Method for enhancing salt tolerance of plant, and alkbhl1 protein used therein and use thereof

Abstract

Provided are a method for enhancing the salt tolerance of a plant, and an ALKBHL1 protein used therein and a use thereof. Specifically provided are a method for enhancing the salt tolerance of a plant, and an ALKBHL1 protein used therein and a use thereof. The ALKBHL1 protein can regulate and control the salt tolerance of a plant, the salt tolerance of Oryza sativa can be significantly improved by overexpressing a coding gene of the ALKBHL1 protein in Oryza sativa, a product for regulating and controlling the salt tolerance of a plant is prepared, and a salt-tolerant plant is cultivated. The ALKBHL1 protein in Oryza sativa has the function of positively regulating and controlling the salt tolerance of Oryza sativa, and has a broad application scope and market prospect in the field of agriculture.

Description

Method for enhancing salt tolerance of plants, ALKBHL1 protein used in the method and use thereof TECHNICAL FIELD

[0001] The present application relates to a method for enhancing salt tolerance of plants, ALKBHL1 protein used in the method and use thereof. BACKGROUND

[0002] Rice (Oryza sativa) is one of the important food crops, which provides staple food for more than half of the world's population. In recent years, soil salinization caused by climate change has become a global problem that needs to be solved urgently. With the continuous growth of population and the decrease of available fresh water, the imbalance between food supply and demand is further aggravated. Influenced by high temperature, soil secondary salinization has an increasingly aggravating trend. The comprehensive utilization potential of saline-alkali land is huge. Identifying important stress-tolerant genetic resources and then breeding new rice varieties with stress tolerance and stable yield is an effective way to solve the problem.

[0003] SUMMARY

[0004] The technical problem to be solved by the present application is how to enhance the salt tolerance of plants.

[0005] In order to solve the problems existing in the prior art, the present application provides the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in regulating the salt tolerance of plants.

[0006] The application provided by the present application is the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in any of the following:

[0007] 1) the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in regulating the salt tolerance of plants;

[0008] 2) the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in the preparation of a product for regulating the salt tolerance of plants;

[0009] 3) the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in the breeding of plants with changed salt tolerance traits;

[0010] 4) the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in the preparation of a product for breeding plants with changed salt tolerance traits;

[0011] 5) the use of a protein or an expression substance of a regulatory gene or a substance for regulating the activity or content of the protein in plant breeding;

[0012] The protein can be any of the following proteins:

[0013] (a1) a protein having an amino acid sequence of SEQ ID No. 1;

[0014] (a2) a protein having 80% or more identity to the protein of a1) and having a function of regulating plant salt tolerance, which is obtained by substitution, deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1;

[0015] (a3) a fusion protein obtained by linking a tag to the terminal of the protein defined in any one of (a1) or (a2).

[0016] The amino acid sequence of the protein of (a2) can be SEQ ID No. 1.

[0017] The protein of (a1) is named ALKBHL1. The protein of (a2) can be an ALKBHL1 mutant.

[0018] In order to facilitate purification or detection of the protein of (a1), a tag protein can be linked to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence of SEQ ID No. 1 in the sequence listing.

[0019] The protein can be artificially synthesized or obtained by first synthesizing a gene encoding the protein and then performing biological expression.

[0020] In the protein, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using DNA in vitro recombination technology, so as to facilitate expression, detection, tracking and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0021] The protein in the above-mentioned application is derived from rice (Oryza sativa).

[0022] In this context, the substance that regulates the activity and / or content of the protein can be a substance that regulates the expression of a gene encoding the protein ALKBHL1.

[0023] In the above, the substance that regulates the expression of the gene can be a substance that performs at least one of the following 6 kinds of regulation: 1) regulation performed at the transcription level of the gene; 2) regulation performed after the transcription of the gene (that is, regulation performed on the splicing or processing of the primary transcript of the gene); 3) regulation performed on the RNA transport of the gene (that is, regulation performed on the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation performed on the translation of the gene; 5) regulation performed on the degradation of the mRNA of the gene; and 6) post-translational regulation of the gene (that is, regulation performed on the activity of the protein translated from the gene).

