CN112961230B - OsFLP protein related to plant salt tolerance, related biological material and application thereof - Google Patents

Abstract

The invention discloses an OsFLP protein related to plant salt tolerance, as well as related biological materials and applications. The OsFLP protein can specifically be a protein of the following A1), A2) or A3): A1) the amino acid sequence is the protein of sequence 1 in the sequence listing; A2) the protein of A1) is substituted by one or several amino acid residues and / or deletion and / or addition of the protein shown in A1) has more than 90% identity and has a protein that improves salt tolerance activity; A3) connects the protein at the N-terminal or/and C-terminal of A1) or A2) Label the resulting fusion protein. OsFLP protein and its related biomaterials can be used to improve the salt tolerance of plants.

Description

An OsFLP protein related to plant salt tolerance and its related biological materials and application

technical field

The invention relates to an OsFLP protein related to plant salt tolerance and related biological materials and applications in the field of biotechnology.

Background technique

With global climate change, the impact of abiotic stress on plant growth has become one of the major challenges faced by human beings, seriously restricting the growth and development of plants. Abiotic stress refers to the adverse effects of any abiotic factors on plants in a specific environment, including drought, low temperature, salinity and other factors. Drought and salinity are the two most important stress factors restricting plant growth. Especially as the degree of salinization of the global land is deepening and the area of arable land is decreasing, the research on the response genes and their mechanisms of action to plant responses to abiotic stress has become one of the hottest topics today. It is very important to screen and breed genetic varieties related to plant salt tolerance to improve agricultural production.

As one of the most important food crops in the world, rice has a smaller genome than other crops such as corn and wheat, with a genome length of about 420Mb. It has become another important model plant after Arabidopsis thaliana. In recent years, rice has been widely used in the research of plant genetics, developmental biology and molecular biology. In the face of the increasingly serious soil salinization problem in crop production, cloning rice salt-tolerant genes and studying their functions has important practical significance for cultivating salt-tolerant varieties of crops as soon as possible.

Salt stress can cause osmotic stress and ion stress, which can inhibit the normal cell growth and division of plants. In response to adverse environments, plants maintain osmotic and ionic homeostasis through rapid osmotic and ionic signaling. High-salt osmotic stress can rapidly increase the biosynthesis of abscisic acid (ABA), thereby regulating ABA-dependent stress response pathways. Genes of protein kinases, transcription factors, miRNAs and ROS-related proteins can all participate in the response to salt stress through ABA-dependent pathways; there are also some salt-induced genes that are independent of ABA. In addition, since salt stress can cause the accumulation of osmoprotective substances in plants, the genes related to the synthesis of these osmoprotective macromolecules also play a role in the salt stress response; hormones, as molecules that transmit stress signals to cells, their synthesis and transmission Genes in the pathway were also involved in salt stress. The plant stress regulation network is intricate, and its research is full of challenges and significance. The exploration and application of rice salt-tolerant genes provides a new idea for solving the current problems in agricultural production, and is of great significance to molecular breeding and the development of salt-tolerant varieties. .

Contents of the invention

The technical problem to be solved by the invention is how to improve the salt tolerance of plants.

The present invention provides a protein named OsFLP, which is the following A1), A2) or A3) protein:

A1) The amino acid sequence is the protein of sequence 1 in the sequence listing;

A2) A protein having more than 90% identity with the protein shown in A1) obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein of A1) and having a salt-tolerance-improving activity;

A3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of A1) or A2).

The above-mentioned proteins can be synthesized artificially, or their coding genes can be synthesized first, and then biologically expressed.

Among the above proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the above-mentioned proteins, the identity refers to the identity of amino acid sequences. Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, set Gap existence cost, Perresidue gap cost and Lambda ratio to 11 respectively , 1 and 0.85 (the default value) and search for the identity of a pair of amino acid sequences for calculation, and then the value (%) of the identity can be obtained.

Among the above proteins, the above 90% identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biological materials related to OsFLP also belong to the protection scope of the present invention.

The biological material related to the protein OsFLP provided by the present invention is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding OsFLP;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3);

B5) The transgenic plant cell line containing the nucleic acid molecule described in B1), or the transgenic plant cell line containing the expression cassette described in B2), or the transgenic plant cell line containing the recombinant vector described in B3).

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

In the above-mentioned biological material, the nucleic acid molecule described in B1) is the gene shown in b1) or b2) below:

b1) The coding sequence (CDS) of the coding strand is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;

b2) The nucleotides of the coding strand are cDNA molecules or DNA molecules of sequence 2 in the sequence listing.

Wherein, sequence 2 in the sequence listing consists of 1617 nucleotides, encoding the protein shown in sequence 1 in the sequence listing.

Among the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule (OsFLP gene expression cassette) described in B2) refers to a nucleic acid molecule capable of expressing OsFLP in a host cell. A terminator that terminates transcription of OsFLP may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato Protease Inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S. Patent 5,057,422 ); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin ( Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, e.g.: Odell et al. ( ¹⁹⁸⁵ ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res., 15:9627).

