Application of ALKBHL1 protein or substance for regulating expression of ALKBHL1 protein in regulation of salt tolerance character of rice

Abstract

The invention discloses application of an ALKBHL1 protein or a substance for regulating expression of the ALKBHL1 protein in regulating the salt tolerance character of rice. The invention belongs to the technical field of biology, and particularly relates to application of ALKBHL1 protein or a substance for regulating and controlling expression of the ALKBHL1 protein in regulating and controlling the salt tolerance character of rice. The ALKBHL1 protein disclosed by the invention can be applied to the aspects of regulating and controlling the salt tolerance of plants, preparing products for regulating and controlling the salt tolerance of the plants, cultivating salt-tolerant plants and the like. The rice ALKBHL1 protein has the function of positively regulating and controlling the salt tolerance of rice, the salt tolerance of the rice can be remarkably improved by over-expressing the coding gene of the ALKBHL1 protein in the rice, and the rice ALKBHL1 protein has wide application space and market prospect in the field of agriculture.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of ALKBHL1 protein or a substance for regulating expression of the ALKBHL1 protein in regulation of salt tolerance of rice. BACKGROUND

[0002] Rice (Oryza sativa) is one of the important food crops, and 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 the population (it is estimated that the population will reach about 10 billion in 2050), and the decrease of available fresh water (it is estimated that the amount of available fresh water will decrease by 50%), the imbalance between food supply and demand is further aggravated. According to data investigation, there are about 100 million mu of salt-alkali land that can be developed and utilized in China. Due to the influence of high temperature, the secondary salinization of soil is also increasing. These salt-alkali lands are part of the important land resources in China, and have great potential for comprehensive utilization. Identifying important stress-resistant gene resources and then breeding new rice varieties with stress resistance and stable yield is an effective way to solve the problem. SUMMARY

[0003] The technical problem to be solved by the present application is how to enhance the salt tolerance of plants.

[0004] In order to solve the problems in the prior art, the present application provides application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in regulation of salt tolerance of plants.

[0005] The application provided by the present application is application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in any one of the following:

[0006] 1) application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in regulation of salt tolerance of plants;

[0007] 2) application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in preparation of a product for regulating salt tolerance of plants;

[0008] 3) application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in breeding of plants with changed salt tolerance;

[0009] 4) application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in preparation of a product for breeding of plants with changed salt tolerance;

[0010] 5) application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in plant breeding;

[0011] The protein can be any one of the following proteins:

[0012] (a1) a protein having an amino acid sequence of SEQ ID No. 1;

[0013] (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 of the amino acid sequence of SEQ ID No. 1;

[0014] (a3) a fusion protein obtained by linking a tag to the terminal of the protein defined in any one of (a1) or (a2).

[0015] The amino acid sequence of the protein of (a2) can be SEQ ID No. 1.

[0016] The protein of (a1) is named ALKBHL1. The protein of (a2) can be an ALKBHL1 mutant.

[0017] 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.

[0018] The above-mentioned protein can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0019] In the above-mentioned protein, the tag refers to a polypeptide or protein 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.

[0020] The protein in the above-mentioned application is derived from rice (Oryza sativa).

[0021] In this context, the substance regulating the activity and / or content of the protein can be a substance regulating the expression of a gene encoding the protein ALKBHL1.

[0022] 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).

[0023] 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:

[0024] B1) a nucleic acid molecule encoding the aforementioned protein;

[0025] B2) an expression cassette containing the nucleic acid molecule of B1);

[0026] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0027] 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);

[0028] 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);

[0029] B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

[0030] B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2).

[0031] In the above biological material, the nucleic acid molecule of B1) can be any one of the following DNA molecules:

[0032] C1) a DNA molecule whose nucleotide sequence is SEQ ID No. 3;

[0033] C2) a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No. 2;

[0034] C3) a cDNA molecule or a DNA molecule whose coding sequence is from 79th to 1179th in SEQ ID No. 4 in the sequence listing;

[0035] 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;

[0036] C5) a DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined in C1), C2) or C3), and encoding the protein described supra.

