Use of the FSLE1 gene or its encoded protein in regulating the waterlogging tolerance of plants

Abstract

The present invention discloses FSLE1 the use of a gene or its encoded protein in regulating the waterlogging tolerance of plants. The present invention obtained fsm1 mutants in a large-capacity rice mutant library, which showed abnormal aerenchyma development. Experiments found that fsm1 the mutants were waterlogging-sensitive mutants; map-based cloning revealed that fsm1 the mutants were FSLE1 mutants obtained by gene deletion. By complementing fsm1 the FSLE1 gene in the mutants, it was found that the lines complemented with FSLE1 the gene restored the phenotype of wild-type rice. The present invention further constructed overexpression FSLE1 gene lines and simulated waterlogging treatment of wild-type rice WT and overexpression FSLE1 gene lines at the tillering stage. It was found that compared with wild-type rice WT, the yield reduction of the overexpression FSLE1 gene lines was significantly reduced. The present invention has application prospects in improving the waterlogging tolerance of plants and cultivating rice varieties with strong waterlogging tolerance, etc.

Description

Technical Field

[0001] The present invention relates to genes related to submergence tolerance isolated from rice and their uses, and particularly to the uses of genes or their encoded proteins isolated from rice in regulating the submergence tolerance of rice, belonging to the field of FSLE1 genes and their uses. FSLE1 Background Art

[0002] Flooding disaster is one of the common abiotic stresses in rice production, and 25 - 30% of the world's rice planting area is vulnerable to flooding stress. When plants are submerged for a long time, they will be subjected to oxidative stress, generating excessive reactive oxygen species (ROS) that damage cells. A large amount of mineral elements and main intermediate metabolites in plant roots will dissolve and be lost. At the same time, anaerobic respiration will produce toxic secondary metabolites that are adverse to plant growth, such as ethanol, acetaldehyde, etc. Eventually, it will lead to hypoxia in plant roots and even the whole plant cells, and in severe cases, it will cause plant death.

[0003] In recent years, some remarkable progress has been made in the molecular mechanism of waterlogging tolerance in rice. SUB1A is the first major QTL locus discovered to control rice submergence tolerance. Rice varieties containing the SUB1A gene can induce the expression of fermentation genes such as alcohol dehydrogenase under flooding conditions, inhibit the growth of plants underwater, and improve the submergence tolerance of plants and their survival ability after the flood recedes (Xu, K., Xu, X., Fukao, T., et al. (2006). Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice, Nature , 442: 705 - 8). Yoko Hattori et al. used the line C9285 that can survive in deep water to map and clone the SK1 gene and SK2 gene. SK1 The expression products of the SK2 gene and Nature ​, 460:1026-U116). Protein kinase CIPK15 regulates the plant energy and stress sensor SnRK1A and links the hypoxia signal to the SNRK1-dependent sugar sensing cascade, thereby regulating energy production and enabling rice to continue growing in deep water environments (Lee, K. W., Chen, P. W., Lu, C. A., et al. (2009). Coordinated Responsesto Oxygenand Sugar Deficiency Allow Rice Seedlings to Tolerate Flooding, Science Signaling , 2).

[0004] Aerenchyma is a continuous interconnected gas space formed by some cavities within plant parenchyma. The formation of aerenchyma is also an adaptation to waterlogging stress and is beneficial for the transport of oxygen from the upper part to the lower part of the plant. The classical view is that aerenchyma provides a diffusion pathway for plants, enabling oxygen to be transported from the above-ground parts of the plant to the hypoxic or anoxic roots to ensure the normal metabolic needs of the roots. Rice leaves are the main sites of photosynthesis. Through the aerenchyma (air cavities) in the midrib of rice leaves, the oxygen produced by photosynthesis is continuously transported to other tissue cells, especially the roots of rice, ensuring the normal growth of the plant under waterlogging stress.

[0005] Current research on rice aerenchyma mainly focuses on the formation of lysigenous aerenchyma in the roots, and the molecular mechanism of aerenchyma formation in rice leaves is still unclear. Therefore, exploring genes that regulate the development of rice leaf aerenchyma has important application value in breeding new rice varieties tolerant to waterlogging. Summary of the Invention

[0006] The main object of the present invention is to apply FSLE1 genes, FSLE1 protein, expression cassettes containing FSLE1 genes, or recombinant plant expression vectors containing FSLE1 genes to regulate the waterlogging tolerance of plants or to breed plant varieties tolerant to waterlogging.