An existing plant expression vector can be used to construct a recombinant expression vector containing the OsFLP gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pCAMBIA1391-Xb (CAMBIA Company), pH7WG2D.1, etc. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated regions transcribed at the 3' ends of storage protein genes have similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be 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 vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to metharexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

Among the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

The application of the above-mentioned proteins or the above-mentioned biological materials in regulating the salt tolerance of plants also belongs to the protection scope of the present invention.

In order to solve the above technical problems, the present invention also provides a plant agent, which is used to improve the salt tolerance of plants.

The plant reagent provided by the present invention contains the protein or/and the biological material related to the protein.

The active ingredient of the above-mentioned plant reagent can be the protein or/and the biological material related to the protein, the active component of the above-mentioned plant reagent can also contain other biological components or/and non-biological components, the other active components of the above-mentioned plant reagent Those skilled in the art can determine according to the nitrogen uptake effect of plants.

In order to solve the above technical problems, the present invention also provides a method for cultivating salt-tolerant plants.

The method for cultivating salt-tolerant plants provided by the present invention includes introducing the nucleic acid molecule encoding the protein into a target plant to obtain a salt-tolerant plant; the tolerance of the salt-tolerant plant to salt is better than that of the target plant Salt tolerance.

The present invention also provides a protein that is sensitive to salt, said protein being a protein of X1), X2) or X3) as follows:

X1) The amino acid sequence is a protein obtained by replacing the 118th amino acid residue of Sequence 1 in the sequence listing with an alanine residue with a threonine residue and keeping other sequences unchanged;

X2) A protein having more than 90% identity with the protein shown in X1) obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein of X1) and having an activity of improving salt sensitivity;

X3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of X1) or X2).

The present invention also provides a biological material related to the above-mentioned salt-sensitive protein, which is any one of the following Y1) to Y5):

Y1) a nucleic acid molecule encoding the salt-sensitive protein;

Y2) an expression cassette containing the nucleic acid molecule of Y1);

Y3) a recombinant vector containing the nucleic acid molecule described in Y1), or a recombinant vector containing the expression cassette described in Y1);

Y4) A recombinant microorganism containing the nucleic acid molecule described in Y1), or a recombinant microorganism containing the expression cassette described in Y2), or a recombinant microorganism containing a recombinant vector described in Y3);

Y5) the transgenic plant cell line containing the nucleic acid molecule of Y1), or the transgenic plant cell line containing the expression cassette of Y2), or the transgenic plant cell line containing the recombinant vector of Y3).

The present invention also provides a method for cultivating salt-sensitive plants, comprising introducing the nucleic acid molecule encoding the salt-sensitive protein into a target plant to obtain a salt-sensitive plant; the sensitivity of the salt-sensitive plant to salt is higher than that of the target plant Sensitivity of plants to salt.

The target plant of the present invention may be a monocotyledonous plant or a dicotyledonous plant not containing the nucleic acid molecule encoding the protein. The monocot can be millet, rice.

In the method of the present invention, the nucleic acid molecule can be modified as follows first, and then introduced into the target plant to achieve a better expression effect:

1) modifying the gene sequence adjacent to the starting methionine to allow efficient initiation of translation; for example, using sequences known to be effective in plants for modification;

2) Linking with various plant-expressed promoters to facilitate its expression in plants; said promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; e.g. a tissue or organ-specific expression promoter, depending on what stage of development the recipient is desired; although proven source Many promoters for dicots are functional in monocots and vice versa, but ideally, dicot promoters are chosen for expression in dicots and monocot promoters are used for Expression in monocots;

3) be connected with suitable transcription terminator, also can improve the expression efficiency of gene of the present invention; For example derive from the tml of CaMV, derive from the E9 of rbcS; Any obtainable terminator that is known to work in the plant all can be combined with The gene of the present invention is connected;

4) Introduce enhancer sequences, such as intron sequences (eg derived from Adhl and bronze) and viral leader sequences (eg derived from TMV, MCMV and AMV).

The nucleic acid molecule can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus carrier, direct DNA transformation, microinjection, electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

The salt-tolerant plant or the salt-sensitive plant of the present invention can be a transgenic plant, or a plant obtained by conventional breeding techniques such as hybridization.

The transgenic plants of the present invention are understood to include not only the first to second generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

The transgenic experiment of introducing the OsFLP gene into rice proved that the transgenic rice expressing the OsFLP gene significantly promoted the tolerance to salt compared with the recipient rice, indicating that the OsFLP gene is a gene related to salt tolerance, and the OsFLP gene can be used to improve the tolerance of plants. Salt tolerance, improve the stress resistance of plants.

Description of drawings

Fig. 1 is a sequence comparison diagram of rice OsFLP protein in Example 1 and known R2R3-MYB family proteins.

Fig. 2 is the GUS staining figure of the plant tissue of the OsFLP-positive transformation plant in Example 1, wherein, A is the rice seedling that germinates for 3 days, B is the main root, C is the central column of the root, D is the mature stomata, and E is the neck node of the seedling, F is a partial enlarged view of the neck node of E.

Figure 3 is a histogram of OsFLP gene expression in wild-type rice under different NaCl treatment durations in Example 1, the data are expressed as mean ± standard deviation, the number of repetitions is 3, and the Student's t test is used for significance analysis, ** indicates The significant analysis result was P<0.01.

Fig. 4 is an analysis diagram of the mutation site of the osflp-1 mutant in Example 2.

