CN116751270A

CN116751270A - Method for improving salt tolerance of plants, protein used by method and related biological material

Info

Publication number: CN116751270A
Application number: CN202310687584.3A
Authority: CN
Inventors: 姚琴芳; 姚松泉; 张卫良; 邱芬; 慎思杰; 倪佳希
Original assignee: Huzhou Songquan Agricultural Technology Co ltd
Current assignee: Huzhou Songquan Agricultural Technology Co ltd
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2023-09-15

Abstract

The application discloses a method for improving salt tolerance of plants, and protein and related biological materials used by the method. The method for improving the salt tolerance of the plants comprises the following steps: enhancing the expression of the protein RIP1 in the recipient plant, and obtaining the target plant with salt tolerance higher than that of the recipient plant. Salt stress experiments of arabidopsis mutants introduced with RIP1 genes prove that the expression of the RIP1 genes is enhanced, the salt tolerance can be improved, and NaCl salt stress with the concentration of up to 450mM can be tolerated. Therefore, the salt tolerance can be regulated and controlled by reasonably utilizing the RIP1 gene, and the effective utilization of saline-alkali soil resources is facilitated.

Description

Method for improving salt tolerance of plants, protein used by method and related biological material

Technical Field

The application relates to a method for improving plant salt tolerance in the field of biotechnology, and protein and related biological materials used by the method.

Background

Salt stress is one of the major limiting factors in plant growth and yield, and can cause a series of physiological and metabolic reactions in plants, resulting in plant yield loss and even death. The damage of cultivated land resources and the loss of agricultural production caused by soil salinization become a worldwide ecological problem. Since most plants, especially crops, are sensitive to salt stress, soil salinization has become an important factor limiting global agricultural productivity. The practice proves that the improvement of the saline soil is a complex, difficult and long-time work, so that the mechanism of the plant for coping with the salt stress is revealed, and the salt tolerance of the plant is improved accordingly, and the improvement of the saline soil has become an important foundation for promoting agricultural production.

The complexity of plant salt tolerance makes it very difficult to improve crop salt tolerance by traditional breeding methods, and research in recent years shows that plant salt tolerance is complex, multiple mechanisms of cell adaptation and multiple metabolic pathways are involved, and in order for plants to reach ion balance, three aspects of interrelated regulation must be performed, firstly plants must be prevented from being poisoned, secondly plants must reestablish a balanced in-vivo environment in adverse conditions, and finally growth must be restored. Thus, genetically designing salt tolerant crops using biotechnology methods is a research hotspot in the current agricultural field.

Many genes related to plant salt tolerance have been cloned and studied, including genes encoding various organic solute synthases: such as the P5cs gene involved in proline synthesis, the gene involved in betaine synthesis, the gene involved in mannitol synthesis; a LEA protein gene; peroxidase genes with antioxidant stress effects, etc., but most of them need to act together with other salt-tolerant genes to achieve a desirable salt-tolerant effect. It is desirable to purposefully clone other salt tolerance genes from different species of plants that do not affect the normal growth of the plant in order to obtain crops with good salt tolerance.

Disclosure of Invention

The application aims to solve the technical problem of improving the saline-alkali resistance of plants.

In order to solve the technical problems, the application aims to provide a method for improving salt tolerance of plants through protein RIP1, which comprises the following steps: enhancing expression of protein RIP1 in a recipient plant to obtain a target plant with salt tolerance higher than that of the recipient plant; the protein RIP1 is a protein of the following A1), A2) or A3):

a1 Amino acid sequence is protein with SEQ ID NO. 2 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), is derived from sorghum and has the same activity;

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

In the method, SEQ ID No. 2 of the sequence Listing consists of 126 amino acid residues.

In the above method, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with the 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 method, 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, per residue 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 method, the 90% identity or more may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above method, the enhanced expression of the RIP1 protein is achieved by introducing a nucleic acid molecule encoding said protein into a recipient plant.

In the above method, the nucleic acid molecule may be modified as follows before being introduced into the recipient plant to achieve better expression:

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 application 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 application;

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,Plant Molecular Biology (2 nd Edition).

