CN113061172B - Plant salt tolerance related LIP1 protein and related biological material and application thereof - Google Patents

Abstract

The invention discloses a plant salt tolerance related LIP1 protein and related biological materials and application thereof. The LIP1 protein may be specifically the following A1), A2) or A3): A1) the amino acid sequence is the protein of SEQ ID NO 2 in the sequence table; A2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein A1), which has 90% or more of identity with the protein A1) and has the same activity; A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2). The LIP1 protein and its related biological material can be used for regulating plant salt tolerance.

Description

Plant salt tolerance related LIP1 protein and related biological material and application thereof

Technical Field

The invention relates to a plant salt tolerance related LIP1 protein and related biological materials and application thereof in the technical field of biology.

Background

According to incomplete statistics, at least 20% of cultivated land and over 50% of irrigated land in the world are affected by salt stress to varying degrees, and salt stress, one of the abiotic stresses, is a major limiting factor in plant growth and yield and can cause a series of physiological and metabolic reactions in plants, resulting in reduced yield and even death of the plants. Since most plants, especially crops, are susceptible to salt stress, salt stress has severely affected global food yield. Practice proves that the improvement of saline soil is a complex, difficult and long-term work, so that a mechanism of the plant for coping with salt stress is disclosed, and the salt tolerance of the plant is improved, so that the improvement of the saline soil becomes an important foundation for promoting agricultural production.

In recent years, the salt tolerance of plants is complex and involves multiple mechanisms of cellular adaptation and multiple metabolic pathways, three aspects of interconnected regulation are necessary for achieving the ionic equilibrium, firstly, the plants must be protected from toxicity, secondly, the plants must reestablish a balanced in vivo environment under stress, and finally, the growth must be recovered. The complexity of plant salt tolerance makes it very difficult to improve the salt tolerance of crops by using traditional breeding methods, so that the design of salt tolerant crops by using biotechnology from heredity becomes a research hotspot in the current agricultural field.

To date, many genes associated with plant salt tolerance have been cloned and studied, including genes encoding various organic solute synthetases: 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 the function of resisting oxidative stress and the like, but most of them can obtain ideal salt-resistant effect only by the combined action with other multiple salt-resistant genes. Therefore, it is necessary to purposefully clone other salt-tolerant genes which do not affect the normal growth of plants from different kinds of plants in order to obtain crops with good salt tolerance.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the saline-alkali resistance of plants.

The invention provides a protein, which is named LIP1 and is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of SEQ ID NO 2 in the sequence table;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein A1), has more than 90% of identity with the protein A1), is derived from sorghum, and has the same activity;

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

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

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home web site. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Perresilute Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

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

Substances for regulating the expression level of LIP1 protein-encoding gene are also included in the scope of the present invention.

The substance for regulating the expression level of the LIP1 protein coding gene provided by the invention is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding LIP 1;

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

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

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

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

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 substances, the nucleic acid molecule of B1) is a gene represented by any one of the following B1) -B4):

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

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

b3) a cDNA or DNA molecule having 80% or more identity with the nucleotide sequence defined in b1) or b2) and encoding the LIP1 protein;

b4) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the defined nucleotide sequence of any one of b1) -b3) and encodes the LIP1 protein.

Wherein, SEQ ID NO. 1 in the sequence table consists of 414 nucleotides and codes the protein shown in SEQ ID NO. 2 in the sequence table.

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

Of the above, B2) the expression cassette containing the nucleic acid molecule of B1) (LIP1 gene expression cassette) means a nucleic acid molecule capable of expressing LIP1 in a host cell, and the nucleic acid molecule may include a promoter for promoting transcription of the LIP1 gene. 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: the constitutive promoter 35S of cauliflower mosaic virus (CAMV); ubiquitin (Ubiquitin) gene promoter (pUbi); 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-carbothioic acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (us patent 5,187,267); tetracycline inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleocin, and soybean beta conglycin (Beach 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 incorporated by reference in their entirety.

Of the above, the expression cassette containing the nucleic acid molecule described in B2) may further comprise a terminator for terminating transcription of LIP 1. 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 terminators (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; 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 Acid Res, 15: 9627).