Figure 5 is the plasmid map of pH7WG2D.1 in Example 3.

Fig. 6 is the photo and the bar graph of the survival rate of the 4 kinds of materials in Example 3 after being treated with salt for 21 days. Wherein, the A picture of Fig. 6 is the photo of the control group without salt treatment of 4 kinds of materials, the B picture of Fig. 6 is the photo of the experimental group after the salt treatment of 4 kinds of materials, and the C picture of Fig. 6 is the photo of the 4 kinds of material treatment groups and the contrast Statistical histogram of the survival rate of the group, the data is expressed as the mean ± standard deviation, the number of repetitions is 3, and the Student's t test is used for significant analysis. ** indicates that the significant analysis result is P<0.01.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples, unless otherwise specified, are conventional biochemical reagents, which can be obtained from commercial sources.

1 carrier

The vector pCAMBIA1301 in the following examples is a product of Beijing Huabo Deyi Biotechnology Co., Ltd., product number: VT3013.

The vector pH7WG2D.1 in the following examples is a product of China Plasmid Vector Strain Cell Strain Gene Collection Center (BioVector), product number: BioVector921816.

The carrier TOPOvector (article number: CV0402) in the following examples is a product of Beijing Aide Technology Co., Ltd.

2 plant strains

The rice variety ZH11 (Oryza sativa L.ssp. Japonica cv. Zhonghua11, ZH11) in the following examples is described in the non-patent literature "OsPGIP1-Mediated Resistance to Bacterial Leaf Streakin Rice is Beyond Responsive to the Polygalacturonase of Xanthomonas oryzaepv.oryzicola" . The public can obtain it from the Institute of Botany, Chinese Academy of Sciences to repeat the experiment of this application, and it cannot be used for other purposes.

4 reagents

Gateway LR ClonaseⅡenzyme mix (Product No.: 11791019) is a product of Thermo Fisher Scientific (China) Co., Ltd.

In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

Example 1. Acquisition of plant salt-tolerance-related gene OsFLP

1. Sequence analysis of rice OsFLP protein

Using the amino acid sequence of Arabidopsis AtFLP in NCBI (http://www.ncbi.nlm.nih.gov/) and the rice genome database (http://rice.plantbiology.msu.edu/), to find Homologous proteins of Arabidopsis AtFLP. As a result, a protein having 33.89% similarity to AtFLP's amino acid sequence was obtained and named as OsFLP, and its amino acid sequence is shown in Sequence 1 in the Sequence Listing.

By comparing and analyzing several known R2R3-MYB family protein sequences, including Arabidopsis AtFLP, AtMYB88, AtMYB121, ATMYB0 (ATGL1) and rice OsMPS protein sequences, the results are shown in Figure 1, and it is found that there is a R2 and R3 two MYB domains. Among them, R2 contains three conserved motifs of H1, H2 and H3, and R3 contains three conserved motifs of H1, H2 and H3, which proves that OsFLP is a R2R3-MYB family protein.

2. Cloning of rice OsFLP gene

The name of the gene encoding the OsFLP protein is OsFLP, which is coded as Os07g0627300 in the rice genome database, and is derived from the rice Nipponbare ecotype. The OsFLP gene is located on chromosome 7 and has 12 exons.

Take 100-200 mg of rice seedlings, use RNA extraction kit to extract total RNA, and synthesize a certain amount of single-stranded cDNA by reverse transcription according to the concentration of extracted RNA after nitration. OsFLP gene amplification conditions are as follows, the synthesized single-stranded cDNA was diluted 4 times, and used as a template for PCR reaction: the full-length cDNA of OsFLP was amplified with high-fidelity DNA polymerase KOD-Plus from TOYOBO, and the amplification system was as follows: 50 μL, containing 5 μL of 10×KOD buffer, 5 μL of 2mM dNTP mix, 1.5 μL of Primer1 and Primer2, 0.5 μL of KOD-Plus DNA polymerase, 1-5 μL of template, supplemented with sterilized ultrapure water to 50 μL, and the reaction system preparation process was on ice operate.

Primer 1: 5'-ATGGCGACCGGACCCGATCTG-3';

Primer 2: 5'-TCATAGGCTATTAAGGAGAGC-3'.

Amplification was carried out on a BIO-RAD PCR amplification instrument, with pre-denaturation for 5 minutes, denaturation at 94°C for 30s, 30s at 54°C, 2min at 68°C, 32 cycles, and extension at 68°C for 10 minutes. The amplified fragment was subjected to electrophoresis on 1% agarose gel, and the amplified fragment was recovered, connected to the vector Blunt, subjected to enzyme digestion and sequencing identification, and obtained the correct OsFLP gene cDNA (see sequence 2 in the sequence listing).

3. Analysis of rice OsFLP expression pattern

Using the plant tissue pattern expression vector pGUS1301 and the OsFLP gene cDNA obtained in step 2, construct the OsFLP:OsFLP-GUS expression vector, transform ZH11 wild-type rice, and perform GUS staining on the positively transformed plants to analyze the OsFLP expression pattern.

The staining results are shown in Figure 2. OsFLP can be expressed at the base of germinated rice seeds, as well as at the shoots and roots. It is expressed in the root tip and mature stomata, and in the shoot, the expression level is high in the neck node.