[0007] To achieve the above object, the main technical solutions adopted by the present invention include:

[0008] On the one hand, the present invention applies FSLE1 genes, FSLE1 protein, expression cassettes containing FSLE1 genes, or recombinant plant expression vectors containing FSLE1 genes to regulate the waterlogging tolerance of plants.

[0009] A preferred specific embodiment of the present invention, wherein the plant is a gramineous plant.

[0010] A preferred specific embodiment of the present invention, wherein regulating the waterlogging tolerance of plants is to improve the waterlogging tolerance of plants; wherein, improving the waterlogging tolerance of plants is to promote the development of plant aerenchyma or reduce the yield reduction under waterlogging stress.

[0011] As a reference, the present invention provides an embodiment. That is, by overexpressing the coding gene of the FSLE1 protein related to the waterlogging tolerance of plants in plants, the expression level or activity of the FSLE1 protein related to the waterlogging tolerance of plants is increased, thereby improving the waterlogging tolerance of plants, and further promoting the development of plant aerenchyma or reducing the yield reduction under waterlogging stress.

[0012] A preferred specific embodiment of the present invention, a method for cultivating waterlogging-tolerant plant varieties, comprising: overexpressing FSLE1 gene in plants to enhance the expression level of FSLE1 gene or enhance the function or activity of the FSLE1 protein; for example, connecting the FSLE1 gene derived from rice with an expression regulatory element to obtain a recombinant plant expression vector for expressing the gene in plants; transforming the recombinant plant expression vector into plants to overexpress the FSLE1 gene in plants, and the waterlogging tolerance of the obtained transgenic plants is improved.

[0013] As a reference, the present invention provides a FSLE1 gene plant recombinant expression vector, comprising: connecting the FSLE1 gene derived from rice with an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector can be composed of a 5′-untranslated region, the nucleotide shown in SEQ ID NO.2, and a 3′-untranslated region; wherein, the 5′-untranslated region may include a promoter sequence, an enhancer sequence, or / and a translation enhancer sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue- or organ-specific promoter; the 3′-untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

[0014] The recombinant plant expression vector may further contain a selectable marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds, etc. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins, etc.

[0015] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell available for transformation. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment, etc. In certain embodiments, various transient transformation methods can be utilized to provide the FSLE1 gene to the plant. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0016] Another aspect of the present invention is to provide FSLE1 gene, FSLE1 protein, an expression cassette containing FSLE1 gene or a recombinant plant expression vector containing FSLE1 gene in regulating the tiller number, plant height, leaf length or width of rice; wherein, the regulation of the tiller number of rice is to increase the tiller number of rice; the regulation of the plant height of rice is to reduce the plant height; the regulation of the leaf length or width of rice is to reduce the leaf length or width of rice; including: mutating the FSLE1 gene in rice to reduce the expression level of the FSLE1 gene or to cause defects in the normal function of the FSLE1 protein.

[0017] The mutation includes substitution, deletion and / or addition of one or more nucleotides in the nucleotide sequence of the FSLE1 gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes but is not limited to radiation mutagenesis, space breeding, etc.; chemical mutagenesis methods include mutagenesis caused by treating with mutagens such as EMS, etc.; the gene editing methods include but are not limited to ZFN, TALE, and / or CRISPR / Cas, etc.

[0018] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing techniques to knockout the FSLE1 gene in plants, for example, constructing a FSLE1 gene knockout vector or using gene editing techniques to construct a FSLE1 CRISPR / Cas9 gene editing vector of the FSLE1 gene to knockout or mutate the FSLE1 gene in plants, and these methods are all proficiently mastered by those skilled in the art.

[0019] Those skilled in the art are aware that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the position in the host genome where gene editing is to be performed through a nucleic acid fragment called guide RNA (gRNA), that is, the target DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to proteins such as Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14.