[0024] In the above application, the substance that regulates the expression of the gene and the substance that regulates the activity or content of the protein can be a biological material related to the protein, and the biological material can be any one of the following:

[0025] B1) a nucleic acid molecule encoding the aforementioned protein;

[0026] B2) an expression cassette containing the nucleic acid molecule of B1);

[0027] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0028] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0029] B5) a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);

[0030] B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

[0031] B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2).

[0032] In the above biological material, the nucleic acid molecule of B1) can be any one of the following DNA molecules:

[0033] C1) a DNA molecule whose nucleotide sequence is SEQ ID No. 3;

[0034] C2) a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No. 2;

[0035] C3) a cDNA molecule or a DNA molecule whose coding sequence is from the 79th to 1179th in SEQ ID No. 4 in the sequence listing;

[0036] C4) a DNA molecule having 90% or more identity to the nucleotide sequence defined in C1), C2) or C3), and encoding the protein described supra;

[0037] C5) a DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined in C1), C2) or C3), and encoding the protein described supra.

[0038] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0039] The nucleotide sequence encoding the protein ALKBHL1 of the present application can be easily mutated by those of ordinary skill in the art using known methods, such as methods of directed evolution or point mutation. Those nucleotides artificially modified, having 75% or more identity to the nucleotide sequence of the protein ALKBHL1 isolated in the present application, as long as encoding the protein ALKBHL1 and having the function of the protein ALKBHL1, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0040] Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. Identity of an amino acid sequence or a nucleotide sequence can be determined using homology search sites on the internet, such as BLAST page of NCBI homepage. For example, identity (%) can be obtained by searching in Advanced BLAST 2.1 using blastp as a program, setting 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 value) respectively, and then calculating identity of an amino acid sequence or a nucleotide sequence.

[0041] The above 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0042] Herein, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0043] The vectors described herein are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corporation), etc. Specifically, the vector can be pCAMBIA1307 (or simply referred to as pC1307-3flag) vector.

[0044] The recombinant expression vector containing the ALKBHL1 gene can be constructed using existing plant expression vectors. The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector can also comprise a 3' untranslated region of the foreign gene, i.e., comprising a polyadenylation signal and any other DNA segment that is involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylation of the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium tumefaciens Ti plasmid genes (e.g., the nopaline synthase Nos gene), plant genes (e.g., the soybean storage protein gene), etc.

[0045] When the ALKBHL1 gene of the present application is used to construct a recombinant plant expression vector, any kind of enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to, a cauliflower mosaic virus (CAMV) 35S promoter, a maize ubiquitin promoter, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be an ATG initiation codon or an adjacent region initiation codon, but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the initiation codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0046] In a specific embodiment, the recombinant expression vector pCAMBIA1307-ALKBHL1 can be a recombinant vector obtained by replacing the fragment between the restriction enzymes XbaI and BamHI of the pCAMBIA1307 vector with the DNA molecule of SEQ ID No. 2, while keeping the other sequences of the pCAMBIA1307 vector unchanged.

[0047] Further, the nucleotide sequence of the recombinant expression vector pCAMBIA1307-ALKBHL1 is SEQ ID No. 4.

[0048] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.), or anti-chemical reagent marker genes (such as anti-herbicide genes), etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0049] The present application also provides a method for regulating the salt tolerance of plants.

[0050] The present application provides a method for regulating the salt tolerance of plants, which comprises regulating the expression of the coding gene of the above-mentioned protein or regulating the activity or content of the above-mentioned protein to regulate the salt tolerance of plants.

[0051] The present application also provides a method for enhancing the salt tolerance of plants, which comprises increasing and / or increasing the expression amount of the coding gene of the above-mentioned protein in the target plant, or / and increasing and / or increasing the activity and / or content of the coding gene of the above-mentioned protein to enhance the salt tolerance of plants.

[0052] The application also provides a method for breeding salt-tolerant plants.