In the above method, the recipient plant may be a transgenic plant, or a plant obtained by conventional breeding techniques such as crossing.

In the above methods, the transgenic plants 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.

In the above method, the recipient plant may be a monocot or dicot. The monocot plant may be sorghum, maize, rice or wheat; the dicot may be arabidopsis, soybean or cotton.

The application also protects the protein RIP1.

The present application also protects a biomaterial related to the protein RIP1 described above, which is any one of the following B1) to B4):

b1 Nucleic acid molecules encoding the protein RIP 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), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism 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) may specifically be a nucleic acid molecule as shown in any one of the following B1) to B4):

b1 The coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID NO. 1 in the sequence table;

b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID NO. 1 in the sequence table;

b3 A cDNA molecule or a DNA molecule having 80% or more identity to the nucleotide sequence defined in b 1) or b 2) and encoding a RIP1 protein;

b4 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the defined nucleotide sequence of any of b 1) to b 3) and which codes for a RIP1 protein.

Wherein, SEQ ID NO. 1 in the sequence table consists of 381 nucleotides, and codes for protein shown in SEQ ID NO. 2 in the sequence table.

The stringent conditions may be hybridization in 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS, respectively.

In the above biological material, B2) the expression cassette (RIP 1 gene expression cassette) containing the nucleic acid molecule of B1) means a nucleic acid molecule capable of expressing RIP1 in a host cell, and the nucleic acid molecule may include a promoter for initiating transcription of the RIP1 gene. Promoters useful in the present application 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: constitutive promoter 35S of cauliflower mosaic virus (CAMV); ubiquitin (Ubiquitin) gene promoter (pUbi); wound-inducible promoter 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 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin and soybean beta-glycin (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.

In the above biological material, the expression cassette of B2) containing the nucleic acid molecule may further comprise a terminator that terminates RIP1 transcription. Further, the expression cassette may also include an enhancer sequence. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S 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 RIP1 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 pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin (CAMBIA Co.) and the like. 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 application 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 can produce a color change (GUS gene, GFP 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 the antibiotic hygromycin, dhfr gene conferring resistance to methatrexa, EPSPS gene conferring resistance to glyphosate) or anti-chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. 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 also protects the application of the method in salt-tolerant plant breeding.

The application also protects the application of the protein and the biological material in salt-tolerant plant breeding or improving the salt tolerance of plants.

The application also provides the use of a cell line, which is a transgenic plant cell line comprising a nucleic acid molecule of the protein RIP1, in the breeding of salt tolerant plants or for improving the salt tolerance of plants.

In the above application, the plant may be a monocot or dicot. The monocot plant may be sorghum, maize, rice or wheat; the dicot may be arabidopsis, soybean or cotton.

In order to solve the technical problems, the application also provides a plant reagent which is used for improving the salt tolerance of plants. The plant reagent provided by the application contains the protein RIP1 and/or the biological material.

The application also provides application of the plant reagent in salt-tolerant planting of plants.

Salt stress experiments of arabidopsis mutants introduced with RIP1 genes prove that the expression of the RIP1 genes is enhanced, the salt tolerance can be improved, and NaCl salt stress with the concentration of up to 450mM can be tolerated. Therefore, the salt tolerance can be regulated and controlled by reasonably utilizing the RIP1 gene, and the effective utilization of saline-alkali soil resources is facilitated.

Drawings

FIG. 1 is a chart showing total RNA electrophoresis of sorghum tissues under salt stress in example 1 of the present application.

FIG. 2 is a flow chart showing construction of a recombinant expression vector SQKJ-Sb of sorghum cDNA library fragments under salt stress in example 1 of the present application.

FIG. 3 is an electrophoretogram of the present application after PCR amplification of cDNA inserts in sorghum library plasmids under salt stress in example 1.

FIG. 4 shows transgenic positive seedlings of sorghum library plasmid transformed Arabidopsis under salt stress in example 2 of the present application.