The recombinant expression vector containing the LIP1 gene expression cassette can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector 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 a 3' untranslated region of the foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct plant expression vectors, enhancers, including translational enhancers or transcriptional enhancers, may also be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and selection of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, GFP gene, luciferase gene, etc.), marker genes for antibiotics (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhfr gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate) which are expressible in plants, or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above-mentioned material, the recombinant microorganism can be specifically yeast, bacteria, algae and fungi.

The invention also provides a method for improving the salt tolerance of plants, which comprises the following steps: enhancing the expression of LIP1 protein in a receptor plant to obtain a target plant with higher salt tolerance than the receptor plant.

In the above method, the enhanced expression of the LIP1 protein is achieved by introducing a nucleic acid molecule encoding said protein into the plant of interest.

In the above method, the nucleic acid molecule may be modified as follows and then introduced into the target plant to achieve better expression effect:

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

2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; the promoters may include constitutive, inducible, temporal regulated, developmental regulated, chemical regulated, tissue preferred and tissue specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

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

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

In the above method, the plant of interest may be a transgenic plant or a plant obtained by a conventional breeding technique such as crossing.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

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

In order to solve the above technical problems, the present invention also provides a plant agent which functions to improve salt tolerance of plants. The plant reagent of the present invention comprises the LIP1 protein and/or the substance capable of regulating the expression level of the gene encoding the LIP1 protein.

The invention also provides application of the LIP1 protein or the substance for regulating the expression level of the gene coding the LIP1 protein or the content or the activity of the LIP1 protein in improving the salt tolerance of plants.

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

Salt stress experiments of the Arabidopsis mutant introduced with the LIP1 gene prove that the expression of the LIP1 gene is enhanced, the salt tolerance of plants can be improved, and NaCl salt stress with the concentration as high as 2.63 percent can be tolerated. Therefore, the LIP1 gene can be reasonably utilized to regulate and control the salt tolerance of plants, and is beneficial to the effective utilization of saline-alkali soil resources.

Drawings

FIG. 1 is an electrophoretogram of total RNA of sorghum tissues under salt stress in example 1 of the present invention.

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

FIG. 3 is an electrophoresis diagram of the PCR amplification of cDNA insert in sorghum library plasmid under salt stress in example 1 of the present invention.

FIG. 4 is a diagram showing the salt tolerance effect of Arabidopsis thaliana plants transformed with LIP1 gene under salt stress in library screening in example 3 of the present invention.

FIG. 5 is a diagram showing the salt tolerance effect of Arabidopsis thaliana plants transformed with LIP1 gene under salt stress in example 4 of the present invention.

FIG. 6 is a diagram showing the salt tolerance effect of the Arabidopsis thaliana plant transformed with the LIP1 gene under salt stress in example 4 of the present invention when verified again.

FIG. 7 is an electrophoretogram after PCR amplification of an exogenous insert in a transgenic Arabidopsis plant according to example 5 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

1 vectors and strains

The following examples describe the vector pCAMBIA2300-35S-OCS in the non-patent literature, "Xiyan et al, the building blocks of pCAMBIO2300-betA-BADH bivalent plant expression vector, Chinese agricultural bulletin, 2009, 25 (09): 47-50 ", publicly available from lake Songquan agricultural science and technology, Inc. to repeat the experiments of this application, and not for other uses.

In the following examples, the vector SQKJ was modified from the vector pCAMBIA2300-35S-OCS backbone, the vector pCAMBIA2300-35S-OCS and the vector pCAMBIA3301(VT1386, Youbao organism) were digested with restriction enzyme Xho I, and the Bar gene fragment of the vector pCAMBIA3301 was used to replace the NPT II gene fragment of the vector pCAMBIA2300-35S-OCS, to construct a novel SQKJ vector.

Coli XL1-Blue competent cell (cat. No. BTN90504) was a product of the company Baiolaibo in the examples described below.

In the following examples, the competent cells of Agrobacterium GV3101 (Cat. Waryong GT707) were a product of Beijing Huayuyo Biol.