[0053] The application provides a method for breeding salt-tolerant plants, which comprises increasing and / or enhancing the expression of the gene encoding the protein in the target plant, or / and increasing and / or enhancing the activity and / or content of the gene encoding the protein, so as to obtain a salt-tolerant plant.

[0054] In the breeding method, the activity and / or content of the protein in the target plant can be enhanced or increased or up-regulated, or / and the expression of the gene encoding the protein can be increased, by introducing the ALKBHL1 gene into the recipient plant, so as to obtain a target plant with higher salt tolerance than the recipient plant. The ALKBHL1 gene encodes the ALKBHL1 protein.

[0055] In an embodiment of the application, the method for breeding salt-tolerant plants comprises the following steps:

[0056] (1) constructing a recombinant expression vector comprising the DNA molecule of SEQ ID No. 2;

[0057] (2) transforming the recombinant expression vector constructed in step (1) into a recipient cell;

[0058] (3) screening and identifying to obtain a salt-tolerant plant with higher salt tolerance than the recipient plant.

[0059] The introduction refers to introduction by recombination means, including but not limited to Agrobacterium-mediated transformation, biolistic method, electroporation, in planta technology, etc.

[0060] The ALKBHL1 gene or fragment of the gene provided by the application is introduced into a recipient cell or a recipient plant by using any vector that can guide the expression of an exogenous gene in a plant, so as to obtain a transgenic cell line and a transgenic plant with enhanced salt tolerance. The expression vector carrying the ALKBHL1 gene can be transformed into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. conventional biological methods, and the transformed plant tissue is cultivated into a plant.

[0061] The microorganism described herein can be a yeast, a bacterium, an alga or a fungus. Among them, the bacterium can be from Escherichia, Erwinia, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it can be Agrobacterium tumefaciens EHA105.

[0062] In the present application, the recombinant microorganism (or the recombinant recipient cell) refers to the manipulation and modification of the genes of the target microorganism (or the target recipient cell), thereby obtaining a recombinant microorganism (or a recombinant recipient cell) with changed functions.

[0063] The recipient cell (also referred to as the host cell) described herein can be a plant cell. The host cell can be understood to refer not only to the particular recipient cell, but also to the progeny of such a cell, and since natural, accidental or deliberate mutations and / or alterations can occur, the progeny can not necessarily be identical to the original parent cell, but are nevertheless included within the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc.

[0064] The transgenic plant produced according to the above method also falls within the scope of protection of the present application.

[0065] In any of the above-mentioned applications or methods, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the ALKBHL1 gene into the recipient plant, but also its progeny. For the transgenic plant, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species using conventional breeding techniques, particularly including commercial varieties. The transgenic plant includes seeds, callus, whole plants and cells.

[0066] In the present application, the regulation can be up-regulation or enhancement or increase. The regulation can also be down-regulation or weakening or decrease.

[0067] In the present application, the purpose of plant breeding can include breeding salt-tolerant plants.

[0068] In the present application, the plant can be any of the following:

[0069] E1) a monocotyledonous plant or a dicotyledonous plant;

[0070] E2) a plant of the order Poales;

[0071] E3) a plant of the family Poaceae;

[0072] E4) a plant of the genus Oryza;

[0073] E5) rice.

[0074] The protein described herein or the biological material described herein also falls within the scope of protection claimed in the present application. Attached Figure Description

[0075] Figure 1 is a schematic diagram of the structure of plasmid pCAMBIA1307.

[0076] Figure 2 shows the results of quantitative real-time PCR identification of transgenic rice.

[0077] Figure 3 shows the statistical results of salt tolerance phenotypes and survival rates in rice. A represents the phenotypes of different Nip and ALKBHL1-OE lines before treatment; B represents the phenotypes of different Nip and ALKBHL1-OE lines after treatment; C represents the phenotypes of different Nip and ALKBHL1-OE lines after recovery; and D represents the survival rates of different Nip and ALKBHL1-OE lines.

[0078] Figure 4 shows the statistical results of growth and yield per plant under saline-alkali soil conditions. A represents the phenotype of different lines of Nip and ALKBHL1-OE at the tillering stage; B represents the yield per plant of different lines of Nip and ALKBHL1-OE.