[0037] 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.

[0038] 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 which are artificially modified, having 75% or more identity to the nucleotide sequence of the protein ALKBHL1 isolated in the present application, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application, as long as they encode the protein ALKBHL1 and have the function of the protein ALKBHL1.

[0039] In the present application, identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence or the nucleotide sequence can be determined using the homology search site on the internet, such as the BLAST page of the NCBI homepage. For example, the value of identity (%) can be obtained by calculating the identity of the amino acid sequence or the nucleotide sequence in the Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing the search.

[0040] The above-mentioned 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0041] In the present context, the above 80% 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 above 85% identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The above 95% identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0042] 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 pC1307-3flag) vector.

[0043] 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 contain a 3' untranslated region of the foreign gene, i.e., a DNA fragment containing a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase Nos gene), plant gene (such as the soybean storage protein gene), etc.

[0044] 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.

[0045] 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.

[0046] 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 genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, 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.

[0047] The present application also provides a method for regulating the salt tolerance of plants.

[0048] 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.

[0049] 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.

[0050] The present application also provides a method for breeding salt-tolerant plants.

[0051] The application provides a method for breeding salt-tolerant plants, which comprises increasing and / or enhancing the expression of a gene encoding the protein in a 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.

[0052] 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 an ALKBHL1 gene into a receptor plant, so as to obtain a target plant with higher salt tolerance than the receptor plant. The ALKBHL1 gene encodes the ALKBHL1 protein.

[0053] In an embodiment of the application, the method for breeding salt-tolerant plants comprises the following steps:

[0054] (1) constructing a recombinant expression vector comprising a DNA molecule of SEQ ID No. 2;

[0055] (2) transforming the recombinant expression vector constructed in step (1) into a receptor cell;

[0056] (3) screening and identifying to obtain a salt-tolerant plant with higher salt tolerance than the receptor plant.

[0057] The introduction refers to introduction by recombination means, including but not limited to Agrobacterium-mediated transformation, biolistic method, electroporation, in planta technology and the like.

[0058] The ALKBHL1 gene or a fragment of the gene provided by the application is introduced into a receptor cell or a receptor 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 a Ti plasmid, a Ri plasmid, a plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation and the like conventional biological methods, and the transformed plant tissue is cultivated into a plant.

[0059] The microorganism described herein can be a yeast, a bacterium, an algae or a fungus. Among them, the bacterium can be from Escherichia, Erwinia, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus and the like. Specifically, it can be Agrobacterium tumefaciens EHA105.

[0060] In the present context, the recombinant microorganism (or recombinant recipient cell) refers to the manipulation and modification of the genes of the microorganism of interest (or the recipient cell of interest) to obtain a recombinant microorganism (or a recombinant recipient cell) with changed function.

[0061] The recipient cell (also referred to as host cell) described herein can be a plant cell. The host cell is understood to refer not only to the particular subject cell but also to the progeny of the same, and a progeny can not necessarily be identical to the original parent cell, due to natural and accidental or deliberate mutation and / or change, but still falls 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, a plant cell of Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc.

[0062] 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, especially commercial varieties, using conventional breeding techniques. The transgenic plant includes seeds, callus, whole plants and cells.

[0063] 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.

[0064] In the present application, the purpose of plant breeding can include breeding salt-tolerant plants.

[0065] In the present application, the plant can be any of the following:

[0066] E1) a monocotyledonous plant or a dicotyledonous plant;

[0067] E2) a plant of the order Poales;

[0068] E3) a plant of the family Poaceae;

[0069] E4) a plant of the genus Oryza;

[0070] E5) rice.

[0071] The protein described herein or the biological material described herein also falls within the scope of the present application.