[0020] The interference FSLE1 The normal expression or normal function of a gene or promoter can use RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the FSLE1 protein or its promoter or cause defects in its normal function. RNA interference technology is a conventional technology in this field. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short double-stranded RNA (siRNA) or a long double-stranded RNA (dsRNA; double-strand RNA), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0021] Another aspect of the present invention is to provide a FSLE1 gene or its encoded protein derived from rice that can regulate the waterlogging tolerance of plants.

[0022] (a) The polynucleotide sequence shown in SEQ ID NO.2;

[0023] (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1;

[0024] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and this polynucleotide sequence still has the function of regulating the waterlogging tolerance of plants;

[0025] (d) A polynucleotide sequence having at least 95% or more identity with the polynucleotide sequence shown in any one of (a)-(c), and this polynucleotide sequence still has the function of regulating the waterlogging tolerance of plants;

[0026] (e) A polynucleotide sequence that can be complementary to the polynucleotide sequence described in any one of (a)-(d), and this polynucleotide sequence still has the function of regulating the waterlogging tolerance of plants.

[0027] The percentage of sequence identity described in the present invention can be obtained through well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the algorithm of Karlin and Altschul, which are well-known to those skilled in the art.

[0028] In addition, those skilled in the art can also optimize the nucleotides shown in SEQ ID NO.2 to enhance the expression efficiency in plants.

[0029] The present invention can also be achieved by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO.2, and / or performing missense mutations of one or several base pairs.

[0030] Those of ordinary skill in the art can easily use known methods, such as directed evolution or point mutation methods, to FSLE1 mutate the nucleotide sequence of the gene. Those artificially modified nucleotides having 75% or higher identity with the FSLE1 nucleotide sequence of the gene, as long as the encoded protein has the function of regulating the waterlogging tolerance of plants, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0031] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA, etc.

[0032] The amino acid sequence of the FSLE1 protein described in the present invention is selected from any one of the following (a)-(d) amino acid sequences:

[0033] (a) The amino acid sequence shown in SEQ ID NO.1;

[0034] (b) A protein variant obtained by deleting or substituting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating the waterlogging tolerance of plants;

[0035] (c) A protein variant obtained by inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and this protein variant still has the function or activity of regulating the waterlogging tolerance of plants;

[0036] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, and this protein still has the function or activity of regulating the waterlogging tolerance of plants.

[0037] The present invention obtained fsm1 mutants in a large - scale rice mutant library, which showed abnormal aerenchyma development. Experiments found that fsm1 the mutants were waterlogging - sensitive mutants; further map - based cloning found that fsm1 the mutants were FSLE1 mutants obtained by gene deletion, and their phenotypes were reduced plant height, decreased leaf length and width, increased tiller number, increased parenchyma cells, decreased relative aerenchyma area, etc. By complementing fsm1 the FSLE1 gene into the FSLE1 mutants, it was found that the complemented FSLE1 gene lines restored the phenotypes of wild - type rice. The present invention further constructed over - expression FSLE1 gene lines, and simulated waterlogging treatment on wild - type rice WT and over - expression FSLE1 gene lines at the tillering stage. It was found that compared with wild - type rice WT, the over - expression FSLE1 gene lines had a significantly reduced yield reduction after waterlogging treatment. The present invention has application prospects in improving the waterlogging tolerance of plants, and can be used to cultivate rice varieties with strong waterlogging tolerance and apply them to the actual production process.

[0038] Term definitions related to the present invention

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in single - stranded or double - stranded form and their polymers. Unless specifically restricted, the terms cover nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the terms also mean oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (such as phosphorothioates, phosphoroamidates, etc.) used in antisense technology. Unless otherwise specified, a particular nucleic acid sequence also implicitly covers its conservatively modified variants (including (but not limited to) degenerate codon substitutions) and complementary sequences as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0041] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The terms apply to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

[0042] The term "recombinant host cell line" or "host cell" means a cell that contains a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-factor or other methods known in the art. The exogenous polynucleotide may remain as a non-integrated vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0043] The term "operably linked" refers to a functional linkage between two or more elements, and the elements that are operably linked may be adjacent or non-adjacent.

[0044] The term "recombinant plant expression vector" means one or more DNA vectors for achieving plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors having helper plasmids, are most commonly used for Agrobacterium tumefaciens-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be able to express in plant cells, heterologous DNA sequences to be transcribed, etc.

[0045] The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism.