2 plant lines

In the following examples, Sorghum variety BTx623 is described in the non-patent literature "Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Guidlach H, Haber G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharak, Chapman J, Feltus FA, Gowik U, Grigorifiev IV, Lyons E, Maher CA, Martis M, Narechania A, Obillar RP, Pennling BW, Salamov AA, Wang Y, Zhang L, Carpita NC, Freeling M, Gihsle AR, Hash CT, Keller B, Klein P, Kresover S, Canovin, Ming R, Javing R, Heart D, Mahonia D-29, and lawn Fahrysolk K J, and lawn Fa K D, and lawn K KF K, Inc. 29, Inc. of Legend, Inc. Fahren K D, and Massach J; 457(7229) doi 10.1038/nature07723.PMID 19189423 ", publicly available from Songquan agricultural science, Inc., Hu, to repeat the experiments, and not available for other uses.

In the following examples, wild type Arabidopsis thaliana (Arabidopsis thaliana) is of the Columbia-0 subtype, abbreviated as wild type Arabidopsis thaliana Col-0, 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 engineering columns and floor working time through FVE in Arabidopsis thaliana. Nature genetics.2004, 36: 167-.

3. Culture medium and plate

The formula of LB liquid culture medium in the following examples is: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl10g/L, adjusted to pH 7.5 with NaOH.

In the following examples, LB solid plates containing kanamycin (50mg/L) were plates prepared using LB solid medium containing kanamycin (50mg/L) and the formulation of the LB solid medium containing kanamycin (50mg/L) was: kanamycin (50mg/L), tryptone (10 g/L), yeast extract (5 g/L), NaCl (10 g/L) and agar (15 g/L) are added, and the pH value is adjusted to 7.5 by NaOH.

The following examples were prepared using LB liquid medium containing kanamycin (50 mg/L): kanamycin (50mg/L), tryptone (10 g/L), yeast extract (5 g/L) and NaCl (10 g/L) and the pH value is adjusted to 7.5 by NaOH.

The LB solid plates containing 50mg/L of Rifampicin (Rifamicin) and 50mg/L of kanamycin in the following examples were plates prepared with LB solid medium containing 50mg/L of Rifampicin (Rifamicin) and 50mg/L of kanamycin, and the formulation of the LB solid medium containing 50mg/L of Rifampicin (Rifamicin) and 50mg/L of kanamycin was: rifampicin 50mg/L, kanamycin 50mg/L, tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjusted to pH 7.5 with NaOH.

The formulation of YEP broth in the following examples was: 5g/L of peptone, 1g/L of yeast extract, 5g/L of beef extract, 5g/L of sucrose and 0.5g/L of magnesium sulfate.

In the following examples, YEP liquid medium containing rifampicin (10mg/L) and kanamycin (50mg/L) was a medium obtained by adding rifampicin and kanamycin to a YEP liquid medium as a basal medium so that the rifampicin content was 10mg/L and the kanamycin content was 50 mg/L.

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

The total RNA of the sorghum variety BTx623 tissue subjected to salt treatment (sorghum is subjected to water culture by using a Mucun culture solution, the formulation of the Mucun culture solution refers to Ishikawa et al BMC Plant Biology 2011,11:172, and after the sorghum is normally cultured for three weeks, the sorghum variety BTx623 tissue is put into 2.05% NaCl to be subjected to water culture for 3 hours, 6 hours, 12 hours and 24 hours, and then sampling and mixed extraction is carried out) to prepare the total RNA, the integrity of the total RNA is shown in figure 1, the ratio of the total RNA to 260nm/280nm is 1.8-2.0, and the purity is high. Then separating mRNA, dephosphorylating, uncapping and connecting CAP Tag, synthesizing cDNA first chain from purified mRNA, further synthesizing double-chain DNA, carrying out size classification of fragments by agarose gel electrophoresis after the double-chain DNA is enzyme-digested, and respectively tapping and recovering according to the following fragment sizes: 2-12kb, 1-2kb, 0.5-1kb, <0.5 kb.

The above-mentioned fragment and expression vector SQKJ after the rubber tapping recovery are respectively enzyme-cut by using restriction endonucleases Kpn I and BamH I, the above-mentioned gene fragment after enzyme-cut is inserted into the multiple cloning site of expression vector SQKJ, and the vector is constructed by utilizing the enzyme-cutting method known by skilled person in the art, so as to construct the recombinant expression vector SQKJ-Sb mixture, and its construction process is shown in figure 2 (Kanamycin (R) in figure 2: kanamycin gene; prCaMV35 s: cauliflower virus promoter (SEQ ID NO: 3); Sorghumbol gene: sorghum library gene; terminator (SEQ ID NO: 4); BarOCS: glufosinate resistance gene (SEQ ID NO: 5)).