4. Expression analysis of rice OsFLP gene after salt treatment

Take ZH11 wild-type rice seedlings that have grown for 2 weeks, and treat the roots of the seedlings with 200Mm NaCl in a hydroponic tube for 0h, 2h, 4h, 6h, 12h, and 24h, then extract plant total RNA, set up 3 replicates, and detect OsFLP by qRT-PCR The relative expression levels of OsFLP primers and internal reference primers are as follows (OsFLP primers are Primer3 and Primer4; internal reference primers are Primer5 and Primer6).

Primer 3: 5'-AACTGGACAATTATAGCGGCAC-3';

Primer 4: 5'-GGAATGTTGGGACTGTATGAGC-3'.

Primer 5: 5'-GGATCCATCTTGGCATCTCTCA-3';

Primer 6: 5'-GGGCCAGACTCGTCGTACTC-3'.

The results are shown in Figure 3. The expression of OsFLP gene was induced and up-regulated by salt stress. The relative content of OsFLP in rice was significantly higher than normal under different salt treatment time, and the expression level was the highest at 6 hours after treatment.

Embodiment 2, the acquisition of gene OsFLP mutant

1. Using TILLING technology to obtain osflp-1 mutants

TILLING (Targeting Induced Local Lesions IN Genomes) technology is a new reverse genetics research method, using the chemical mutagen ethyl methanesulfonate to mutate the seeds, and then using high-throughput Sequencing methods quickly and efficiently identify point mutations from mutagenized populations. In order to better analyze the function of OsFLP in rice, we used TILLING technology to obtain osflp mutant lines at different mutation sites, see Table 1 for details. We observed and compared the obtained strains at different mutation sites, and found a strain A2349-2 with a different stomatal phenotype from the wild type, and named the mutant osflp-1 mutant.

Table 1

2. Mutation site analysis of osflp-1 mutant

We designed corresponding primers to amplify OsFLP genomic DNA in ZH11 and osflp-1 mutants. It was found by sequencing (Figure 4), that the nucleotide sequence of the OsFLP genomic DNA of ZH11 is shown in sequence 3 of the sequence table, at the 2126th position after the start codon ATG of the OsFLP gene in the osflp-1 mutant, the base G is mutated into a base A, the mutation site is located on the sixth exon of the gene. That is, the 352nd base of OsFLP CDS (sequence listing sequence 2) is mutated from G to A, resulting in the substitution of alanine residue (Ala) for threonine residue (Thr ), the amino acid changes, the polarity of the amino acid changes from non-polar to polar, the hydrophobic amino acid becomes hydrophilic, and the properties of the amino acid change.

Embodiment 3, the acquisition of transgenic OsFLP rice

1. Construction of OsFLP gene overexpression vector and acquisition of rice transgenic materials

The OsFLP gene overexpression vector uses pH7WG2D.1 (see Figure 5 for the plasmid map) as the starting vector, and adopts the gateway system method to construct. The specific implementation is as follows: amplify the cDNA fragment of OsFLP (the nucleotide sequence is sequence 2), and gel recovery Afterwards, the cDNA fragment of OsFLP was connected to the entry vector TOPOvector, and the resulting plasmid was named OsFLP-TOPO. Digest the plasmid OsFLP-TOPO with PVUⅠ restriction endonuclease (recognition site: CGATCG, the cut fragment is sticky, it belongs to restriction endonuclease type II) at 37°C for 3 hours, put The enzyme products were separated by agarose gel electrophoresis and recovered by cutting the gel. Mix 1.5 μL of recovered product, 0.5 μL of pH7WG2D.1 plasmid, and 0.5 μL of Gateway LR ClonaseⅡenzyme mix, and react at 25°C for 4 hours for ligation. After the reaction was terminated, the mixture was transformed into competent cells of Escherichia coli DH5α to obtain single colony clones. The single colony was propagated and the plasmid was extracted, and the plasmid with the correct sequence was obtained after PCR identification of the plasmid. The obtained recombinant expression vector was named Super-pH7WG2D.1-OsFLP. Super-pH7WG2D.1-OsFLP contains the OsFLP gene whose nucleotide sequence is sequence 2, and can express the protein OsFLP whose amino acid sequence is sequence 1.

The plasmid Super-pH7WG2D.1-OsFLP was transformed into competent cells of Agrobacterium tumefaciens strain EHA105, and the positive clones transferred to Super-pH7WG2D.1-OsFLP were identified by colony PCR and enzyme digestion (named as EHA105/ Super-pH7WG2D.1-OsFLP), using EHA105/Super-pH7WG2D.1-OsFLP to transform rice wild-type ZH11 to obtain OsFLP gene overexpressed rice, named OE.

2. OsFLP gene complementary osflp-1 vector construction and positive plants obtained

The rice complementation vector was constructed with pCAMBIA1301 as the starting vector, transformed into the osflp-1 mutant in Example 2, and the obtained transformed seedlings were screened on a hydroponic medium containing 50 μg/μL hygromycin, and the _T2 generation Seeds also need to be screened with hygromycin, and the positive plants obtained are complementary materials, named COM.

