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 and a related biological material and application thereof. The OsFLP protein can be specifically protein of the following A1), A2) or A3): a1 Amino acid sequence is protein of sequence 1 in a sequence table; a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 90 percent of identity with the protein shown in A1) and has the activity of improving salt tolerance; a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2). The OsFLP protein and related biological materials can be used for improving the salt tolerance of plants.

Description

OsFLP protein related to plant salt tolerance, related biological material and application thereof

Technical Field

The invention relates to an OsFLP protein related to plant salt tolerance and a related biological material and application thereof in the field of biotechnology.

Background

With global climate change, the effect of abiotic stress on plant growth has become one of the major challenges facing humans, severely restricting plant growth and development. Abiotic stress refers to the adverse effects of any abiotic factor on plants in a particular environment, including drought, low temperature, salt and alkali. Drought and salt-alkali are two of the most dominant stress factors that limit plant growth. Especially in the field of global land salinization, the response gene and the action mechanism research of the response gene to plant response to abiotic stress become one of the most hot problems at present, and the application of the response gene to plant response to abiotic stress is today that the cultivated land is less and less. Screening and breeding of genetic varieties related to plant salt tolerance is important for improving agricultural production.

Rice, one of the most important food crops in the world, is smaller than the genome of other crops such as corn, wheat, etc., has a genome length of about 420Mb, and is another important class of pattern plants following arabidopsis (Arabidopsis thaliana). In recent years, rice has been widely used in research of plant genetics, developmental biology and molecular biology. In order to solve the problem of salt-leaching of soil which is serious in crop production, the salt-tolerant gene of rice is cloned and the functions of the salt-tolerant gene are researched, so that the method has important practical significance for cultivating salt-tolerant varieties of crops as soon as possible.

Salt stress can cause osmotic stress and ionic stress, inhibiting normal cell growth and division in plants. To cope with adverse circumstances, plants maintain osmotic and ionic homeostasis through rapid osmotic and ionic signals. High salt osmotic stress can rapidly increase abscisic acid (ABA) biosynthesis, thereby modulating ABA-dependent stress response pathways. Genes for protein kinases, transcription factors, mirnas and reactive oxygen species related proteins can all be involved in salt stress responses through ABA dependent pathways; still other salt-inducing genes are ABA independent. In addition, since salt stress can cause accumulation of osmoprotectant substances in plants, genes related to synthesis of these osmoprotectant macromolecules also play a role in salt stress response; hormones act as molecules that transmit stress signals into cells, and genes in their synthetic and transmission pathways are also involved in salt stress. The plant adversity regulation network is intricate, is full of challenges and significance for research, provides a new thought for solving the problems in the current agricultural production for exploration and application of the rice salt tolerance genes, and has important significance for molecular breeding and research and development of salt tolerance varieties.

Disclosure of Invention

The invention aims to solve the technical problem of improving the salt tolerance of plants.

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

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

a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 90 percent of identity with the protein shown in A1) and has the activity of improving salt tolerance;

a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2).

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, perresidue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biological materials related to OsFLP are also within the scope of the present invention.

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

b1 A nucleic acid molecule encoding an OsFLP;

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 B1);

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).

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.

In the above biological material, the nucleic acid molecule of B1) is a gene represented by B1) or B2) as follows:

b1 A coding sequence (CDS) of the coding strand 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.

Wherein, the sequence 2 in the sequence table consists of 1617 nucleotides, and codes the protein shown in the sequence 1 in the sequence table.

In the above biological material, the expression cassette (OsFLP gene expression cassette) containing the nucleic acid molecule described in B2) refers to a nucleic acid molecule capable of expressing OsFLP in a host cell, and the nucleic acid molecule may include not only a promoter for initiating transcription of the OsFLP gene but also a terminator for terminating transcription of the OsFLP gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful 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: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiology 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are notThe method is 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 terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (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 Acid Res., 15:9627).

The recombinant expression vector containing the OsFLP gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which 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 Co.), pH7WG2D.1, etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos), plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methtrexa, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolizing ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi.

The application of the protein or the biological material in regulating and controlling the salt tolerance of plants also belongs to the protection scope of the invention.