Then the recombinant expression vector SQKJ-Sb mixture is transformed into escherichia coli XL1-Blue competent cells by an electric shock method, wherein the electric shock conditions are as follows: mu.L of E.coli XL1-Blue competent cells, 1. mu.L of library plasmid DNA (recombinant expression vector SQKJ-Sb mixture), after electroporation under the pre-established program Ec2(2.5kV, 6.0ms), were cultured with shaking in LB liquid medium at 37 ℃ for 1 hour (shaking table at 200 rpm), grown overnight on LB solid plate containing kanamycin (50 mg/L). Selecting white clone, carrying out PCR identification on the transformed recombinant escherichia coli by using a primer pair consisting of a primer 1 and a primer 2,

primer 1: 5'-ccaaccacgtcttcaaagca-3' (shown in SEQ ID NO: 6);

primer 2: 5'-tcatgcgatcataggcgtct-3' (shown in SEQ ID NO: 7).

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

EXAMPLE 2 Arabidopsis transformation of sorghum full-Length cDNA library

1. Extraction of full-Length cDNA library plasmid

All the plaques of the sorghum library of example 1 were scraped and cultured in 500mL of LB liquid medium containing kanamycin (50mg/L) at 37 ℃ for 30 min. Extracting plasmids by a tubule alkali method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ L ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 200. mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 5 times, mix, and place on ice for 3-5 min; adding 150 μ L ice-cold solution III (3M potassium acetate, 5M acetic acid), mixing well immediately, and standing on ice for 5-10 min; centrifuging at 4 deg.C and 12000rpm for 5min, collecting appropriate amount of supernatant, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; each tube was added with 50. mu.L of TE (10mM Tris-HCl, 1mM EDTA, pH8.0) containing RNase (20. mu.g/mL) to dissolve the pellet; bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C to obtain full-length cDNA library plasmid.

2. Full-length cDNA library plasmid Agrobacterium transformation

Transforming the full-length cDNA library plasmid constructed in the step 1 into agrobacterium GV3101 by an electric shock method, wherein the transformation conditions are as follows: taking 1 μ L of full-length cDNA library plasmid and 50 μ L of Agrobacterium GV3101 competent cells, mixing well, standing on ice for 5min, transferring into a precooled electric shocking cup, shocking and transforming under a preset program Ec2(2.5kV, 6.0ms), inoculating the transformed Agrobacterium GV3101 into LB liquid medium, culturing at 28 ℃ and 200rpm for 1 h, spreading on LB solid plate containing 50mg/L Rifampicin (Rifampicin) and 50mg/L kanamycin until positive single clone grows out. Selecting white clone, carrying out PCR identification on the transformed recombinant agrobacterium by 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 carrying out electrophoresis detection on the size of the cDNA insert.

3. Arabidopsis transformation of full-length cDNA library plasmids

Wild type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were stored at 4 ℃ for 2 days to complete the need for dormancy to ensure synchronous germination of the seeds. Horse dung soil was mixed with vermiculite and irrigated with water until the soil was wet 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 keeping the light intensity at a constant temperature (22 ℃) and a constant humidity (40-50%) of 120-150 mu mol/m ² The culture was carried out under long-day conditions (16 hours light/8 hours dark) for a second. Irrigating plants with Hoagland nutrient solution, and then with deionized waterKeep the soil moist but not wet thoroughly.

Scraping all the recombinant agrobacterium tumefaciens bacterial plaques obtained in the step 2, and transforming arabidopsis thaliana by using a flower soaking method. Seeds were harvested about 4 weeks after the soaking transformation.

4. Positive rate detection of transgenic Arabidopsis thaliana

Arabidopsis seed (T) after infection with recombinant Agrobacterium containing full-length cDNA library plasmid ₀ Generation seed) contains a large number of transgenic seeds, and the transgenic proportion, namely the positive rate of transgenic arabidopsis thaliana, needs to be determined through resistance screening. Through glufosinate resistance screening, the 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 the transgenic arabidopsis is basically about 2 percent. Harvesting seeds of transgenic plantlets with glufosinate-ammonium resistance as T ₁ Generation positive seeds.