[0079] Figure 5 shows the changes in DNA methylation 6mA modification detected by LC-MS / MS.

[0080] The best way to implement an invention

[0081] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0083] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0084] The pCAMBIA1307 vector used in the following examples has been described in Cui X, Zhang Z, Wang Y, Wu J, Han X, Gu X, Lu T. TWI1 regulates cell-to-cell movement of OSH15 to control leaf cell fate. The New Phytologist, 2018, 221(1): 326-340. The biological material is available from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and can only be used for repeating the experiments related to the present application, and cannot be used for other purposes.

[0085] The rice variety Nip used in the following examples has been described in Zhang Q, Liang Z, Cui X, Ji C, Zhang P, Liu J, Riaz A, Yao P, Liu M, Wang Y, Lu T, Yu H, Zheng H, and Gu X* (2018) N6-Methyladenine DNA Methylation in Japonica and Indica Rice Genomes and Its Association with Gene Expression, Plant Development and Stress Responses; Molecular Plant, 11(12): 1492-1508. The biological material is available from the applicant, and can only be used for repeating the experiments related to the present application, and cannot be used for other purposes.

[0086] Example 1, Study on the role of ALKBHL1 protein in regulating salt tolerance of rice

[0087] 1. Obtaining of rice ALKBHL1 gene

[0088] The leaf RNA of rice variety NIP was extracted and reverse transcribed into cDNA. The cDNA was used as a template, and primers ALKBHL1-F: 5'-ATGTACGGCGACACCGAG-3' (SEQ ID No. 5); ALKBHL1-R: 5'-GTAGACTTGTCTGATGTT-3' (SEQ ID No. 6) were used for amplification. PCR amplification was performed using Max Super-Fidelity DNA Polymerase (Cat No: P505-d1, Vazyme), and the amplification product (i.e. the coding region of ALKBHL1 gene) was obtained.

[0089] ​The coding sequence of the ALKBHL1 gene in the rice variety NIP is the nucleotide sequence of SEQ ID No. 2, and the amino acid sequence of the encoded protein is SEQ ID No. 1. In the genomic DNA of the rice NIP, the nucleotide sequence of the gene encoding the ALKBHL1 protein is SEQ ID No. 3. The 2055-2170th position of SEQ ID No. 3 is the first exon, the 2269-2438th position is the second exon, the 3067-3321th position is the third exon, the 4263-4401th position is the fourth exon, the 4499-4565th position is the fifth exon, the 4647-4768th position is the sixth exon, and the 4872-5011th position is the seventh exon.

[0090] The amino acid sequence of the rice ALKBHL1 protein is as follows:

[0091]

[0092]

[0093] 2. Construction of recombinant plasmid pCAMBIA1307-ALKBHL1

[0094] ALKBHL1 coding region sequence was obtained by PCR amplification using cDNA of rice variety Nip as template and primer set ALKBHL1-F and ALKBHL1-R. The ALKBHL1 target gene fragment was recovered by gel.

[0095] The target gene and the vector pCAMBIA1307 were digested by the same restriction enzymes XbaI and BamHI, linked and transformed into E. coli, and then verified by one-generation sequencing and sequence alignment to obtain the pCAMBIA1307-ALKBHL1 overexpression vector.

[0096] The structural diagram of the starting vector pCAMBIA1307 is shown in Figure 1. The recombinant overexpression vector pCAMBIA1307-ALKBHL1 is obtained by replacing the fragment between the restriction enzymes XbaI and BamHI of the pCAMBIA1307 vector with the DNA molecule of SEQ ID No. 2, while keeping other sequences of the pCAMBIA1307 vector unchanged. The nucleotide sequence of the overexpression vector pCAMBIA1307-ALKBHL1 is SEQ ID No. 4 by whole plasmid sequencing.

[0097] 3. Obtaining of transgenic rice

[0098] The overexpression vector pCAMBIA1307-ALKBHL1 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain the recombinant Agrobacterium EHA105 / pCAMBIA1307-ALKBHL1.