In order to solve the technical problems, the invention also provides a plant reagent which is used for improving the salt tolerance of plants.

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

The active ingredient of the plant agent may be the protein or/and the biological material related to the protein, and the active ingredient of the plant agent may further contain other biological components or/and non-biological components, and the other active components of the plant agent may be determined by those skilled in the art according to the nitrogen absorption effect of plants.

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

The method for cultivating salt-tolerant plants provided by the invention comprises the steps of introducing nucleic acid molecules encoding the protein into target plants to obtain salt-tolerant plants; the salt tolerant plants have a salt tolerance that is better than the salt tolerance of the plant of interest.

The invention also provides a protein which is salt-sensitive, said protein being a protein of the following X1), X2) or X3):

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 protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein of X1) and having an identity of 90% or more to the protein represented by X1) and having an activity of enhancing salt sensitivity;

x3) a fusion protein obtained by ligating a protein tag to the N-terminus or/and the C-terminus of X1) or X2).

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

y1) a nucleic acid molecule encoding said salt-sensitive protein;

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 Y1);

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).

The invention also provides a method for cultivating salt-sensitive plants, which comprises the steps of introducing nucleic acid molecules encoding the salt-sensitive proteins into target plants to obtain salt-sensitive plants; the salt-sensitive plant has a higher sensitivity to salt than the plant of interest.

The plant of interest according to the invention may be a monocot or a dicot which does not contain nucleic acid molecules encoding the proteins. The monocotyledonous plant may be millet, rice.

In the method of the invention, the nucleic acid molecule can be modified as follows before being introduced into the target plant to achieve better expression effect:

1) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

3) The expression efficiency of the gene of the invention can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present invention;

4) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

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

The salt tolerant plants or the salt sensitive plants of the invention may be transgenic plants, or plants obtained by conventional breeding techniques such as crossing.

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

Transgenic experiments of introducing the OsFLP gene into rice prove that compared with receptor rice, transgenic rice expressing the OsFLP gene remarkably promotes salt tolerance, which indicates that the OsFLP gene is a gene related to salt tolerance, and can be used for improving the salt tolerance of plants and improving the stress resistance of the plants.

Drawings

FIG. 1 is a sequence comparison of OsFLP protein from oryza sativa with known R2R3-MYB family proteins in example 1.

FIG. 2 is a GUS staining chart of plant tissues of an OsFLP positive transformed plant in example 1, wherein A is a 3D germinated rice seedling, B is a main root, C is a center pillar of the root, D is a mature stomata, E is a seedling neck node, and F is a neck node partial enlarged view of E.

FIG. 3 is a bar graph showing the OsFLP gene expression level in wild-type rice at different NaCl treatment periods in example 1, wherein the data are expressed as mean value.+ -. Standard deviation, the repetition number is 3, and the significance analysis is carried out by using Student's t test, and the significance analysis result is P < 0.01.

FIG. 4 is a graph showing analysis of mutation sites of the osflp-1 mutant in example 2.

FIG. 5 is a plasmid map of pH7WG2D.1 in example 3.

Fig. 6 is a photograph and survival bar graph of example 3 after 21 days of salt treatment of 4 materials. Wherein, fig. 6 a is a photograph of a control group of 4 materials which are not subjected to salt treatment, fig. 6B is a photograph of an experimental group of 4 materials which are subjected to salt treatment, fig. 6C is a statistical histogram of survival rates of the 4 materials treated group and the control group, data are represented as mean value ± standard deviation, the number of repetitions is 3, and significant analysis is performed by using Student's t test, wherein x represents that the result of the significant analysis is P < 0.01.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples described below are all conventional biochemical reagents, unless otherwise specified, and are commercially available.

1 vector

The vector pCAMBIA1301 in the following examples is a product from the biological technology limited company, bordetella, beijing, cat No.: VT3013.

The carrier pH7WG2D.1 in the following examples is a product of the China plasmid vector strain cell line Gene Collection (Biovector), cat#: bioVector921816.

The carrier TOPO vector (cat# CV 0402) in the following examples is a product of Beijing Aide technologies Co., ltd.