3. Salt-tolerant phenotype analysis of rice osflp-1 mutants and OsFLP gene overexpression lines

Using wild-type ZH11, mutant osflp-1 (see Example 2), complementary material COM (construction of 2 in Example 3) and plant OE (1 in Example 3) with OsFLP gene overexpression, totally 4 kinds of rice materials The T3 generation plants were used as experimental materials.

In the rice field growth environment, the experimental group treated the rice seedlings grown for 20 days with salt, poured a salt solution to the seedlings, and the salt concentration was 200mM NaCl, observed the phenotype and counted the survival rate after the salt treatment, and the control group (control) Replace the saline solution with water. Each material has 12 plants per treatment, and the number of repetitions is 4.

The results showed that after 15 days of salt treatment, there was no difference in the growth status of the seedlings of the four materials in the control group, while the seedlings in the salt treatment group showed stress states such as yellowing of leaves, curling and drooping, and growth stagnation. Under the same salt concentration treatment, the four seedlings responded differently to the stress. The osflp-1 mutant had more obvious yellow and dry leaves, while the OsFLP overexpressed plants had better growth height and overall plant growth status. The results after 21 days of salt treatment are shown in Figure 6. The control groups of the four materials still showed normal growth, and there was no difference among them; while in the experimental group, osflp-1 almost all died, and there were no new leaves, showing that it was resistant to salt treatment. The leaves of wild-type ZH11 and complementary plant COM were yellow overall, and the number of dead plants was less than that of osflp-1, while the overexpression plants of OsFLP gene showed stronger tolerance to salt environment, higher plant height, and relatively It is more stretched, and the number of new leaves is also large. Statistics on the survival rate of rice seedlings treated for 21 days showed that compared with the wild-type ZH11, the survival rate of the osflp-1 mutant was significantly decreased, and the survival rate of the overexpressed plants was significantly increased.

At the same time, we also verified the expression levels of some genes related to salt stress response in ZH11 and osflp-1 mutants, and will also verify the differentially expressed genes.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

sequence listing

<120> An OsFLP protein related to plant salt tolerance and its related biomaterials and applications

<110> Institute of Botany, Chinese Academy of Sciences

<130>GNCSY210905

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 538

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Thr Gly Pro Asp Leu Thr Pro Pro Ala Ala Ala Ala Ser Ala