FIG. 5 is a graph showing the salt tolerance of Arabidopsis plants transformed with RIP1 gene under salt stress in example 3 of the present application during library screening.

FIG. 6 is a graph showing the salt tolerance of Arabidopsis plants transformed with RIP1 gene under salt stress in example 4 of the present application, upon preliminary verification.

FIG. 7 is a graph showing the salt tolerance of Arabidopsis plants transformed with RIP1 gene under salt stress in example 4 of the present application when they were re-verified.

FIG. 8 is an electrophoretogram of the transgenic Arabidopsis plant of example 5 of the present application after PCR amplification of the exogenous insert.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application 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 application 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 and Strain

In the following examples, vector pCAMBIA2300-35S-OCS is described in non-patent literature, "salty ocean et al," construction of pCAMBIO2300-betA-BADH bivalent plant expression vector, chinese agronomic bulletin 2009, 25 (09): 47-50', publicly available from Song spring agricultural technologies Inc. of Huzhou to repeat the experiments of the present application, are not available for other uses.

In the following examples, vector SQKJ was modified on the backbone of vector pCAMBIA2300-35S-OCS, vector pCAMBIA2300-35S-OCS and vector pCAMBIA3301 (VT 1386, ubao organism) were digested with restriction enzyme XhoI, and the NPT II gene fragment on vector pCAMBIA2300-35S-OCS was replaced with the Bar gene fragment on vector pCAMBIA3301 to construct a new SQKJ vector.

Coli XL1-Blue competent cells (accession number BTN 90504) were produced by the company Bai-Lai in the examples described below.

Agrobacterium GV3101 competent cells (product number Waryong GT 707) were obtained from Beijing Walker biological company in the examples described below.

2 plant lines

In the examples described below, sorghum cultivar BTx623 is described in the non-patent literature "Paterson AH, bowers JE, bruggmann R, dubchak I, grimwood J, gundlach H, haber G, hellsten U, mitros T, poliakov A, schmutz J, spannagl M, tang H, wang X, wicker T, bharti AK, chapman J, feltus FA, gowik U, grigoriev IV, lyons E, maher CA, martis M, narechania A, otillar RP, penning BW, salamov AA, wang Y, zhang L, carpita NC, freeling M, gille AR, hash CT, keller B, klein P, kresovich S, mcCann MC, ming R, peterson DG, mehboob-ur-Rahman, ware D, westhoff P, mayer KF, messaging J, rokhsar DS.the Sorghum bicolor genome and the diversification of gratings.Nature.2009 Jan29;457 (7229) 551-6.doi:10.1038/nature07723.PMID:19189423 ", publicly available from Songquan agricultural technologies Inc. of Huzhou, to repeat the experiments of the present application, are not useful for other purposes.

In the examples described below, wild type Arabidopsis thaliana (Arabidopsis thaliana) is a Columbia-0 subtype, abbreviated as wild type Arabidopsis thaliana Col-0, and is described in the non-patent literature "Kim H, hyun Y, park J, park M, kim M, kim H, lee M, moon J, lee I, kim J.A genetic link between cold responses and flowering time through FVE in Arabidopsis thiana. Nature genetics 2004, 36:167-171", available to the public from Song spring agricultural technologies Inc. of Huzhou, to repeat the experiments of the present application, and is not useful for other applications.

3. Culture medium and plate

The formulation of the LB liquid medium in the following examples is: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of NaCl and adjusting the pH to 7.5 with NaOH.

The LB solid plates containing kanamycin (50 mg/L) in the following examples were plates prepared with LB solid medium containing kanamycin (50 mg/L) in the following formulation: kanamycin 50mg/L, tryptone 10g/L, yeast extract 5g/L, naCl10g/L, agar 15g/L, and NaOH was used to adjust the pH to 7.5.

The formulation of LB liquid medium containing kanamycin (50 mg/L) in the following examples was: kanamycin 50mg/L, tryptone 10g/L, yeast extract 5g/L, naCl10g/L, and NaOH was used to adjust the pH to 7.5.