2 plant lines

The rice variety ZH11 (Oryza sativa L.ssp. Japonica cv. Zhonghua 11, ZH 11) in the following examples is described in non-patent document "OsPGIP1-Mediated Resistance to Bacterial Leaf Streak in Rice is Beyond Responsive to the Polygalacturonase of Xanthomonas oryzae pv.oryzicola". The public is available from plants of the chinese sciences to repeat the experiments of the present application and cannot use it as other uses.

4 reagent

Gateway LR Clonase II enzyme mix (product number: 11791019) is manufactured by Semer Feishmania technology (China) Co.

In the following examples, unless otherwise specified, the 1 st 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 obtaining plant salt tolerance-related Gene OsFLP

1. Rice OsFLP protein sequence analysis

The amino acid sequences of the Arabidopsis AtFLP were used for alignment in NCBI (http:// www.ncbi.nlm.nih.gov /) and rice genome database (http:// rice. Plant biology. Msu. Edu /), and homologous proteins of the Arabidopsis AtFLP were found. As a result, a protein having 33.89% similarity with the amino acid sequence of AtFLP was obtained, and designated as OsFLP, the amino acid sequence of which is shown as sequence 1 in the sequence table.

By comparison analysis with several known R2R3-MYB family protein sequences, including Arabidopsis AtFLP, atMYB88, atMYB121, ATMYB0 (ATGL 1) and rice OsMPS protein sequences, the results are shown in FIG. 1, and it was found that two MYB domains of R2 and R3 are present at the N-terminal of the OsFLP protein. Wherein R2 comprises three conserved motifs of H1, H2 and H3, and R3 comprises three conserved motifs of H1, H2 and H3, and the OsFLP is proved to be an R2R3-MYB family protein.

2. Cloning of Rice OsFLP Gene

The gene for encoding the OsFLP protein is named as OsFLP, and the gene is coded as Os07g0627300 in a rice genome database and is derived from the rice Nipponbare ecotype. The OsFLP gene is located on chromosome seven and has 12 exons in total.

100-200mg of young rice is taken, total RNA is extracted by using an RNA extraction kit, and a certain amount of single-stranded cDNA is synthesized by reverse transcription according to the concentration of the extracted RNA after nitrification. The OsFLP gene amplification conditions were as follows, and the synthesized single-stranded cDNA was diluted 4-fold as a template for PCR reaction: the full length of cDNA of OsFLP was amplified using high-fidelity DNA polymerase KOD-Plus from TOYOBO Co, in an amplification system of 50. Mu.L containing 10 XKOD buffer 5. Mu.L, 2mM dNTP mix 5. Mu.L, 1.5. Mu.L each of Primer1 and Primer2, 0.5. Mu.L of KOD-Plus DNA polymerase, and 1-5. Mu.L of template, supplemented with sterilized ultrapure water to 50. Mu.L, and the reaction system preparation was performed on ice.

Primer 1：5’-ATGGCGACCGGACCCGATCTG-3’；

Primer 2：5’-TCATAGGCTATTAAGGAGAGC-3’。

The amplification was performed on a BIO-RAD PCR amplification apparatus, pre-denatured for 5min, denatured for 30s at 94℃for 30s, and for 2min at 54℃for 68℃for 32 cycles, and extended for 10min at 68 ℃. And (3) carrying out electrophoresis on the amplified fragments by using 1% agarose gel, recovering the amplified fragments, connecting the amplified fragments to a carrier Blunt, and carrying out enzyme digestion and sequencing identification to obtain the correct OsFLP gene cDNA (see sequence 2 of a sequence table).

3. Rice OsFLP expression pattern analysis

Constructing an OsFLP-GUS expression vector by using a plant tissue pattern expression vector pGUS1301 and the OsFLP gene cDNA obtained in the step 2, transforming ZH11 wild rice, performing GUS staining on a positive transformed plant, and analyzing the OsFLP expression pattern.

The staining results are shown in FIG. 2, and OsFLP can be expressed at the basal part of germinated rice seeds and expressed at the aerial parts and the root parts. Expressed in root tip and mature stomata, and expressed in overground part, the expression quantity at neck node is high.