EXAMPLE 3 Arabidopsis screening of sorghum full-Length cDNA library

Will T ₁ The generation-positive seeds were suspended in 0.1% agarose solution and stored at 4 ℃ for 2 days. Horse dung soil was mixed with vermiculite and irrigated with water until the soil was wet 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. Seeds are germinated and the light intensity is 120-150 mu mol/m at constant temperature (22 ℃) and constant humidity (40-50 percent) ² The culture was carried out under long-day conditions of second (16 hours of light/8 hours of darkness). When arabidopsis thaliana grows for 7 days, the solution of Baustda (Bayer company) with the dilution ratio of 1:400 is used for foliage spraying to kill the non-transgenic arabidopsis thaliana. When the remaining transgenic arabidopsis grows to 12-14 days, watering to saturation, and then stopping watering; and (3) starting to measure the water content when the growth reaches 18-20 days, and carrying out salt treatment on the transgenic arabidopsis thaliana when the water content is reduced to 25-45%, namely irrigating once by using NaCl with the mass percentage of 2.05%. After 7-10 days of salt treatment, most of transgenic arabidopsis thaliana are observed to die by visual inspection, the result is shown in figure 4, the arabidopsis thaliana plant with the LIP1 gene still survives, the fact that the arabidopsis thaliana plant with the LIP1 gene is provided with certain salt tolerance is shown, the arabidopsis thaliana plant with the LIP1 gene is transferred to normal soil to grow and harvest,to obtain T ₂ Seed (LIP 1-T) ₂ A seed).

The recombinant agrobacterium used for the Arabidopsis thaliana plant transgene transferred with the LIP1 gene is marked as GV3101/SQKJ-LIP1, the recombinant bacterium is obtained by introducing a recombinant plasmid SQKJ-LIP1 into Agrobacterium GV3101, SQKJ-LIP1 is a recombinant vector obtained by replacing a DNA fragment between KpnI and BamHI recognition sequences of SQKJ with a LIP1 gene shown by SEQ ID NO. 1 in a sequence table and keeping other sequences of the vector unchanged, and SQKJ-LIP1 can express the LIP1 protein shown by SEQ ID NO. 2 in the sequence table.

Example 4 salt tolerance Gene validation

1. Preliminary verification of salt-tolerant effect of transgenic arabidopsis

T of the Arabidopsis thaliana plant transformed with the LIP1 gene harvested in example 3 ₂ Seed (LIP 1-T) ₂ Seeds) were dried at room temperature for 7 days. Subjecting the LIP1-T ₂ The seeds and wild type seeds (CK) are suspended in 0.1% agarose solution and stored at 4 ℃ for 2 days, and then sown in the same 28cm x 55cm seedling pot, 14 plants are sown in each material, and the light intensity is 150 mu mol/m at constant temperature (22 ℃) and constant humidity (40-50%) after the seeds germinate ² And culturing under long-day conditions of seconds. Watering to saturation after two weeks of growth, and then stopping watering. The water content was measured 5 days after the termination of the watering, when the soil water content was about 35%, the soil was salted by 2.05% (mass percentage) NaCl solution, and after 8-10 days of salting, the Arabidopsis thaliana plants into which the LIP1 gene (No. 100014) had been transferred were observed ₂ Phenotype of the plants (as shown in FIG. 5).

The results of fig. 5 show that: after 2.05% NaCl solution treatment, most of wild type Arabidopsis thaliana plants were whitened and dead, while Arabidopsis thaliana plants with transferred LIP1 gene had T ₂ The plant can normally bolt, flower and fruit. T of Arabidopsis thaliana plant transformed with LIP1 gene (No. 100014) compared to wild type Arabidopsis thaliana plant ₂ The salt tolerance of the plant is obviously improved.