[0046] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Phenotype observation results of wild-type rice WT and fsm1 mutants; wherein, Figure 1 -A is the plant type diagram of wild-type rice WT and fsm1 mutants at the mature stage; Figure 1 -B is the flag leaf morphology diagram of wild-type rice WT and fsm1 mutants at the mature stage; Figure 1 -C is the transverse section of a hand-cut slice of the leaves of wild-type rice WT and fsm1 mutants at the seedling stage, the asterisk indicates the air cavity, and the arrow indicates the parenchyma cells; Figure 1-D is a transverse section of a paraffin section of the flag leaf of wild-type rice WT at the mature stage. Since the midrib area is relatively large, in order to achieve higher clarity, this figure is composed of multiple high-magnification microscope photos; Figure 1 -E is fsm1 A transverse section of a paraffin section of the flag leaf of the mutant at the mature stage. Since the midrib area is relatively large, in order to achieve higher clarity, this figure is composed of multiple high-magnification microscope photos; Figure 1 -F is the leaf curling degree map of wild-type rice WT and fsm1 The mutant.

[0048] Figure 2 is FSLE1 The gene cloning schematic diagram of the Figure 2 -A is FSLE1 The map-based cloning schematic diagram of the Figure 2 -B is FSLE1 The schematic diagram of the gene structure and mutation position.

[0049] Figure 3 is the construction of the complementation vector and the identification result map of the lines complementing the FSLE1 gene; among them, Figure 3 -A is the schematic diagram of the pCam23A-FSLE1 vector structure; Figure 3 -B is the result map of the lines complementing the FSLE1 gene identified by the primers pCam23A-F and FSLE1-R; Figure 3 -C is the result map of the lines complementing the ACTIN gene amplified by the primers RICE-actin-F and RICE-actin-R of the internal reference gene FSLE1 ;

[0050] Figure 4 is the phenotypic observation and statistical result of the lines complementing the FSLE1 gene; among them, Figure 4 -A is the transverse section of the freehand section of the seedling leaves of wild-type rice WT, fsm1 the mutant, CP1, and CP2; Figure 4 -B is the statistical analysis result of the leaf length of wild-type rice WT, fsm1 the mutant, CP1, and CP2; Figure 4 -C is the statistical analysis result of the leaf width of wild-type rice WT, fsm1 the mutant, CP1, and CP2; Figure 4 -D is the statistical analysis result of the relative air cavity area of wild-type rice WT, fsm1 the mutant, CP1, and CP2; Figure 4 -E is the statistical analysis result of the plant height of wild-type rice WT, fsm1 the mutant, CP1, and CP2; Figure 4-F is the statistical analysis result of leaf rolling degree of wild-type rice WT, fsm1 mutants, CP1 and CP2; Figure 4 -G is the statistical analysis result of tiller number of wild-type rice WT, fsm1 mutants, CP1 and CP2.

[0051] Figure 5 It is the experimental result diagram of waterlogging treatment of wild-type rice WT and FSLE1 mutants of the gene; among them, Figure 5 -A is the seedlings diagram of wild-type rice WT and fsm1 mutants that have grown for 7 days without waterlogging treatment; Figure 5 -B is the seedlings diagram of wild-type rice WT and fsm1 mutants after 7 days of waterlogging treatment; Figure 5 -C is the statistical analysis result of the seedling length of wild-type rice WT and fsm1 mutants after waterlogging treatment; Figure 5 -D is the statistical analysis result of the root length of wild-type rice WT and fsm1 mutants after waterlogging treatment; Figure 5 -E is the detection result of the relative expression level of the FSLE1 gene in wild-type rice WT and overexpressed FSLE1 gene lines; Figure 5 -F is the statistical analysis result of the reduction amplitude of the yield per plant of wild-type rice WT and overexpressed FSLE1 gene lines after waterlogging treatment. Specific Embodiments

[0052] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0053] Vectors, Strains and Experimental Materials

[0054] The pCam23A vector is preserved by the Biotechnology Research Institute of the Chinese Academy of Agricultural Sciences; Agrobacterium tumefaciens AGL1 is purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., product number ZK296; the wild-type rice is Nipponbare rice ( Oryza sativa L.ssp. Japonica variety Nipponbare ; hereinafter also referred to as wild-type rice WT), and is preserved by the Crop High Photosynthetic Efficiency Functional Genomics Team of the Biotechnology Research Institute of the Chinese Academy of Agricultural Sciences.