4. Expression analysis of rice OsFLP gene after salt treatment

The ZH11 wild rice seedlings growing for 2 weeks are taken, the root parts of the seedlings are treated with 200Mm NaCl for 0h, 2h, 4h, 6h, 12h and 24h respectively in a water culture tube, total RNA of the plants is extracted, 3 repeats are arranged, qRT-PCR is carried out to detect the relative expression level of the OsFLP, and the OsFLP primers and the internal reference primers are as follows (the OsFLP primers are Primer3 and Primer4; and the internal reference primers are Primer5 and Primer 6).

Primer 3：5’-AACTGGACAATTATAGCGGCAC-3’；

Primer 4：5’-GGAATGTTGGGACTGTATGAGC-3’。

Primer 5：5’-GGATCCATCTTGGCATCTCTCA-3’；

Primer 6：5’-GGGCCAGACTCGTCGTACTC-3’。

As a result, as shown in FIG. 3, the OsFLP gene expression was induced to be up-regulated by salt stress, and the relative content of OsFLP in rice was significantly higher than that in normal condition at different salt treatment times, and the expression level was highest at 6h treatment time.

Example 2 acquisition of Gene OsFLP mutant

1. Obtaining osflp-1 mutant by TILLING technology

TILLING (Targeting Induced Local Lesions IN Genomes, directed induction of local genome mutations) is a novel reverse genetics research method, which utilizes the chemical mutagen ethyl methylsulfonate to mutagenize seeds, and then rapidly and effectively identifies point mutations from the mutagenized population by means of high-throughput sequencing. In order to better analyze the function of OsFLP in rice, we obtained OsFLP mutant strains with different mutation sites by using TILLING technology, and the specific results are shown in Table 1. We observed and compared the obtained strains with different mutation sites, and found a strain A2349-2 with a phenotype different from that of wild type stomata, and named as osflp-1 mutant.

TABLE 1

2. Analysis of mutant mutation sites of osflp-1

We designed the corresponding primers to amplify the OsFLP genomic DNA in ZH11 and OsFLP-1 mutants. Through sequencing, the nucleotide sequence of the genomic DNA of the OsFLP of ZH11 is shown as sequence 3 of a sequence table, and the 2126 th position after the ATG of the start codon of the OsFLP gene in the OsFLP-1 mutant is mutated into a base A, and the mutation site is positioned on the sixth exon of the gene. Namely, the 352 th base of the OsFLP CDS (sequence 2 of the sequence table) is mutated from G to A, so that the 118 th amino acid residue of the sequence 1 in the sequence table is replaced by threonine residue (Thr) from alanine residue (Ala), the amino acid is changed, the polarity of the amino acid is changed from nonpolar to polar, the hydrophobic amino acid is changed into hydrophilic amino acid, and the amino acid quality is changed.

Example 3 obtaining transgenic OsFLP Rice

1. Construction of OsFLP gene over-expression vector and acquisition of rice transgenic material

The OsFLP gene overexpression vector is constructed by taking pH7WG2D.1 (plasmid map is shown in figure 5) as a starting vector and adopting a gateway system method, and is specifically implemented as follows: cDNA fragments of OsFLP (nucleotide sequence is sequence 2) were amplified, and after gel recovery, the cDNA fragments of OsFLP were ligated to an entry vector TOPO vector, and the resulting plasmid was named OsFLP-TOPO. Plasmid OsFLP-TOPO is digested for 3 hours at 37 ℃ by PVU I restriction enzyme (recognition site: CGATCG, the cut fragment has viscosity, which belongs to restriction enzyme II), and the enzyme product is separated by agarose gel electrophoresis and cut into gel for recovery. The recovered 1.5. Mu.L of the product, 0.5. Mu.L of pH7WG2D.1 plasmid and 0.5. Mu.L of Gateway LR Clonase II enzyme mix were mixed and reacted at 25℃for 4 hours for ligation. After termination of the reaction, the mixture was transformed into competent cells of E.coli DH 5. Alpha. To obtain monoclonal colonies. And breeding single colonies, extracting plasmids, and carrying out PCR identification on the plasmids to obtain plasmids with correct sequences. The resulting recombinant expression vector was designated Super-pH7WG2D.1-OsFLP. Super-pH7WG2D.1-OsFLP contains an OsFLP gene with a nucleotide sequence of sequence 2, and can express a protein OsFLP with an amino acid sequence of sequence 1.