1 5 10 15

Glu Ala Pro Ser Ala Ser Ala Ala Lys Lys Asp Arg His Ile Val Ser

20 25 30

Trp Ser Thr Glu Glu Asp Asp Val Leu Arg Thr Gln Ile Ala Leu His

35 40 45

Gly Thr Asp Asn Trp Thr Ile Ile Ala Ala Gln Phe Lys Asp Lys Thr

50 55 60

Ala Arg Gln Cys Arg Arg Arg Trp Tyr Asn Tyr Leu Asn Ser Glu Cys

65 70 75 80

Lys Lys Gly Gly Trp Ser Arg Glu Glu Asp Leu Leu Leu Cys Glu Ala

85 90 95

Gln Lys Val Leu Gly Asn Lys Trp Thr Glu Ile Ala Lys Val Val Ser

100 105 110

Gly Arg Thr Asp Asn Ala Val Lys Asn Arg Phe Ser Thr Leu Cys Lys

115 120 125

Arg Arg Ala Lys Asp Asp Glu Leu Phe Lys Glu Asn Gly Ser Leu Cys

130 135 140

Ser Ser Thr Ser Ser Ser Lys Arg Ala Leu Val Gln Thr Gly Cys Leu Thr

145 150 155 160

Ser Gly Ala Ser Gly Ser Ala Pro Pro Ile Lys Gln Met Arg Pro Cys

165 170 175

Asn Ser Asp Phe Lys Glu Asn Met Thr Pro Asn Met Arg Leu Val Gly

180 185 190

Gln Asp Lys Ser Thr Gln Asp Ser Arg Gln Pro Leu Ala Ile Val Tyr

195 200 205

Gln Asn Asn Gln Asp Asn Met Asn Thr Met Asp Thr Gln Asn Leu Val

210 215 220

Ala Lys Thr Ala Ala Lys Gln Leu Phe Ala Gly Glu Gln Asn Cys Val

225 230 235 240

Lys His Glu Gly Asn Phe Leu Asn Lys Asp Asp Pro Lys Ile Ala Thr

245 250 255

Leu Leu Gln Arg Ala Asp Leu Leu Cys Ser Leu Ala Thr Lys Ile Asn

260 265 270

Thr Glu Asn Thr Ser Gln Ser Met Asp Glu Ala Trp Gln Gln Leu Gln

275 280 285

His His Leu Asp Lys Lys Asp Asp Asn Asp Met Ser Glu Ser Ser Ser Met

290 295 300

Ser Gly Met Ala Ser Leu Leu Glu Asp Leu Asp Asp Leu Ile Val Asp

305 310 315 320

Pro Tyr Glu Asn Glu Glu Glu Glu Asp Gln Asp Leu Arg Glu Gln Thr

325 330 335

Glu Gln Ile Asp Val Glu Asn Lys Gln Asn Ser Ser Gln Thr Ser Met

340 345 350

Glu Val Thr Ser Gln Met Val Pro Asp Asn Lys Met Glu Asp Cys Pro

355 360 365

Asn Asp Lys Ser Thr Glu Asp Asn Asn Met Glu Pro Cys Pro Gly Glu

370 375 380

Asp Ile Pro Thr Ser Glu Asn Leu Thr Glu Ala Ala Ile Glu Asp Ser

385 390 395 400

Leu Leu Gln Cys Val Glu Tyr Ser Ser Pro Val His Thr Val Ile Gln

405 410 415

Ala Lys Thr Asp Ala Glu Ile Ala Ala Ser Glu Asn Leu Ser Glu Val

420 425 430

Leu Glu His Asn Arg Leu Gln Cys Ile Gln Leu Ala Ser Pro Ala Gln

435 440 445

Thr Thr Thr Pro Val Glu Ala Asn Ala Glu Thr Pro Ala Ser Glu Lys

450 455 460

Leu Arg Glu Val Val Lys Cys Asn Asn Pro Ser Cys Ile Glu Phe Thr

465 470 475 480

Ser Pro Ala His Thr Val Pro Thr Phe Leu Pro Tyr Ala Asp Asp Met

485 490 495

Pro Thr Pro Lys Phe Thr Ala Ser Glu Arg Asn Phe Leu Leu Ser Val

500 505 510

Leu Glu Leu Thr Ser Pro Gly Ser Arg Pro Asp Thr Ser Gln Gln Pro

515 520 525

Ser Cys Lys Arg Ala Leu Leu Asn Ser Leu

530 535

<210> 2

<211> 1617

<212>DNA

<213> Rice (Oryza sativa)