[0099] The embryonic callus of rice NIP was genetically transformed by Agrobacterium infection method using the recombinant Agrobacterium EHA105 / pCAMBIA1307-ALKBHL1, and then resistant callus was screened (resistance screening used 100 mg / L hygromycin), then differentiation and regeneration culture was carried out, and then rooting culture was carried out to obtain regenerated plants.

[0100] The specific steps are as follows:

[0101] (1) Take mature seeds of rice NIP, remove the shell, and pick full and clean seeds without sterile spots for disinfection.

[0102] (2) The disinfected rice NIP seeds were inoculated on the induction medium, and cultured at 28°C in the dark for about 14 days, and then good-looking and good-growing callus was selected.

[0103] (3) Take the recombinant vector pCAMBIA1307-ALKBHL1 constructed in step 2 and introduce it into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium, which is named EHA105 / pCAMBIA1307-ALKBHL1.

[0104] (4) Take the recombinant bacterium obtained in step (3) and resuspend the bacterium in an infection medium (MS liquid medium + 50 g / L sucrose + 50 μL / L Silwet L-77) to obtain a bacterium suspension of EHA105 / pCAMBIA1307-ALKBHL1.

[0105] (5) Soak the NIP callus of step (2) in the bacterium suspension of EHA105 / pCAMBIA1307-ALKBHL1 prepared in step (4) for 20 min. After the infection, pour off the bacterium suspension, take the callus, dry the callus with sterile filter paper, and then place the callus on a co-culture medium (MS basic medium) added with acetosyringone and glucose and incubate the callus at 28°C in the dark for 50-55 h.

[0106] (6) After step (5) is completed, select the callus without obvious Agrobacterium on the surface and transfer the callus to an antibiotic medium (MS basic medium) added with cephalosporin and incubate the callus at 28°C in the dark for 3-4 days.

[0107] (7) Transfer the callus after the above incubation to a selection medium (MS basic medium) added with hygromycin and cephalosporin and incubate the callus at 28°C in the dark for 30 days, and subculture the callus every 10 days.

[0108] (8) After step (7) is completed, take the fresh hygromycin-resistant callus, inoculate the callus in a pre-regeneration medium (MS basic medium), incubate the callus at 28°C in the dark for 7 days, then place the callus in a light incubation room (12 h light / 12 h dark) and continue to incubate the callus for 7 days, and then transfer the callus to a regeneration medium (MS basic medium) and continue to incubate the callus under light until the regenerated plant grows, and obtain the transgenic plant.

[0109] The positive transformed rice obtained by introducing the recombinant vector pCAMBIA1307-ALKBHL1 is recorded as ALKBHL1 positive transformed line.

[0110] The medium and formula used for genetic transformation: the formula of the induction medium and the differentiation medium is MS medium (Phytotech, M519-100L).

[0111] Example 2, identification of the ALKBHL1 positive transformed line of rice

[0112] 1. Identification of the ALKBHL1 positive transformed line of rice

[0113] Tested plants: rice NIP (referred to as CK) and ALKBHL1 positive transformation lines obtained from Example 1.

[0114] Genomic DNA of the tested plants was extracted, and the genomic DNA was used as a template to perform PCR amplification with a primer pair composed of primer ALKBHL1-F and primer ALKBHL1-R, using pCAMBIA1307-ALKBHL1 plasmid as a positive control (referred to as V) and the receptor variety Nip as a negative control (referred to as CK). Then the obtained products were sequenced, and the product sequencing result was the nucleotide sequence of SEQ ID No. 2 (i.e. 79 to 1179 of SEQ ID No. 4).

[0115] Through the above identification, 5 T3 generation positive transformation lines overexpressing ALKBHL1 gene were obtained, and were named ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3, ALKBHL1-OE#4 and ALKBHL1-OE#5, respectively.

[0116] The specific steps for detecting the expression of ALKBHL1 gene are as follows: the extraction of total RNA was performed according to the instructions of Bioteke plant total RNA extraction kit (Bioteke, RP3302). 1 μg of total RNA was taken and reversed to cDNA using M-MLV reverse transcriptase (Invitrogen).