Competent cells of the agrobacterium tumefaciens strain EHA105 are transformed by the plasmid Super-pH7WG2D.1-OsFLP, positive clones transferred into the Super-pH7WG2D.1-OsFLP (named EHA 105/Super-pH7WG2D.1-OsFLP) are identified by a colony PCR and enzyme digestion method, and rice wild type ZH11 is transformed by the EHA105/Super-pH7WG2D.1-OsFLP, so that the OsFLP gene over-expressed rice is named OE.

2. Vector construction of OsFLP gene complementary OsFLP-1 and positive plant acquisition

The rice complementation vector was constructed by using pCAMBIA1301 as a starting vector, the osflp-1 mutant in example 2 was transformed, and the obtained transformed seedlings were screened on a hydroponic medium containing 50. Mu.g/. Mu.L hygromycin, T ₂ The seeds of the generation also need to be screened by hygromycin, and the obtained positive plants are complementary materials and named as COM.

3. Phenotypic analysis of salt tolerance of rice OsFLP-1 mutant and OsFLP gene overexpression strain

T3 generation plants of 4 rice materials in total were used as experimental materials, with wild ZH11, mutant OsFLP-1 (see example 2), complementary material COM (construction 2 in example 3) and plant OE overexpressing the OsFLP gene (1 in example 3).

In a paddy field growth environment, salt treatment is carried out on paddy seedlings growing for 20 days by an experimental group, salt solution is poured into the seedlings, the salt concentration is 200mM NaCl, phenotypes are observed after the salt pouring treatment, the survival rate is counted, and the salt solution is replaced by water by a control group (control). Each material was treated with 12 strains per treatment, with a repetition number of 4.

The results showed that after 15 days of salt-watering treatment, there was no difference in the growth state of the 4 material seedlings in the control group, whereas the salt-treated group seedlings showed stress states of yellowing of the leaves, curling sagging, growth arrest, and the like. Under the treatment of the same salt concentration, 4 seedlings have different stress responses, the phenomena of yellowing and drying out of leaves of the OsFLP-1 mutant are more obvious, and the growth height and the growth state of the whole plant of the OsFLP over-expression plant are better. The results after 21 days of salt treatment are shown in fig. 6, and the control groups of 4 materials still show normal growth states without difference; in the experimental group, osflp-1 almost all died, and no new leaf was grown, showing high sensitivity to salt treatment; the wild ZH11 and the complementary plant COM have yellow leaf, the death plant number is smaller than osflp-1, the OsFLP gene over-expression plant shows stronger tolerance to salt environment, the plant height is higher, the leaf is relatively more stretched, and the number of new leaf is more. Statistical observation of the survival rate of rice seedlings treated for 21 days shows that compared with wild-type ZH11, the survival rate of the osflp-1 mutant is remarkably reduced, and the survival rate of the over-expressed plants is remarkably increased.

Meanwhile, we also verify the expression levels of some genes related to salt stress response in ZH11 and osflp-1 mutants, and also verify the genes differentially expressed therein.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<120> an OsFLP protein related to plant salt tolerance, and biological material and application thereof

<110> institute of plant Material at national 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 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 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 acagattgat 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 ctccaccccc 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 tttaggcgat 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 gtgggatatt 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 tttttcttct tattattttg 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 agacccctat gagaatgaag 3240

aggaagaaga ccaggattta aggtaataat catcatctgg tcatcatttt ggttaaatct 3300

attttctcag ttgttgacac tatctaccat tactgtgcaa acagggagca gaccgaacag 3360

attgatgtgg agaacaagca aaattcttca caaactagca tggaagttac atcacaaatg 3420

gttcctgaca ataaaatgga ggactgccca aatgataaga gcacagaaga caataatatg 3480

gaaccgtgcc ctggtaacct atgctcttaa ttattacttt 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

ttattggtgt 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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