LB solid plates containing 50mg/L of Rifampicin and 50mg/L of kanamycin in the following examples were plates prepared with LB solid medium containing 50mg/L of Rifampicin (Rifampicin) and 50mg/L of kanamycin, which had the following formulation: rifampicin 50mg/L, kanamycin 50mg/L, tryptone 10g/L, yeast extract 5g/L, naCl10g/L, agar 15g/L, and pH adjusted to 7.5 with NaOH.

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 construction of full-length sorghum cDNA library

Taking salt-treated (sorghum is subjected to water culture through a Mucun culture solution, the formula of the Mucun culture solution is referred to Ishikawa et al BMC Plant Biology 2011,11:172, after three weeks of normal culture, the sorghum is put into saline water with 350mM NaCl for water culture for 3 hours, 6 hours, 12 hours and 24 hours, and then sampling and mixing are carried out to prepare total RNA of the sorghum variety BTx623 tissue, wherein the total RNA integrity is shown in figure 1, the ratio of the total RNA at 260nm/280nm is 1.8-2.0, and the purity is higher. Then, after mRNA separation, dephosphorylation, CAP removal and CAP Tag connection treatment, the first strand of cDNA is synthesized from the purified mRNA, and then double-stranded DNA is synthesized, and after enzyme digestion, the double-stranded DNA is subjected to size classification by agarose gel electrophoresis, and the following fragment sizes are respectively recovered by tapping: 2-12kb,1-2kb,0.5-1kb, <0.5kb.

The above fragment and the expression vector SQKJ recovered by the restriction enzymes KpnI and BamHI, respectively, were digested, the digested gene fragment was inserted into the multiple cloning site of the expression vector SQKJ, and the vector was constructed by the digestion method known to those skilled in the art to construct a recombinant expression vector SQKJ-Sb mixture, the construction procedure of which is shown in FIG. 2 (FIG. 2), kanamycin (R): kanamycin gene, prCaMV35s: cauliflower virus promoter (SEQ ID No: 3), sorgum bicolor: sorghum library gene, OCS: terminator (SEQ ID No: 4), bar: glufosinate-resistance gene (SEQ ID No: 5).

Then the recombinant expression vector SQKJ-Sb mixture is used for transforming competent cells of the escherichia coli XL1-Blue by a shock method, and the shock conditions are as follows: 50. Mu.L of E.coli XL1-Blue competent cells, 1. Mu.L of library plasmid DNA (recombinant expression vector SQKJ-Sb mixture), after electric shock transformation under the preset program Ec2 (2.5 kV,6.0 ms), were cultured in LB liquid medium at 37℃for 1 hour with shaking (shaking of shaking table at 200 rpm), and grown overnight on LB solid plates containing kanamycin (50 mg/L). White clones were picked and PCR identification was performed on the transformed recombinant e.coli with primer pairs consisting of primer 1 and primer 2:

primer 1:5'-ccaaccacgtcttcaaagca-3' (SEQ ID No. 6);

primer 2:5'-tcatgcgatcataggcgtct-3' (SEQ ID No: 7).

The size of the cDNA insert was detected by electrophoresis, the proportion of recombinant clones was calculated, and 5' -end sequencing of cDNA was performed simultaneously, and the proportion of full-length cDNA in the library was analyzed. Obtained byThe library capacity of sorghum library was 2X 10 ⁶ Library recombination rate was 96.1% and full length was 95.2%, as shown in FIG. 3.

Example 2 transformation of full-length sorghum cDNA library with Arabidopsis thaliana

1. Extraction of full-Length cDNA library plasmid

All sorghum library plaques from example 1 were scraped and incubated in 500mL LB liquid medium containing kanamycin (50 mg/L) at 37℃for 30min. Extracting plasmids by a small tube alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial cells with 100 μl of ice-precooled solution I (25 mM Tris-HCl,10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH 8.0); 200. Mu.L of freshly prepared solution II (0.2M NaOH,1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 5 times, mixed, and placed on ice for 3-5min; adding 150 μl ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 4deg.C and 12000rpm for 5min, collecting appropriate amount of supernatant, adding 2 times volume of absolute ethanol into the supernatant, mixing, and standing at room temperature for 5min; centrifuging at 4deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; 50. Mu.L of TE (10 mM Tris-HCl,1mM EDTA,pH8.0) containing RNase (20. Mu.g/mL) was added per vial to dissolve the pellet; digesting RNA in a water bath at 37 ℃ for 30 min; preserving at-20 ℃ for standby to obtain the full-length cDNA library plasmid.