[0072] The application provides an ALKBHL1 protein and a coding gene thereof, the gene is introduced into rice, and a rice plant overexpressing the ALKBHL1 gene is obtained; a salt tolerance experiment is carried out on the transgenic rice, and it is found that, compared with wild-type rice, the salt tolerance of the overexpression strain is enhanced. The results show that the ALKBHL1 gene and the protein encoded by the ALKBHL1 gene play an important role in the salt tolerance of plants, have important application value in the research of enhancing the salt tolerance of plants, and have wide application space and market prospect in the field of agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 It is a schematic diagram of plasmid pCAMBIA1307 structure.

[0074] Figure 2 It is the identification result of the fluorescent quantitative PCR of the transgenic rice.

[0075] Figure 3 It is the statistical result of the salt tolerance phenotype and survival rate of rice. Wherein A is the phenotype of Nip and ALKBHL1-OE different strains before treatment; B is the phenotype of Nip and ALKBHL1-OE different strains after treatment; C is the phenotype of Nip and ALKBHL1-OE different strains after recovery; D is the survival rate of Nip and ALKBHL1-OE different strains.

[0076] Figure 4 It is the statistical result of the growth and yield per plant under saline-alkali conditions. Wherein A is the phenotype of Nip and ALKBHL1-OE different strains at the tillering stage; B is the yield per plant of Nip and ALKBHL1-OE different strains.

[0077] Figure 5 It is the LC-MS / MS detection of DNA methylation 6mA modification changes. DETAILED DESCRIPTION

[0078] The application will be further described in detail in combination with specific embodiments, and the examples given are only for illustrating the application, rather than limiting the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0079] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0080] In the quantitative test in the following examples, three repeated experiments are set, and the average value is taken, unless otherwise specified.

[0081] 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.

[0082] 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.

[0083] Example 1, Study on the ALKBHL1 protein in regulating the salt tolerance of rice

[0084] 1. Obtaining of the ALKBHL1 gene of rice

[0085] The leaf RNA of the rice variety NIP was extracted and reverse transcribed into cDNA, and the cDNA was used as a template to amplify using the primers ALKBHL1-F: 5'-ATGTACGGCGACACCGAG-3' (SEQ ID No. 5); ALKBHL1-R: 5'-GTAGACTTGTCTGATGTT-3' (SEQ ID No. 6). The 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 the ALKBHL1 gene) was obtained. Max Super-Fidelity DNA Polymerase (Cat. No. P505-d1, Vazyme), and the amplification product (i.e. the coding region of the ALKBHL1 gene) was obtained.

[0086] 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. The nucleotide sequence of the gene encoding the ALKBHL1 protein in the genomic DNA of the rice NIP is SEQ ID No. 3. The 2055-2170 of SEQ ID No. 3 is the first exon, the 2269-2438 is the second exon, the 3067-3321 is the third exon, the 4263-4401 is the fourth exon, the 4499-4565 is the fifth exon, the 4647-4768 is the sixth exon, and the 4872-5011 is the seventh exon.

[0087] 2. Construction of recombinant plasmid pCAMBIA1307-ALKBHL1

[0088] The cDNA of the rice variety NIP was used as a template for PCR amplification using the primer set ALKBHL1-F and ALKBHL1-R, and the coding region sequence of ALKBHL1 was obtained. The target gene fragment of ALKBHL1 was recovered by gel.

[0089] The target gene and the vector pCAMBIA1307 were digested with the same restriction enzymes XbaI and BamHI, respectively, and then transformed into E. coli. After one-generation sequencing verification and sequence alignment, the pCAMBIA1307-ALKBHL1 overexpression vector was obtained.

[0090] The structure diagram of the starting vector pCAMBIA1307 is 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 the other sequences of the pCAMBIA1307 vector unchanged. The nucleotide sequence of the overexpression vector pCAMBIA1307-ALKBHL1 is SEQ ID No. 4, as determined by whole plasmid sequencing.

[0091] 3. Obtaining of transgenic rice

[0092] The overexpression vector pCAMBIA1307-ALKBHL1 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105, and the recombinant Agrobacterium EHA105 / pCAMBIA1307-ALKBHL1 was obtained.