[0055] Experimental Reagents

[0056] The primers used in PCR were synthesized by Beijing Tsingke Biotechnology Co., Ltd., and the sequencing was completed by Beijing Tsingke Biotechnology Co., Ltd. Restriction endonucleases Sma I and Xba I, Infusion recombinase, and high-fidelity enzyme were all purchased from Beijing Lihetong Trading Co., Ltd. (TaKaRa); antibiotics were purchased from SIGMA Company in the United States; the rest of the reagents were all domestic analytical pure.

[0057] The reagent formulas used in the experiment are as follows:

[0058] 2,4-D (2 mg / mL): First, dissolve 2,4-D by heating it in a microwave oven with 5 - 10 mL of 1 N KOH, and then make up the volume to a certain value with ultrapure water. Store at room temperature.

[0059] 6-BA (3 mg / mL): Weigh 150 mg of 6-BA, first dissolve it with 5 mL of 1 N KOH, and then make up the volume to 50 mL with sterile water. Filter sterilize.

[0060] Timentin Tim (200 mg / mL): Dissolve 2 g of Timentin with sterile water and make up the volume to 10 mL, then filter sterilize.

[0061] G418 (150 mg / mL): Dissolve it with sterile water, make up the volume, and filter sterilize.

[0062] Rifampicin Rif (25 mg / mL): First, dissolve 0.5 g of rifampicin (Rif) with 1 N NaOH, and then make up the volume to 10 mL with methanol; or directly make up the volume to 10 mL with DMSO, and then filter sterilize. Store at -20°C, and the working concentration is 25 μg / mL.

[0063] Kanamycin (50 mg / mL): Weigh 0.5 g of kanamycin sulfate powder, dissolve it with 10 mL of ultrapure water, filter sterilize, and the working concentration is 50 μg / mL.

[0064] Data processing

[0065] The following test examples used GraphPad Prism 8 statistical software to process the data. The significance analysis of a set of data used the Students t test. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0066] Test example 1 fsm1 Phenotypic analysis of mutants and cloning experiment of regulatory genes

[0067] 1 fsm1 Phenotypic and genetic analysis of mutants

[0068] Using the large-scale rice mutant library established in our laboratory previously (Wan et al., 2008), mutants were obtained fsm1 ( flooding sensitive mutant 1 ) that showed abnormal phenotypes such as abnormal aerenchyma development. Subsequently, the mutants were analyzed from two aspects: phenotypic characteristics and genetics.

[0069] After several years of multi-generation cultivation, the mutant phenotypes were stably inherited. Compared with the wild-type Nipponbare rice, fsm1 the plant height of the mutants was significantly reduced ( Figure 1 -A, Figure 4 -E), the leaf length was significantly decreased ( Figure 1 -B, Figure 4 -B), and the leaf width was significantly decreased ( Figure 1 -B, Figure 4 -C); both the wild-type and fsm1 mutant seedlings had two aerenchyma (indicated by asterisks in Figure 1 -C), and the relative aerenchyma area was significantly reduced ( Figure 1 -C, Figure 4 -D). At the same time, fsm1 the number of parenchyma cells in the mutants (the cells in the part indicated by the arrow in Figure 1 -C) was significantly increased compared with the wild-type; the flag leaves of the wild-type at the mature stage had four aerenchyma, while fsm1 the mutants only had two aerenchyma, with a significantly reduced area and a significant increase in parenchyma cells ( Figure 1 -D, Figure 1 -E); there was also a significant increase in leaf rolling degree ( Figure 1 -F, Figure 4 -F) and a significant increase in tiller number ( Figure 1 -A, Figure 4 -G).

[0070] The mutants were reciprocally crossed with the wild-type Nipponbare rice. The F fsm1 1 generation plants showed wild-type phenotypes, suggesting that these phenotypes were controlled by recessive genes. The F 1 1 generation plants were self-crossed to produce the F 1 2 generation, and the F 2 2 generation population was phenotypically analyzed and observed. The segregation ratio of the F 2 2 generation plants was subjected to chi-square test. It was found that the ratio of the number of plants showing wild-type rice phenotypes to the number of plants with mutant phenotypes conforms to Mendel's segregation law (Table 1), indicating that 2 the mutant phenotypes shown by the mutants are controlled by a pair of single recessive nuclear genes. fsm1 The mutant phenotypes shown by the mutants are controlled by a pair of single recessive nuclear genes.