2. Full-length cDNA library plasmid Agrobacterium transformation

The full-length cDNA library plasmid constructed in the step 1 is transformed into agrobacterium GV3101 by an electric shock method, and the transformation conditions are as follows: 1 μl of full-length cDNA library plasmid was mixed with 50 μl of Agrobacterium GV3101 competent cells, allowed to stand on ice for 5min, transferred to a pre-chilled electric shock cup, subjected to electric shock transformation under a preset program Ec2 (2.5 kV,6.0 ms), inoculated with the transformed Agrobacterium GV3101 in LB liquid medium, cultured at 28℃and 200rpm for 1 hour, and plated on LB solid plates containing 50mg/L rifampicin and 50mg/L kanamycin until positive monoclonal was developed. White clones were picked, and the transformed recombinant Agrobacterium was also PCR-identified using a primer pair consisting of the primer 1 (shown as SEQ ID No: 6) and the primer 2 (shown as SEQ ID No: 7), and the size of the cDNA insert was detected by electrophoresis.

3. Arabidopsis transformation of full-length cDNA library plasmid

Wild-type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were kept at 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds. The horsemanure was mixed with vermiculite and irrigated with water to soil moisture, and the soil mixture was allowed to drain for 24 hours. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 4 days. Germinating the seeds and providing a constant humidity (40-50%) at a constant temperature (22deg.C) with a light intensity of 120-150 μmol/m ² Culturing under long-day conditions (16 hours light/8 hours dark) for seconds. Plants were initially irrigated with Hoagland's nutrient solution followed by deionized water to keep the soil moist but not wet.

All the recombinant Agrobacterium plaques obtained in step 2 were scraped off and the Arabidopsis was transformed using the floral dip method. Seeds were harvested approximately 4 weeks after soaking transformation.

4. Positive rate detection of transgenic arabidopsis thaliana

Arabidopsis seeds (T) infected with recombinant Agrobacterium containing full-length cDNA library plasmids ₀ The generation seeds) contain a large number of transgenic seeds, and the transgenic proportion, namely the positive rate of transgenic arabidopsis, is determined through resistance screening. Through glufosinate resistance screening, transgenic seedlings with glufosinate resistance can grow normally, wild seedlings without glufosinate resistance die, the number of the transgenic seedlings is counted, and the positive rate is calculated. The positive rate of transgenic Arabidopsis thaliana was substantially about 2% (as shown in FIG. 4).

EXAMPLE 3 Arabidopsis screening of full-length sorghum cDNA library

To rapidly and efficiently screen salt-resistant Arabidopsis plants in a full-length cDNA library, T in example 2 ₀ After germination of Arabidopsis seeds, a glufosinate resistance screening is carried out at the seedling stage, and then a salt treatment is carried out before bolting so as to obtain the product with the period of T ₀ The salt resistance gene is found. The method comprises the following specific steps:

to make more effective use of temperatureChamber and seedling pot resources, 100-150 transgenic plants are expected to be reserved in each seedling pot, and T for sowing of one pot of arabidopsis is calculated according to the arabidopsis positive rate of about 2 percent ₀ 5000-7500 seeds.

Will T ₀ The seeds of Arabidopsis thaliana were suspended in 0.1% agarose solution and stored at 4℃for 2 days. The horsemanure was mixed with vermiculite and irrigated with water to soil moisture, and the soil mixture was allowed to drain for 24 hours. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 4 days. Germinating the seeds and providing a constant humidity (40-50%) at a constant temperature (22deg.C) with a light intensity of 120-150 μmol/m ² Culturing under long-day conditions (16 hours light/8 hours dark) for seconds. When arabidopsis thaliana grows to 7 days, foliar spray is performed with a pilot (bayer corporation) solution at a dilution ratio of 1:400, allowing non-transgenic arabidopsis thaliana to die (i.e., a glufosinate resistance screening is performed).