[0093] The recombinant Agrobacterium EHA105 / pCAMBIA1307-ALKBHL1 is used to genetically transform the embryogenic callus of rice NIP by Agrobacterium dipping method, and then resistant callus is screened (resistance screening uses 100 mg / L hygromycin), then differentiation and regeneration culture is carried out, and then rooting culture is carried out to obtain regenerated plants.

[0094] The specific steps are as follows:

[0095] (1) Take mature seeds of rice NIP, remove the hull, and pick full and clean seeds without bacterial spots for disinfection.

[0096] (2) The disinfected rice NIP seeds are inoculated on the induction medium, and cultured at 28°C in the dark for about 14 days, and then calli with good appearance and growth are selected.

[0097] (3) The recombinant vector pCAMBIA1307-ALKBHL1 constructed in step 2 is introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium, which is named EHA105 / pCAMBIA1307-ALKBHL1.

[0098] (4) The recombinant bacterium obtained in step (3) is resuspended in the infection medium (MS liquid medium + 50 g / L sucrose + 50 μL / L Silwet L-77) to obtain EHA105 / pCAMBIA1307-ALKBHL1 bacterial suspension.

[0099] (5) The NIP callus of step (2) is soaked in the EHA105 / pCAMBIA1307-ALKBHL1 bacterial suspension prepared in step (4) for 20 min. After infection, the bacterial suspension is poured out, the callus is dried with sterile filter paper, and then placed on the co-culture medium (MS basic medium) containing acetosyringone and glucose, and cultured at 28°C in the dark for 50-55 h.

[0100] (6) After step (5) is completed, the callus without obvious Agrobacterium on the surface is selected and moved to the antibiotic medium (MS basic medium) containing cefotaxime, and cultured at 28°C in the dark for 3-4 days.

[0101] (7) The callus after the above culture is moved to the selection medium (MS basic medium) containing hygromycin and cefotaxime, and cultured at 28°C in the dark for 30 days, and subcultured every 10 days.

[0102] (8) After step (7) is completed, take the fresh hygromycin-resistant callus, inoculate it in the pre-regeneration medium (MS basic medium), and cultivate it in the dark at 28°C for 7 days, then place it in the light cultivation room (12h light / 12h dark) for continuous cultivation for 7 days, and then transfer it to the regeneration medium (MS basic medium), continue to cultivate it in the light until the regenerated plant grows, and obtain the transgenic plant.

[0103] The positive transformation rice obtained by introducing the recombinant vector pCAMBIA1307-ALKBHL1 is denoted as the ALKBHL1 positive transformation line.

[0104] Culture medium and formula for genetic transformation: the induction medium and the differentiation medium are both MS medium (Phytotech, M519-100L).

[0105] Example 2, identification of the ALKBHL1 positive transformation line of rice

[0106] 1. Identification of the ALKBHL1 positive transformation line of rice

[0107] Tested plants: rice NIP (referred to as CK) and the ALKBHL1 positive transformation line obtained in Example 1.

[0108] Genomic DNA of the tested plants is extracted, and the genomic DNA is used as a template to perform PCR amplification with the primer pair composed of primer ALKBHL1-F and primer ALKBHL1-R, with the 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 product is sequenced, and the product sequencing result is the nucleotide sequence of SEQ ID No. 2 (i.e. 79th to 1179th of SEQ ID No. 4).

[0109] Through the above identification, five T3 generation positive transformation lines overexpressing the ALKBHL1 gene are obtained, and are respectively denoted as ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3, ALKBHL1-OE#4 and ALKBHL1-OE#5.

[0110] The specific steps for detecting the expression of the ALKBHL1 gene are as follows: the extraction of total RNA is performed according to the instructions of the Bioteke plant total RNA extraction kit (Bioteke, RP3302). 1 μg of total RNA is taken, and the M-MLV reverse transcriptase (Invitrogen) is used to reverse it into cDNA.

[0111] qRT-PCR was performed according to the SYBR kit (TaKaRa) instructions. The amplification reaction was performed in a Roche LightCycler480 PCR instrument, each sample was repeated mechanically 3 times, and ACT1 was used for relative quantitative analysis. Each experiment was repeated at least 3 times to obtain consistent results, and the representative results were finally selected for display.