[0117] qRT-PCR was performed according to the instructions of SYBR kit (TaKaRa). The amplification reaction was performed in a Roche Light Cycler 480 PCR instrument, each sample was mechanically repeated for 3 times, and ACT1 was used for relative quantitative analysis. Each experiment was repeated at least 3 times to obtain consistent results, and finally the representative results were displayed.

[0118] Through detecting the expression abundance of ALKBHL1 gene, it was determined that in the above 5 lines, ALKBHL1 gene was up-regulated to different degrees (Figure 2), indicating that the exogenous pCAMBIA1307-ALKBHL1 was successfully expressed in rice.

[0119] ALKBHL1-OE#1 hygromycin-resistant plants were selfed and seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. T1 generation plants were selfed and seeds were harvested, which were T2 generation seeds, and T2 generation plants were selfed and seeds were harvested, which were T3 generation seeds.

[0120] ALKBHL1-OE#2 hygromycin resistant plants were selfed and seeds were harvested, which were grown into plants, i.e. T1 generation plants. T1 generation plants were selfed and seeds were harvested, i.e. T2 generation seeds, and T2 generation plants were selfed and seeds were harvested, i.e. T3 generation seeds.

[0121] ALKBHL1-OE#3 hygromycin resistant plants were selfed and seeds were harvested, which were grown into plants, i.e. T1 generation plants. T1 generation plants were selfed and seeds were harvested, i.e. T2 generation seeds, and T2 generation plants were selfed and seeds were harvested, i.e. T3 generation seeds.

[0122] ALKBHL1-OE#4 hygromycin resistant plants were selfed and seeds were harvested, which were grown into plants, i.e. T1 generation plants. T1 generation plants were selfed and seeds were harvested, i.e. T2 generation seeds, and T2 generation plants were selfed and seeds were harvested, i.e. T3 generation seeds.

[0123] ALKBHL1-OE#5 hygromycin resistant plants were selfed and seeds were harvested, which were grown into plants, i.e. T1 generation plants. T1 generation plants were selfed and seeds were harvested, i.e. T2 generation seeds, and T2 generation plants were selfed and seeds were harvested, i.e. T3 generation seeds.

[0124] 2. Salt tolerance test of rice ALKBHL1 positive transformation lines

[0125] The tested plants were: control material Nipponbare rice (Nip), T3 generation homozygous lines of ALKBHL1-OE (ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3 and ALKBHL1-OE#4).

[0126] Salt tolerance test conditions: seeds of each tested line were germinated in a greenhouse and cultured to the three-leaf stage (time point A), treated with 150 mM NaCl solution for 6 days (time point B), and then transferred to nutrient solution without NaCl for continued culture for 7 days (time point C). Photographs were taken at each time point to count the survival rate (at least 30 plants of each tested plant were counted). The greenhouse conditions were: 28°C, 10 hours of light / 14 hours of darkness.

[0127] In Figure 3, panel A shows the growth of plants before 150 mM NaCl treatment (time point A), panel B shows the growth of plants during 150 mM NaCl treatment (corresponding to time point B), and panel C shows the growth of plants after 150 mM NaCl treatment (corresponding to time point C). As can be seen from Figure 3, before 150 mM NaCl treatment, NIP and ALKBHL1-OE grew similarly (Figure 3, panel A), after 150 mM NaCl treatment for 6 days, ALKBHL1-OE grew better than NIP (Figure 3, panel B), and after recovery culture, the number of surviving plants of ALKBHL1-OE was more than that of NIP.

[0128] Survival rate results are shown in Figure 3D. The survival rate of NIP is about 18%, and the survival rate of ALKBHL1-OE plants is 25%-66%. Compared with NIP, the salt tolerance of ALKBHL1-OE is significantly enhanced.