2. Revalidation of salt tolerance genes

In order to further determine the salt tolerance of Arabidopsis thaliana plants transformed with LIP1 gene (100014), the plants were individually subjected to salt toleranceCollecting 3 parts of each of the above materials, and adding LIP1-T ₂ Seeds and wild type seeds, 14 plants each, 6 parts of the seeds were sown as in step 1 in 3 pots, each pot containing 1 part of the LIP1-T ₂ Seed and 1 part of wild type seed. Watering to saturation after two weeks of growth, and then stopping watering. The water content measurement was started after 5 days from the termination of watering, and when the water content was about 35%, the plants were separately watered with 1.46%, 2.05% and 2.63% NaCl aqueous solutions, and after 8-10 days of salt treatment, T-cells of Arabidopsis thaliana plants into which LIP1 gene (100014) had been transferred were observed ₂ Phenotype of the plants (as shown in FIG. 6).

The results of fig. 6 show that: after treatment with 1.46%, 2.05% and 2.63% aqueous NaCl solutions, the wild type Arabidopsis plants were mostly white and dead, while Arabidopsis plants with the LIP1 gene (accession No. 100014) had their T ₂ The plants can normally bolt, flower and fruit. The above results further indicate that the Arabidopsis thaliana plant into which the LIP1 gene (numbered 100014) was transferred has strong salt stress tolerance as compared with the wild type Arabidopsis thaliana plant.

Example 5 obtaining of salt-tolerant Gene sequences

Arabidopsis thaliana plants into which the LIP1 gene (numbered 100014) was transferred and which were verified to have salt stress tolerance were subjected to extraction of genomic DNA by the CTAB method and used as template DNA.

Amplifying the LIP1 gene from template DNA by using a primer pair consisting of the primer 1 and the primer 2 and the following PCR amplification system:

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

The obtained PCR amplification product was electrophoresed on a 1% (w/v) agarose gel to separate a desired fragment having a length of about 1300bp, as shown in FIG. 7. Then sequencing and analyzing the recovered and purified PCR product, and analyzing the sequencing result to obtain the nucleotide sequence (414 nucleotides) of the LIP1 gene, which is shown as SEQ ID NO:1 in the sequence table, and the amino acid sequence (137 amino acids) of the encoded LIP1 protein, which is shown as SEQ ID NO:2 in the sequence table.

In conclusion, the protein LIP1 related to abiotic stress reaction is separated from sorghum for the first time, the protein particularly has tolerance to salt stress, the transgenic plant with the gene encoding the salt-tolerant protein LIP1 can tolerate the salt stress with the concentration of 2.63%, and the protein has important significance for improving, developing and utilizing saline-alkali soil resources, relieving land pressure, increasing reserve arable land and guaranteeing grain safety.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> Huzhou Songquan agricultural science and technology Co., Ltd

<120> plant salt tolerance related LIP1 protein and related biological material and application thereof

<130> GNCSY211074

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 414

<212> DNA

<213> Sorghum (Sorghum bicolor)

<400> 1

atgcaggctg ccgctactgc cgttgggttc tcggccgtgc tgcctgtcaa ggcccggccg 60

gcggcgagga gcacggcggt ggcccgcgtc cctgccacaa ggaggagcgt ccgtaccgcc 120

gctgccgccg tcgtcgtcgc ggagcctgcg gaggtcgact acagctcgag cttctcggtg 180

ttcccgatgg aggcctgcga cctgctcggc ggcgacgcgt gcatcgggaa gatgttcccg 240

gaggccaagc tcgccgcggc ggcgccggag gcgagcagga gggtggacgc ggtggagagg 300

gactacctgt cctacgacgg gccaaaaacg gtgttcccgg gcgaggcgtg cgacgacctc 360

ggcggcgagt tctgcgaggc gccgtacatg gacggcgtct ccagggacgc ctga 414

<210> 2

<211> 137

<212> PRT

<213> Sorghum (Sorghum bicolor)