[0112] By detecting the expression abundance of ALKBHL1 gene, it was determined that ALKBHL1 gene was up-regulated to different degrees in the above five strains Figure 2 ), indicating that the exogenous pCAMBIA1307-ALKBHL1 has been successfully expressed in rice.

[0113] The ALKBHL1-OE#1 hygromycin-resistant plants were selfed and the seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. The T1 generation plants were selfed and the seeds were harvested, which were T2 generation seeds, and the T2 generation plants were selfed and the seeds were harvested, which were T3 generation seeds.

[0114] The ALKBHL1-OE#2 hygromycin-resistant plants were selfed and the seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. The T1 generation plants were selfed and the seeds were harvested, which were T2 generation seeds, and the T2 generation plants were selfed and the seeds were harvested, which were T3 generation seeds.

[0115] The ALKBHL1-OE#3 hygromycin-resistant plants were selfed and the seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. The T1 generation plants were selfed and the seeds were harvested, which were T2 generation seeds, and the T2 generation plants were selfed and the seeds were harvested, which were T3 generation seeds.

[0116] The ALKBHL1-OE#4 hygromycin-resistant plants were selfed and the seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. The T1 generation plants were selfed and the seeds were harvested, which were T2 generation seeds, and the T2 generation plants were selfed and the seeds were harvested, which were T3 generation seeds.

[0117] The ALKBHL1-OE#5 hygromycin-resistant plants were selfed and the seeds were harvested, and the seeds were cultivated into plants, which were T1 generation plants. The T1 generation plants were selfed and the seeds were harvested, which were T2 generation seeds, and the T2 generation plants were selfed and the seeds were harvested, which were T3 generation seeds.

[0118] 2. Salt tolerance test of rice ALKBHL1 positive transformation strain

[0119] The plants to be tested were: T3 generation homozygous strain of rice NIP, ALKBHL1-OE (ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3 and ALKBHL1-OE#4).

[0120] Salt tolerance test conditions: Seeds of each test line were germinated in a greenhouse and cultured to the three-leaf stage (time point A). They were then treated with 150 mM NaCl solution for 6 days (time point B), followed by transfer to a NaCl-free nutrient solution for another 7 days (time point C). Survival rates were recorded at each time point (at least 30 plants of each test line were counted). Greenhouse conditions were: 28℃, 10 hours light / 14 hours darkness.

[0121] Figure 3 In the diagram, Figure A shows the plant growth before treatment with 150 mM NaCl (time point A); Figure B shows the plant growth during treatment with 150 mM NaCl (corresponding time point B); and Figure C shows the plant growth after treatment with 150 mM NaCl (corresponding time point C). Figure 3 It can be seen that before treatment with 150mM NaCl, the growth of NIP and ALKBHL1-OE was consistent. Figure 3 After treatment with 150 mM NaCl for 6 days, the growth of ALKBHL1-OE was superior to that of NIP ( ). Figure 3 In the case of B), the number of ALKBHL1-OE plants that survived after recovery culture was greater than that of NIP.

[0122] Survival rate results are shown in Figure 3 The survival rate of D. NIP was approximately 18%, while the survival rate of ALKBHL1-OE plants ranged from 25% to 66%. Compared to NIP, ALKBHL1-OE exhibited significantly enhanced salt tolerance.

[0123] 3. Rice yield per plant under saline-alkali conditions

[0124] The plants to be tested were T3 generation homozygous lines of rice NIP and ALKBHL1-OE (ALKBHL1-OE#1, ALKBHL1-OE#2, ALKBHL1-OE#3 and ALKBHL1-OE#4).

[0125] Salt tolerance test conditions: Seedlings of each test line were planted in a saline-alkali experimental field (NaCl concentration 0.38%, pH≈9.2). After the rice tillering stabilized, the number of tillers of each line was counted (time point A, photo taken). After the plants matured, the yield per plant of NIP and ALKBHL1-OE was counted (at least 20 plants of each test line were counted).