[0129] 3. Rice single plant yield test under saline-alkali conditions

[0130] The plants to be tested are T3 generation homozygous lines of rice NIP and ALKBHL1-OE (ALKBHL1-OE #1, ALKBHL1-OE #2, ALKBHL1-OE #3, and ALKBHL1-OE #4).

[0131] Salt tolerance test conditions: The seedlings of each test line are planted in a saline-alkali test field (NaCl concentration 0.38%, pH ≈ 9.2), and the tiller number of each line is counted after the rice tillers are stable (time point A, photographing). The single plant yield of NIP and ALKBHL1-OE is counted after the plants mature (at least 20 plants of each test plant are counted).

[0132] The growth of the test plants is shown in Figure 4, the left panel corresponds to the tillering of NIP and ALKBHL1-OE (time point A), and the right panel is the single plant yield of mature plants (corresponding to time point B). As can be seen from Figure 4, under saline-alkali conditions, the tiller number of ALKBHL1-OE is more than that of NIP (Figure 4A), and the single plant yield of mature ALKBHL1-OE is better than that of NIP (Figure 4B).

[0133] 4. DNA 6mA modification level

[0134] The plants to be tested are T3 generation homozygous lines of rice NIP and ALKBHL1-OE (ALKBHL1-OE #1, ALKBHL1-OE #2, ALKBHL1-OE #3, ALKBHL1-OE #4, and ALKBHL1-OE #5).

[0135] The specific experimental steps are as follows: The seeds of each test line are germinated in a greenhouse and cultured to the three-leaf stage, the aboveground parts are taken, the genomic DNA of the test lines is extracted, and the DNA 6mA modification level is determined. The abundance of DNA 6mA modification is detected by triple quadrupole liquid chromatography mass spectrometry (UHPLC-MS / MS). Agilent 6400 triple quadrupole liquid chromatography mass spectrometry instrument is used, distilled water (0.1% formic acid) and acetonitrile (0.1% formic acid) are used as mobile phases, GOLDaQ column (100mm*2.1mm) with pore size 1.9μm, and ion pair injection detection is set. The histone modification level is determined.

[0136] The results are shown in Figure 5. Compared with Nip, the DNA 6mA modification level of ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3, ALKBHL1-OE#4 and ALKBHL1-OE#5 plants was significantly down-regulated.

[0137] Industrial applications

[0138] The present application provides an ALKBHL1 protein and its encoding gene. The encoding gene is introduced into rice to obtain a rice plant overexpressing the ALKBHL1 gene. The transgenic rice is subjected to salt tolerance experiment. It is found that the overexpression strain has enhanced salt tolerance compared with the wild type rice. The results show that the ALKBHL1 gene and the protein encoded thereby play an important role in plant salt tolerance and have important application value in enhancing plant salt tolerance and have wide application space and market prospect in the field of agriculture.

Claims

1. A method for enhancing salt tolerance in plants, characterized in that, The method comprises increasing and / or enhancing the expression of a gene encoding a protein in a plant of interest, or / and increasing and / or enhancing the activity and / or content of the gene encoding the protein, to enhance the salt tolerance of the plant. The protein is any one of the following: (a1) a protein having the amino acid sequence of SEQ ID No. 1, (a2) a protein having more than 80% identity to the protein of (a1) and having the function of regulating the salt tolerance of a plant, which is obtained by substitution, deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1, (a3) a fusion protein obtained by connecting a tag to the end of the protein defined in (a1) or (a2).

2. The method of claim 1, wherein, The plant is any one of the following: E1) a monocotyledonous plant or a dicotyledonous plant; E2) a plant of the order Poales; E3) a plant of the family Poaceae; E4) a plant of the genus Oryza; E5) rice.

3. A method for modulating salt tolerance in plants, characterized in that, The method comprises regulating the salt tolerance of a plant by regulating the expression of a gene encoding a protein or regulating the activity or content of the protein, wherein the protein is any one of the following: (a1) a protein having the amino acid sequence of SEQ ID No. 1, (a2) a protein having more than 80% identity to the protein of (a1) and having the function of regulating the salt tolerance of a plant, which is obtained by substitution, deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1, (a3) a fusion protein obtained by connecting a tag to the end of the protein defined in (a1) or (a2).