<400> 2

Met Gln Ala Ala Ala Thr Ala Val Gly Phe Ser Ala Val Leu Pro Val

1 5 10 15

Lys Ala Arg Pro Ala Ala Arg Ser Thr Ala Val Ala Arg Val Pro Ala

20 25 30

Thr Arg Arg Ser Val Arg Thr Ala Ala Ala Ala Val Val Val Ala Glu

35 40 45

Pro Ala Glu Val Asp Tyr Ser Ser Ser Phe Ser Val Phe Pro Met Glu

50 55 60

Ala Cys Asp Leu Leu Gly Gly Asp Ala Cys Ile Gly Lys Met Phe Pro

65 70 75 80

Glu Ala Lys Leu Ala Ala Ala Ala Pro Glu Ala Ser Arg Arg Val Asp

85 90 95

Ala Val Glu Arg Asp Tyr Leu Ser Tyr Asp Gly Pro Lys Thr Val Phe

100 105 110

Pro Gly Glu Ala Cys Asp Asp Leu Gly Gly Glu Phe Cys Glu Ala Pro

115 120 125

Tyr Met Asp Gly Val Ser Arg Asp Ala

130 135

<210> 3

<211> 530

<212> DNA

<213> Cauliflower mosaic virus (Cauliflower mosaic virus)

<400> 3

ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac 60

agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 120

agcacgacac acttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg 180

caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag 240

ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc 300

attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg 360

gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 420

aagtggattg atgtgatatc tccactgacg taagggatgg cgcacaatcc cactatcctt 480

cgcaagaccc ttcctctata taaggaagtt catttcattt ggagagaaca 530

<210> 4

<211> 235

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggcatgccag ggctctcaat ggagtttgaa tcaaatcttc cagctgcttt aatgagatat 60

gcgagacgcc tatgatcgca tgatatttgc tttcaattct gttgtgcacg ttgtaaaaaa 120

cctgagcatg tgtagctcag atccttaccg ccggtttcgg ttcattctaa tgaatatatc 180

acccgttact atcgtatttt tatgaataat attctccgtt caatttactg attga 235

<210> 5

<211> 552

<212> DNA

<213> Streptomyces hygroscopicus (Streptomyces hygroscopicus)

<400> 5

atgtctcctg agagaagacc tgttgagatt agacctgcta ctgctgctga tatggctgct 60

gtttgtgata ttgttaacca ttacattgag acttctactg ttaacttcag aactgagcct 120

caaactcctc aagagtggat cgatgatctt gagagacttc aagatagata cccttggctt 180

gttgctgagg ttgagggagt tgttgctgga attgcttacg ctggaccttg gaaggctaga 240

aacgcttacg attggactgt tgagtctact gtttacgttt ctcatagaca tcaaagactt 300

ggacttggat ctactcttta cactcatctt cttaagtcta tggaggctca aggattcaag 360

tctgttgttg ctgttattgg acttcctaac gatccttctg ttagacttca tgaggctctt 420

ggatacactg ctagaggaac tcttagagct gctggataca agcatggagg atggcatgat 480

gttggattct ggcaaagaga tttcgagctt cctgctcctc ctagacctgt tagacctgtt 540

actcaaatct ga 552

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccaaccacgt cttcaaagca 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcatgcgatc ataggcgtct 20

Claims (5)

1. The application of the LIP1 protein in improving the salt tolerance of plants, wherein the LIP1 protein is the protein of A1) or A2) as follows:

   A1) the amino acid sequence is the protein of SEQ ID NO 2 in the sequence table;

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

   the plant is Arabidopsis thaliana.
2. 2. Use of a substance for controlling the expression level of the gene encoding the LIP1 protein according to claim 1 or controlling the content or activity of the LIP1 protein according to claim 1 for improving salt tolerance in plants, wherein the substance 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 containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

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

   B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

   the plant is Arabidopsis thaliana.
3. 3. The use according to claim 2, wherein the nucleic acid molecule of B1) is a gene as shown in any one of B1) -B4):

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

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

   b3) a cDNA or DNA molecule having 80% or more identity to the nucleotide sequence defined in b1) or b2) and encoding the protein of claim 1;

   b4) a cDNA molecule or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence as defined in any of b1) -b3) and which encodes a protein as claimed in claim 1.
4. 4. A method for improving the salt tolerance of plants is characterized by comprising the following steps: enhancing expression of the LIP1 protein of claim 1 in a recipient plant to produce a plant of interest with higher salt tolerance than the recipient plant;

   the plant is Arabidopsis thaliana.
5. 5. The method of claim 4, wherein the enhancing the expression of the LIP1 protein is achieved by introducing a nucleic acid molecule encoding the LIP1 protein of claim 1 into a plant of interest.

Images