[0126] The growth status of the plants to be tested can be seen in the following figures. Figure 4 The left figure corresponds to the tillering status of NIP and ALKBHL1-OE (time point A), and the right figure shows the yield per mature plant (time point B). Figure 4 It can be seen that under saline-alkali conditions, ALKBHL1-OE has more tillers than NIP. Figure 4In A), the single plant yield of ALKBHL1-OE mature plants is superior to NIP Figure 4 In B).

[0127] 4. DNA 6mA modification level

[0128] 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).

[0129] The specific experimental steps are as follows: the seeds of each test line are germinated in the greenhouse and cultured to the three-leaf stage, the aboveground parts are taken, the genomic DNA of the test line 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 phase, GOLDaQ column (100mm*2.1mm) with pore size 1.9μm, and ion pair injection detection is set. The histone modification level is determined.

[0130] 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 is significantly down-regulated.

[0131] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. Application, characterized in that, The application is any one of the following: 1) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating plant salt tolerance; 2) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that regulate plant salt tolerance; 3) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the cultivation of plants with altered salt tolerance traits. 4) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that cultivate plants with altered salt tolerance traits. 5) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in plant breeding; The protein is any of the following: (a1) A protein with the amino acid sequence SEQ ID No.

1. (a2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence of SEQ ID No. 1, which has more than 80% identity with the protein of (a1) and has the function of regulating plant salt tolerance. (a3) A fusion protein obtained by attaching a tag to the end of the protein defined in (a1) or (a2).

2. The application according to claim 1, characterized in that, The protein is derived from rice.

3. The application according to claim 1 or 2, characterized in that, The substance that regulates gene expression is a substance that increases or upregulates the expression of the gene.

4. The application according to any one of claims 1 or 2, characterized in that, The substance regulating gene expression and the substance regulating the activity or content of the protein are biological materials related to the protein of claim 1 or 2, wherein the biological material is any one of B1) to B7) below: B1) A nucleic acid molecule encoding the protein described in claim 1; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plants containing the expression cassette described in B2). Because of plant organs official.

5. The application according to claim 4, characterized in that, B1) The nucleic acid molecule is any of the following DNA molecules: C1) The nucleotide sequence is the DNA molecule of SEQ ID No. 3; C2) The coding sequence is the cDNA molecule or DNA molecule of SEQ ID No. 2; C3) The coding sequence is the cDNA molecule or DNA molecule at positions 9593 to 12967 of SEQ ID No. 4 in the sequence listing; C4) has 90% or more identity with the nucleotide sequence defined by C1), C2) or C3), and is derived from rice and is a DNA molecule encoding the protein of claim 1; C5) hybridizes under stringent conditions with a nucleotide sequence defined by C1), C2) or C3) and encodes a DNA molecule that encodes the protein of claim 1.

6. A method for regulating plant salt tolerance, characterized in that, The method includes regulating plant salt tolerance by controlling the expression of the gene encoding the protein of claim 1 or 2, or by controlling the activity or content of the protein of claim 1 or 2.

7. A method for enhancing the salt tolerance of plants, characterized in that, The method includes increasing and / or enhancing the expression level of the gene encoding the protein of claim 1 in the target plant, and / or increasing and / or enhancing the activity and / or content of the gene encoding the protein of claim 1, to improve the salt tolerance of the plant.

8. A method for cultivating salt-tolerant plants, characterized in that, The method includes increasing and / or enhancing the expression level of the gene encoding the protein of claim 1 in the target plant, and / or enhancing and / or enhancing the activity and / or content of the gene encoding the protein of claim 1, to obtain a salt-tolerant plant.

9. The application according to any one of claims 1 or 2, or the method according to any one of claims 6-8, characterized in that, The plant is any of the following: E1) Monocotyledonous or dicotyledonous plants; E2) Plants of the order Poales; E3) Gramineae plants; E4) Plants of the genus *Oryza*; E5) Rice.

10. The protein as described in claim 1 or 2, or the biomaterial as described in claim 4 or 5.

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