When the remaining transgenic arabidopsis grows for 12-14 days, watering is firstly carried out until the transgenic arabidopsis is saturated, and then watering is stopped; the water content measurement is started when the transgenic arabidopsis thaliana grows to 18-20 days, and when the water content is reduced to 25-45%, the transgenic arabidopsis thaliana is subjected to salt treatment, namely, is irrigated once by NaCl with the concentration of 350 mM. Most transgenic Arabidopsis thaliana died visually (i.e., one salt tolerance screening) after 7-10 days of salt treatment.

As shown in FIG. 5, the Arabidopsis plant transformed with RIP1 gene still survived, showing that the Arabidopsis plant has a certain salt tolerance, and the Arabidopsis plant transformed with RIP1 gene is transferred into normal soil to grow and harvest seeds, thus obtaining T ₁ Seed (RIP 1-T) ₁ Seed).

The recombinant agrobacterium used for transferring the RIP1 gene into the arabidopsis plant transgene is GV3101/SQKJ-RIP1, the recombinant agrobacterium is obtained by introducing a recombinant plasmid SQKJ-RIP1 into the agrobacterium GV3101, the SQKJ-RIP1 is a recombinant vector obtained by replacing a DNA fragment between KpnI and BamHI recognition sequences of the SQKJ with the RIP1 gene shown as SEQ ID NO. 1 in a sequence table and keeping other sequences of the vector unchanged, and the SQKJ-RIP1 can express the RIP1 protein shown as SEQ ID NO. 2 in the sequence table.

Example 4 salt tolerance gene validation

1. Preliminary verification of salt tolerance effect of transgenic Arabidopsis thaliana

T of Arabidopsis plant transformed with RIP1 Gene obtained in example 3 ₁ Seed (RIP 1-T) ₁ Seeds) were dried at room temperature for 7 days, theoretically T ₁ The generation seeds contain 3/4 of transgenic positive seeds and 1/4 of transgenic negative seeds. Subjecting the RIP1-T to ₁ The seeds and wild type seeds (CK) are suspended in 0.1% agarose solution and stored at 4deg.C for 2 days, then sowed in the same seedling pot of 28cm×55cm, each material is sowed with 14 plants, and after germination, the seeds are subjected to constant temperature (22deg.C) and constant humidity (40-50%) with light intensity of 120-150 μmol/m ² Culturing under long sunlight condition for second. After one week of growth, foliar spray was performed with a pilot plant (Bayer Co.) solution at a dilution ratio of 1:400 to kill the non-transgenic Arabidopsis thaliana. Watering to saturation after the remaining transgenic arabidopsis grows for two weeks, and stopping watering. After stopping watering for 5 days, starting water content measurement, when the soil water content is about 35%, watering with 350mM NaCl water solution for salt treatment, observing T of Arabidopsis plant transferred with RIP1 gene (number 100057) after 8-10 days of salt treatment ₁ Phenotype of the generation plants (as shown in FIG. 6).

The results of fig. 6 show that: after treatment with 350mM NaCl aqueous solution, most of wild Arabidopsis plants die while Arabidopsis plants transformed with RIP1 gene are T ₁ The plants can normally bolting, flowering and maturing. Compared with the wild type Arabidopsis plant, the T of the Arabidopsis plant transferred with the RIP1 gene (number 100057) ₁ The salt tolerance of the plants is obviously improved.

Acquisition of RUB1-T ₁ Mature grains obtained by selfing on plants are RIP1-T ₂ Seed.