<400> 2

atggcgaccg gacccgatct gaccccaccc gccgccgccg cctccgccga agcgccgtcg 60

gcgtcggcgg ccaagaagga ccgccacatc gtgagttgga gcaccgagga ggatgatgtg 120

cttcgtactc aaattgcgct tcatggaact gataactgga caattatagc ggcacaattc 180

aaggacaaga cggccagaca gtgcaggaga agatggtaca attatttgaa ctcggagtgc 240

aagaaaggag ggtggtccag agaagaggac ttgctattgt gtgaggctca aaaagttctt 300

gggaacaaat ggactgaaat agcaaaggtt gtctcaggca gaactgataa tgcagtgaag 360

aatcggtttt ctactctatg caaaaggcgg gccaaggatg acgagctatt caaggaaaat 420

ggatcattat gttccagtac aagttcaaag agggcattgg tacaaactgg gtgtctcaca 480

tctggtgcaa gtggctctgc accacctatt aagcagatga ggccatgtaa ttctgatttc 540

aaagagaata tgacaccaaa tatgagatta gttggacaag acaagagcac acaagattct 600

cgacagcctc ttgcaattgt ttatcaaaac aatcaagata atatgaatac aatggatacc 660

caaaatcttg ttgctaaaac tgcagcaaaa caattatttg cgggggaaca gaattgcgtt 720

aagcatgagg gtaatttttt gaacaaggat gatccaaaaa ttgctacttt attgcagcga 780

gccgacttgc tttgctccct agcaacgaaa ataaatactg aaaatacaag ccaaagcatg 840

gacgaagcct ggcagcaact acagcatcat ttggataaga aagatgataa tgacatgtca 900

gagagcagta tgtcgggaat ggcttcactc ctagaggatc ttgacgattt aattgtagac 960

ccctatgaga atgaagagga agaagaccag gatttaaggg agcagaccga acagatgat 1020

gtggagaaca agcaaaattc ttcacaaact agcatggaag ttacatcaca aatggttcct 1080

gacaataaaa tggaggactg cccaaatgat aagagcacag aagacaataa tatggaaccg 1140

tgccctggtg aagacatacc aacatctgaa aacttgactg aggctgctat cgaagatagc 1200

ttgcttcaat gtgtggaata cagctctcct gtacacacag ttattcaagc taaaacagat 1260

gcagaaatag cagcatcgga gaatcttagc gaggttctcg aacataacag gcttcagtgt 1320

attcaattag cctcccctgc tcagacaact accccagttg aagcaaatgc agaaacacca 1380

gcttctgaga agttacgcga ggttgtcaaa tgtaacaatc cttcatgtat cgaattcact 1440

tcgcctgctc atacagtccc aacattcctg ccgtacgcag atgacatgcc gactccaaaa 1500

tttactgcta gtgagaggaa ttttttgctg tctgtgcttg agttgacctc gccagggtcg 1560

aggccagaca cttctcagca gccttcttgc aaaagggctc tccttaatag cctatga 1617

<210> 3

<211> 4733

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

ccctttaccc ccatctcaca ctccacccccc cctctctctc gtttcctcct ctccacccaa 60

cccaacccaa ctcggtcgtc gtcgtcgtct tcctctcgaa tccacggttc aaaaattccc 120

tccactcgcg cccatttccc caaaccctag ccggccccgg ctcgccgccg cccgccgccc 180

gccggctgcc tacctcggcc tgccgggatc cgtgcgcgga acatgcggga tcccggacca 240

tggcgaccgg acccgatctg accccacccg ccgccgccgc ctccgccgaa gcgccgtcgg 300

cgtcggcggc caagaaggac cgccacatcg tgagttggag caccgaggtg cgtacgagtt 360

gtcgtggtct atcgagtcgt gtgtttctgg attttgggag tttttttttt gggtgcgttt 420

gcgctttcct gctcgatttg gttgcctgaa attttggggt tccactcctg tagcaatcat 480

tccccttctt tatttttttg ggggctaata tgcagtggat ttttcacgta attaggcatg 540

acctatcgaa atggactttg gttgggctcc tcgcgcctaa tttttgtggt ttaggcgat 600

attcgtgagg attgagaata attggagttt gaatttgtgt ctacttttca agattttggt 660

tgggttcctc tcacctaatt ctatggttta aggcaatatt cttcgtgagc attgaggata 720

attgaaattt gaatctgtgt accttttaga taatggaaaa gtgcatcaca cttcctggaa 780

tttgtgcctt tcccgctcga tttcgatgcc tagaacatgg gggtcgaatt agttctcgac 840

gcctctagca attattcccc ttctttttgc ctaacgaaca gtagattttt cacatagtta 900

ggcataatct atctaaatgg actttggttt gattcgactc ctgcctattt tttttggtct 960

aaggtgatat ttgtgagcat cgagaatagt cgaagtttga atatgtgtcc ctttcttctt 1020

tttgaaaagt gtattgcatt tccaagaact tgtgcctaca taattataat taattataat 1080

tataatataa ttataattgt tagcttcaga tgctgtcgaa cctttccttg ggagtgcgtc 1140

tgcgattttt tgtttaactt ttagaccaaa ttgtttttct gatgtttgca cctttcttcc 1200

tgttaggagg atgatgtgct tcgtactcaa attgcgcttc atggaactga taagtatgaa 1260

ttcttcccac ttcccatctc aattgacttt ttttttctag atttaatcac catattttac 1320

actgtttttc cttttccttt cggtagctgg acaattatag cggcacaatt caaggacaag 1380

acggccagac agtgcaggag aaggtgtgta cagtgtacct cgccatcctc tgactaaaag 1440

agtggtttta ttggaatttc gttcgaattt tttacgaaat atgcatgccg ccctgttcat 1500

tttttctttg cttggttact gctgcagatg gtacaattat ttgaactcgg agtgcaagaa 1560

aggagggtgg tccagagaag aggacttgct attgtgtgag gtggggatatt gatattgtga 1620

cattttaatt atttggtcca gacactgaat ttccctgaag ttaggaattg ctgcataaaa 1680

cacttagatt tctgatttgg caaatatgcg tcagtttcat gaatttggga attttggcag 1740

acactttgat tttattatta ggaaccattt gtgatgtaac ttttgggcca aactttagat 1800

ttctgaattg gcaaatatgc ggtcggttac ttcctcagtg agacaacagg ccaggcatat 1860

tgctatgccc atggaattga gcatgtacgt atgcagcttt tcagattttt gggaaaagaa 1920

tgggattctt ttgtaaatag caggagatag gatttatatt tttgtatata taagagaagt 1980

ctagatgggc ttgcgcattg attagttttg caatgtcata tttttcttcttattattttg 2040

gctgcaagtt ttatgctctg acagacttgg aactccgcat actggaaacc ctaaaccacc 2100

tgttctgcat agatttgtgg ttgttttcat aattatttgt ttctaccatg gattccttaa 2160

caagattgaa tcatgtattc ttctctactt ggaattgtca tgcaggctca aaaagttctt 2220

gggaacaaat ggactgaaat agcaaaggtt gtctcaggca ggtgggtttc ttgtattatg 2280

cagtttactg caagatgaaa aaaatacatg taataaaata tctgacatgt tttttgtttg 2340

tttgtccaca gcagaactga taatgcagtg aagaatcggt tttctactct atgcaaaagg 2400

cgggccaagg atgacgagct attcaaggaa aatggatcat tatgttccag tacaagttca 2460

aagagggcat tggtacaaac tgggtgtctc acatctggtg caagtggctc tgcaccacct 2520

attaagcaga tgaggtacac ataacactag catcactatt ttctattaag catgcgaagc 2580

aacttacatt tttctcttct gcaactaggc catgtaattc tgatttcaaa gagaatatga 2640

caccaaatat gagattagtt ggacaagaca agagcacaca agattctcga cagcctcttg 2700

caattgttta tcaaaacaat caagataata tgaatacaat ggatacccaa aatcttgttg 2760

ctaaaactgc agcaaaacaa ttatttgcgg gggaacagaa ttgtaagcct gttttcctct 2820

