa 1200

agctatggtt atgctccccc tcctcaagat cctgcaatgt atgcctatgg tggctaccct 1260

ggatatggga attataatca acaggtaagc tag 1293

<210> 2

<211> 430

<212> PRT

<213> Artificial sequence

<400> 2

Met Met Gln Pro Thr Ala Gly Val Gly Pro Pro Phe Ala Asn Pro Asn

1 5 10 15

Gln Asn Gln Gln Arg Gln Gln Trp Leu Gln Gln Gln Gln Gln Met Ala

20 25 30

Met Ala Met Gln Gln Gln Gln Gln Pro Pro Gln Gln Gln Ala Asn Gln

35 40 45

Ala Met Ala Met Gln Gln Gln Gln Ala Pro Met Met Ala Gln Gln Tyr

50 55 60

Tyr Ala Gln Gln Pro Gln Tyr Gln Gln Gln Gln Leu Pro Met Val Met

65 70 75 80

Gln Gln His Gln Pro Gln Ser Ser Asp Glu Val Lys Thr Leu Trp Val

85 90 95

Gly Asp Leu Gln Phe Trp Met Asp Glu Gly Tyr Leu His Thr Cys Phe

100 105 110

Ser His Thr Gly Glu Leu Val Ser Ala Lys Ile Ile Arg Asn Lys Tyr

115 120 125

Thr Gly Gln Ser Glu Gly Tyr Gly Phe Met Glu Phe Ile Thr Arg Thr

130 135 140

Ala Ala Glu Lys Ile Met Gln Thr Tyr Asn Gly Thr Leu Met Pro Asn

145 150 155 160

Thr Glu Gln Val Phe Arg Met Asn Trp Ala Thr Phe Ser Met Gly Glu

165 170 175

Arg Arg Leu Asp Gly Gly Pro Asp Phe Ser Ile Phe Val Gly Asp Leu

180 185 190

Asp Ser Asp Val Ser Asp Leu Val Leu Gln Glu Thr Phe Gln Ser Arg

195 200 205

His Ser Ser Val Lys Ala Ala Lys Val Val Met Asp Ala Asn Thr Gly

210 215 220

Arg Ser Lys Gly Tyr Gly Phe Val Arg Phe Gly Glu Glu Ser Glu Arg

225 230 235 240

Ala Arg Ala Met Thr Glu Met Asn Gly Val Tyr Cys Ser Thr Arg Pro

245 250 255

Met Arg Ile Ser Ala Ala Thr Pro Arg Lys Ser Ala Gly Val Gln His

260 265 270

Gln Tyr Ser Gly Arg Ala Gly Asn Gly Gly Ser His Ala Gln Gly Phe

275 280 285

Pro Ser Asp Asn Asp Leu Asn Asn Thr Thr Ile Phe Val Gly Arg Leu

290 295 300

Asp Pro Asn Ala Thr Asp Glu Asp Leu Arg Gln Val Phe Gly Gln Tyr

305 310 315 320

Gly Glu Leu Val Ser Val Lys Ile Pro Val Gly Lys Gly Cys Gly Phe

325 330 335

Val Gln Phe Gly Asn Arg Ala Ser Ala Glu Glu Ala Leu Gln Arg Leu

340 345 350

His Gly Thr Val Ile Arg Gln Gln Thr Val Arg Leu Ser Trp Gly Arg

355 360 365

Ser Pro Ala Asn Lys Gln Gln Pro Gln Pro Gln Gly Gln Gln Pro Gln

370 375 380

Ser Asp Pro Asn Gln Trp Asn Gly Ala Tyr Tyr Gly Gln Gly Tyr Glu

385 390 395 400

Ser Tyr Gly Tyr Ala Pro Pro Pro Gln Asp Pro Ala Met Tyr Ala Tyr

405 410 415

Gly Gly Tyr Pro Gly Tyr Gly Asn Tyr Asn Gln Gln Val Ser

420 425 430

Claims

1. The application of PwRBP1 protein in improving the salt tolerance and/or drought tolerance of plants; or in the cultivation of transgenic plants with improved salt tolerance and/or drought tolerance; the proteins are as follows: a) protein with a sequence shown as SEQ ID NO. 2; or b) a fusion protein with a tag sequence connected with the end of the amino acid shown in SEQ ID NO. 2.

2. Use of a biological material for increasing salt tolerance and/or drought tolerance in a plant; or in the cultivation of transgenic plants with improved salt tolerance and/or drought tolerance;

the biomaterial is any one of the following A1) to A12):

A1) nucleic acid molecules which code for the protein shown in SEQ ID NO. 2;

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).

3. Use according to claim 2, characterized in that: A1) the nucleic acid molecule is a DNA molecule shown in SEQ ID NO. 1.

4. A method for cultivating transgenic plants with improved salt tolerance and/or drought tolerance comprises the steps of improving the expression quantity and/or activity of protein shown as SEQ ID NO.2 in receptor plants to obtain transgenic plants; the recipient plant is a monocot or a dicot.

5. The method of claim 4, wherein: the transgenic plant has higher salt tolerance and/or drought tolerance than the recipient plant in any one of the following (1) to (3):

(1) the seed germination rate of the transgenic plant is higher than that of the receptor plant;

(2) the transgenic plant has a longer root length than the recipient plant;

(3) the survival rate of the transgenic plant is higher than that of the receptor plant.

6. The method according to any one of claims 4-5, wherein: the method for improving the expression quantity and/or activity of the protein shown by the SEQ ID NO.2 in the receptor plant is to express or over-express the protein shown by the SEQ ID NO.2 in the receptor plant.

7. The method of claim 6, wherein: the expression or over-expression method is to introduce the gene shown in SEQ ID NO.1 into a receptor plant.