[0071] Table 1 Statistical results of the F 2 2 generation segregation population

[0072]

[0073] Note: χ 2 0.05 (1) = 3.841.

[0074] 2 Regulation fsm1 Map-based cloning of mutant phenotype genes

[0075] Cross the mutant with indica rice, and self-cross the F fsm1 generation to obtain the F 1 generation, and select the phenotypically positive individual plants in the F 2 generation segregation population for gene mapping. Using more than 150 pairs of preliminary mapping molecular markers evenly distributed throughout the genome stored in the laboratory, perform PCR amplification and electrophoresis on 6 individual plants showing mutant phenotypes in the F 2 population. Screening reveals that the target gene is linked to markers 4-10 and 4-11 on chromosome 4. Subsequently, expand the number of phenotypically positive individual plants for further linkage analysis. The analysis results confirm the linkage between these molecular markers and the target gene, and the target gene is located within an interval of approximately 10.5 cM between markers 4-10 and 4-11 on chromosome 4 ( 2 -A). Figure 2 -A).

[0076] Design multiple molecular markers between markers 4-10 and 4-11. The primer list of the molecular markers is shown in Table 2. Use the phenotypically positive individual plants in the F 2 generation population for fine mapping. Finally, the target gene is located within an interval of approximately 0.2 cM. By sequencing all the genes in this interval, a 72-bp deletion is found in the FSLE1 gene exon, that is, a deletion of bases 105-176 of the CDS ( Figure 2 -B). This mutation leads to the loss of the function of the encoded protein. Therefore, FSLE1 the fsm1 gene is a candidate gene for regulating the

[0077] The amino acid sequence of the FSLE1 protein is shown in SEQ ID NO.1: MDRLNAKLYLQNCYIMKENERLRKKALLLNQENQALLTELKQRLAKTKAAAAAAAATKANGNGNMPAGGGRASLPDLNSAPPAHGHDKAVPKSKKTAAK* (SEQID NO.1).

[0078] FSLE1The nucleotide sequence of the gene is shown in SEQ ID NO.2: ATGGACAGGCTGAACGCGAAGCTGTACCTGCAGAACTGCTACATCATGAAGGAGAACGAGCGGCTGCGCAAGAAGGCGCTGCTGCTGAACCAGGAGAACCAGGCCTTGCTCACCGAGCTCAAGCAGCGGCTCGCCAAGACGAAGGCGGCGGCGGCGGCGGCGGCCGCGACCAAGGCTAACGGCAACGGCAACATGCCCGCCGGCGGCGGCCGCGCGTCCCTCCCCGACCTCAACTCGGCTCCGCCGGCGCACGGCCATGACAAGGCCGTGCCCAAGTCCAAGAAGACGGCCGCCAAGTAA (SEQ ID NO.2).

[0079] Table 2 Molecular marker primer sequences

[0080]

[0081] Experimental Example 2 FSLE1 Experiment on gene complementation and phenotype analysis

[0082] 1 Rice RNA extraction and reverse transcription

[0083] RNA extraction: The total RNA of rice was extracted using the RNA prep pure plant kit with the product number DP432 from Tiangen Biochemical Technology (Beijing) Co., Ltd. The tissue samples used for RNA extraction were quickly placed in tin foil after sampling in the field, frozen in liquid nitrogen, and then taken back to the Beijing laboratory for RNA extraction. The specific steps for RNA extraction are shown in the instruction manual.

[0084] RNA reverse transcription: The FastKing cDNA First Strand Synthesis Kit (genome - removed) with the product number KR116 from Tiangen Biochemical Technology (Beijing) Co., Ltd. was used and operated according to the instruction manual. The specific method is as follows:

[0085] Taking 50 ng - 2 μg of total RNA can establish a 20 μL reaction system. Thaw the template RNA on ice; thaw 5×gDNABuffer, FQ - RT Primer Mix, 10×King RT Bufer, RNase - Free ddH 2 O at room temperature and quickly place it on ice after thawing. Vortex each solution to mix well before use and briefly centrifuge to collect the liquid remaining on the tube wall. The following operation steps need to be carried out on ice.