ataatgtgca tttgagtttg atgtatttgt cttgtcccag ctttaatcac agctatgtgc 2880

ataattgcag gcgttaagca tgagggtaat tttttgaaca aggatgatcc aaaaattgct 2940

actttattgc agcgagccga cttgctttgc tccctagcaa cgaaaataaa tactgaaaat 3000

acaagccaaa gcatggacga agcctggcag gtatgacagc accaacttgt tctaaatttg 3060

gaacttaggc ttgcagcctt gtactctatt tgaaataaac tgtgctatgc ctgtttcagc 3120

aactacagca tcatttggat aagaaagatg ataatgacat gtcagagagc agtatgtcgg 3180

gaatggcttc actcctagag gatcttgacg atttaattgt agaccccctat gagaatgaag 3240

aggaagaagaaga ccaggattta aggtaataat catcatctgg tcatcatttt ggttaaatct 3300

attttctcag ttgttgacac tatctaccat tactgtgcaa acaggggagca gaccgaacag 3360

attgatgtgg agaacaagca aaattcttca caaactagca tggaagttac atcacaaatg 3420

gttcctgaca ataaaatgga ggactgccca aatgataaga gcacagaaga caataatatg 3480

gaaccgtgcc ctggtaacct atgctcttaa ttaattacttt ggtaaaatgc tctttaaacc 3540

acatatagaa aatgaactaa attctttaag ctaaaccagg tgaagacata ccaacatctg 3600

aaaacttgac tgaggctgct atcgaagata gcttgcttca atgtgtggaa tacagctctc 3660

ctgtacacac agttattcaa gctaaaacag atgcagaaat agcagcatcg gagaatctta 3720

gcgaggttct cgaacataac aggcttcagt gtattcaatt agcctcccct gctcagacaa 3780

ctaccccagt tgaagcaaat gcagaaacac cagcttctga gaagttacgc gaggttgtca 3840

aatgtaacaa tccttcatgt atcgaattca cttcgcctgc tcatacagtc ccaacattcc 3900

tgccgtacgc agatgacatg ccgactccaa aatttactgc tagtgtaagc tttcaactgc 3960

ttgacatatg atatggccgg ttcatatcga acatataata cttacctgaa aaaaccatat 4020

gacaaatata tttacattta tatttatttg aatttccagg agaggaattt tttgctgtct 4080

gtgcttgagt tgacctcgcc agggtcgagg ccagacactt ctcagcagcc ttcttgcaaa 4140

agggctctcc ttaatagcct atgaaactgt tataagtata tttcagtttt ttttcctgca 4200

ttatggtgt gcctgggatc taaacccaag cctgccatgc atgccccctc agcatgccta 4260

ctattcagcg tgtgttagga ggttctttga catactccta tttgcatcaa gtttggatat 4320

ctcatatcca agtttagctg tttgtatatg agatctcaat tgttttaaga gtctcatagt 4380

ttgtttggtt gtacatattg cattggcggt gttgtagtag catcatacag gaattaaaag 4440

gggcgatggt gtaccaaaga gtttacagct tgtatccaaa atatagaata agtactattc 4500

tttgtgtaat taactcttgt aacctttcta tgttgtacgg aatatattgg tttggtgtgt 4560

agtgtgattt aacagtttcc ttctgactga acaaatgtgg ctgatcctat acggcgaggt 4620

gatattaggt actactagta ttgtatgtgc tccctgatgt tgggaagtta ccttattgat 4680

gctcactgtg ttgtccagta ctccagtttc aatatacatt cttctatgtg ata 4733

Claims (7)

1. Use of a protein for regulating salt tolerance in rice, characterized in that the protein is a protein of the following A1) or A2):

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1);

the regulation of the salt tolerance of the rice is that the salt tolerance of the rice for up-regulating the expression quantity of the protein is enhanced; the salt tolerance of rice with the expression quantity of the protein is reduced.

2. Use of a biological material related to the protein of claim 1 for regulating and controlling salt tolerance of rice, wherein the regulation of salt tolerance of rice is an enhancement of salt tolerance of rice which up-regulates the expression level of the protein; the salt tolerance of rice with the expression quantity of the protein is reduced;

the biomaterial is any one of the following B1) to B5):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1), or a transgenic plant cell line comprising the expression cassette of B2), or a transgenic plant cell line comprising the recombinant vector of B3).

3. The use according to claim 2, wherein the nucleic acid molecule of B1) is a gene as set forth in B1) or B2) below:

b1 A coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;

b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table.

4. A method for cultivating salt-tolerant rice comprising introducing a nucleic acid molecule encoding the protein of claim 1 into a rice of interest to obtain salt-tolerant rice; the salt tolerance of the salt tolerant rice is better than the salt tolerance of the rice of interest.

5. The method according to claim 4, wherein the rice of interest is a rice not containing a nucleic acid molecule encoding the protein of claim 1.

6. A protein, characterized in that it is a protein of the following X1) or X2):

x1) is protein which is obtained by replacing 118 th amino acid residue of the sequence 1 in the sequence table by threonine residue with alanine residue and keeping other sequences unchanged;

x2) a fusion protein obtained by ligating a protein tag to the N-terminus or/and the C-terminus of X1).

7. A biological material related to the protein according to claim 6, which is any one of the following Y1) to Y5):

y1) a nucleic acid molecule encoding the protein of claim 6;

y2) an expression cassette comprising the nucleic acid molecule of Y1);

y3) a recombinant vector comprising the nucleic acid molecule of Y1) or a recombinant vector comprising the expression cassette of Y2);

y4) a recombinant microorganism comprising the nucleic acid molecule of Y1), or a recombinant microorganism comprising the expression cassette of Y2), or a recombinant microorganism comprising the recombinant vector of Y3);

y5) a transgenic plant cell line containing the nucleic acid molecule of Y1), or a transgenic plant cell line containing the expression cassette of Y2), or a transgenic plant cell line containing the recombinant vector of Y3).

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