2. Re-verification of salt tolerance genes

To further determine the salt tolerance of the Arabidopsis plants transformed with the RIP1 gene (number 100057), 3 parts of each of the RIP1-T were taken ₂ Seeds and wild type seeds, 14 plants each, 6 parts of the seeds were sown in 3 seedling pots according to the method in step 1, each seedling pot containing 1 part of the RIP1-T ₂ Seeds and 1 part of wild type seeds. To two weeks of Arabidopsis thaliana growthAnd (5) watering until the water is saturated, and stopping watering. Measuring water content 5 days after stopping watering, when water content is about 35%, respectively watering with 250mM,350mM, 450mM NaCl water solution, salt treating for 8-10 days, observing T of Arabidopsis plant transferred with RIP1 gene (number 100057) ₂ Phenotype of the generation plants (as shown in FIG. 7).

The results of fig. 7 show that: after treatment with 250mM,350mM and 450mM NaCl aqueous solution, the wild type Arabidopsis plants mostly die white, while the Arabidopsis plants transformed with the RIP1 gene (number 100057) were T ₂ The plants can normally bolting, flowering and fruit bearing. The above results further indicate that the Arabidopsis plant transformed with the RIP1 gene (number 100057) has strong salt stress tolerance as compared with the wild type Arabidopsis plant.

Example 5 obtaining salt-tolerant gene sequences

The genomic DNA of the Arabidopsis plant transformed with the RIP1 gene (number 100057) which has been verified to have salt stress tolerance was extracted by using the CTAB method as a template DNA.

Amplifying RIP1 gene from template DNA using a primer pair consisting of the primer 1 and the primer 2 and the following PCR amplification system:

the PCR reaction conditions were: pre-denaturation at 94℃for 5min; the following cycle is then entered: denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min for 35 cycles; finally, the mixture is extended for 10min at 72 ℃ and cooled to room temperature.

The PCR amplified product thus obtained was electrophoresed on 1% (w/v) agarose gel to isolate a desired fragment having a length of about 1200bp, as shown in FIG. 8. And then sequencing and analyzing the recovered and purified PCR product, and analyzing the sequencing result to obtain the nucleotide sequence (381 nucleotides) of the RIP1 gene, wherein the nucleotide sequence is shown as SEQ ID NO. 1 in a sequence table, and the amino acid sequence (126 amino acids) of the encoded RIP1 protein is shown as SEQ ID NO. 2 in the sequence table.

In summary, the protein RIP1 related to salt tolerance reaction is isolated from sorghum for the first time, the protein is particularly tolerant to salt stress, and the transgenic plant transformed with the gene encoding the salt tolerance protein RIP1 can tolerate the salt stress with the concentration of up to 450mM, so that the method has important significance for improving, developing and utilizing saline-alkali soil resources, relieving soil pressure, increasing reserve of cultivated land and guaranteeing grain safety.

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

Sequence listing

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Claims

1. A method for improving salt tolerance of a plant comprising the steps of: enhancing expression of protein RIP1 in a recipient plant to obtain a target plant with salt tolerance higher than that of the recipient plant; the protein RIP1 is a protein of the following A1), A2) or A3):

a1 Amino acid sequence is protein with SEQ ID NO. 2 in a sequence table;

2. The method of claim 1, wherein the recipient plant is a monocot or dicot.

3. The protein RIP1 as claimed in claim 1.

4. A biomaterial, characterized in that the biomaterial is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding the protein RIP1 of claim 3;

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

5. The biological material according to claim 4, wherein the nucleic acid molecule of B1) is a nucleic acid molecule as shown in any one of the following B1) to B4):

6. Use of the method of any one of claims 1-2 in salt tolerant plant breeding.

7. Use of the protein RIP1 according to claim 3 for breeding salt tolerant plants or for improving salt tolerance of plants.

8. Use of the biomaterial according to claim 4 or 5 in salt tolerant plant breeding or for improving plant salt tolerance.

9. Use of a cell line in the breeding of salt tolerant plants or for improving salt tolerance of plants, characterized in that the cell line is a transgenic plant cell line comprising a nucleic acid molecule encoding a protein RIP1 according to claim 3.

10. The use according to any one of claims 6 to 9, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.