4. The method of claim 3, wherein, The plant is any one of the following: E1) a monocotyledonous plant or a dicotyledonous plant; E2) a plant of the order Poales; E3) a plant of the family Poaceae; E4) a plant of the genus Oryza; E5) rice.

5. A method for breeding salt-tolerant plants, characterized in that, The method comprises increasing and / or enhancing the expression of a gene encoding a protein in a plant of interest, or / and increasing and / or enhancing the activity and / or content of the gene encoding the protein, to enhance the salt tolerance of the plant. The protein is any one of the following: (a1) a protein having the amino acid sequence of SEQ ID No. 1, (a2) a protein having more than 80% identity to the protein of (a1) and having the function of regulating the salt tolerance of a plant, which is obtained by substitution, deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1, 6. The method of claim 5, wherein, (a3) a fusion protein obtained by connecting a tag to the end of the protein defined in (a1) or (a2). The plant is any one of the following: E1) a monocotyledonous plant or a dicotyledonous plant; E2) a plant of the order Poales; E3) a plant of the family Poaceae; E4) a plant of the genus Oryza; 7. Use, characterized in that, E5) rice. The use is any one of the following: 1) a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in the regulation of the salt tolerance of a plant; 2) a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in the preparation of a product for regulating the salt tolerance of a plant; 3) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in breeding plants with altered salt tolerance; 4) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in the manufacture of a product for breeding plants with altered salt tolerance; 5) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in plant breeding; the protein is any one of the following: (a1) a protein having an amino acid sequence of SEQ ID No. 1, (a2) a protein having 80% or more identity to the protein of (a1) and having a function of regulating salt tolerance of a plant, which is obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1, (a3) a fusion protein obtained by linking a tag to the terminal of the protein defined in (a1) or (a2). the plant is any one of the following:

8. Use according to claim 7, characterized in that, E1) a monocotyledonous plant or a dicotyledonous plant; E2) a plant of the order Poales; E3) a plant of the family Poaceae; E4) a plant of the genus Oryza; E5) rice. the substance that regulates expression of a gene is a substance that increases or enhances or up-regulates expression of the gene.

9. Use according to claim 7 or 8, characterized in that, the substance that regulates expression of a gene and the substance that regulates activity or content of the protein are biological materials related to the protein in the use of claim 7, and the biological materials are any one of the following B1) to B7):

10. Use according to any one of claims 7 or 8, characterized in that, B1) a nucleic acid molecule encoding the protein in the use of claim 7; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1) or an expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1) or an expression cassette of B2) or a recombinant vector of B3); B5) a transgenic plant cell line containing the nucleic acid molecule of B1) or an expression cassette of B2); B6) a transgenic plant tissue containing the nucleic acid molecule of B1) or an expression cassette of B2); B7) a transgenic plant organ containing the nucleic acid molecule of B1) or an expression cassette of B2). the nucleic acid molecule of B1) is any one of the following DNA molecules:

11. Use according to claim 7 or 8, characterized in that, C1) a DNA molecule having a nucleotide sequence of SEQ ID No. 3; C2) a cDNA molecule or a DNA molecule having a coding sequence of SEQ ID No. 2; C3) a DNA molecule having 90% or more identity to the nucleotide sequence defined in C1) or C2), derived from rice and encoding the protein in the use of claim 7; C4) a DNA molecule hybridizing to the nucleotide sequence defined in C1) or C2) under stringent conditions and encoding the protein in the use of claim 7. the protein is any one of the following:

12. A protein, characterized in that, (a1) a protein having an amino acid sequence of SEQ ID No. 1, ​ (a2) a protein having 80% or more identity with the protein of a1) and having a function of regulating salt tolerance in plants, which is obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of SEQ ID No. 1, (a3) a fusion protein obtained by attaching a tag to the terminal of the protein defined in (a1) or (a2).

13. A biomaterial, characterized in that, The biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein described in claim 12; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

14. A transgenic plant obtained by the method described in claim 5.

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