[0086] The gDNA removal reaction system is as follows: 2 μL of 5×gDNA Buffer; 50 ng - 2 μg of RNA; RNase-Free ddH 2 O is made up to 10 μL. Incubate at 42°C for 3 min, then place on ice.

[0087] The reverse transcription reaction system is as follows: 2 μL of 10×King RT Bufer; 1 μL of FastKing RT Enzyme Mix; 2 μL of FQ-RTPrimer Mix; RNase-Free ddH 2 O is made up to 10 μL.

[0088] Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step and mix well. Incubate at 42°C for 15 min. Incubate at 95°C for 3 min and then place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0089] 2 FSLE1 Construction of gene complementation vector

[0090] Use the primer design software DNAMAN to design primers FSLE1-23AF and FSLE1-23AR. The nucleotide sequences of the primers are as follows:

[0091] FSLE1-23AF: TTGTAGGTAGAAGAGGTACCCGGGATGGACAGGCTGAACGCGAAG (SEQ ID NO.25);

[0092] FSLE1-23AR: GCATGCCTGCAGGTCGACTCTAGACCACGAAGCTGCATTACTTGG (SEQ ID NO.26).

[0093] Using the obtained cDNA as a template, FSLE1-23AF and FSLE1-23AR as primers, perform PCR amplification with the high-fidelity enzyme PrimeSTAR HS DNA Polymerase with GC Buffer (TaKaRa product number: R044A) purchased from Beijing Liuhetong Economic and Trade Co., Ltd. to obtain a PCR product.

[0094] Perform agarose gel electrophoresis separation, gel cutting and gel recovery on the obtained PCR product. The obtained recovered product and after Sma I and XbaThe pCam23A vector digested with I was recombined using the recombinase In-Fusion Snap Assembly Master Mix (TaKaRa product number: 638948). After sequencing, this recombinant vector pCam23A-FSLE1 was obtained by replacing the nucleotides of the CDS sequence of the FSLE1 gene with the Sma I and Xba I sites of the pCam23A vector ( Figure 3 -A). The arrow in the figure indicates the FSLE1 gene sequence.

[0095] 3 Complementation FSLE1 Preparation of gene lines

[0096] The above-prepared recombinant vector pCam23A-FSLE1 was introduced into Agrobacterium tumefaciens AGL1 to obtain the recombinant bacterium AGL1 / pCam23A-FSLE1. After digestion verification, positive recombinant bacteria were obtained.

[0097] The recombinant bacterium AGL1 / pCam23A-FSLE1 was transformed into fsm1 mutants by rice genetic transformation to obtain T 0 generation complementation FSLE1 gene lines. The specific operation steps are as follows:

[0098] (1) Seed sterilization and callus induction stage: Remove the hulls of mature seeds, select 300 plump and intact dehulled rice seeds and put them into a 50 mL sterilized centrifuge tube, and wash the seeds three times with sterilized ultrapure water. Then surface sterilize with 40 mL of 75% ethanol for 5 min, sterilize with 40 mL of 50% sodium hypochlorite for 5 min, repeat the sterilization once, and finally wash 10 times with sterile water until the water is clear. Blot the sterilized seeds dry with sterile filter paper and place them on the induction medium, 20 seeds per dish, and culture them under dark conditions at 28°C for 28 days until callus the size of shed millet grains grows.

[0099] (2) Subculture and pre-culture stage of callus: Transfer the well-conditioned embryogenic callus to the MS medium. About 100 pieces can be placed in each dish. Culture under dark conditions at 28°C for 7 days. The selected callus can be put back for continued culture. 3-4 times of selection can be carried out for one batch of induction. At the same time, the large pieces of un-shed callus can be placed on the new MS medium to make them shed again and then selected again.

[0100] (3) Preparation of Agrobacterium: One day in advance, culture Agrobacterium on YEP medium (or LB medium) supplemented with the corresponding resistance screening agent, kanamycin (50 mg / mL) and rifampicin (25 mg / mL). Use a spreading stick to scrape the Agrobacterium colonies on the original culture dish and spread them evenly on the new culture medium. If it is a bacterial liquid, pour a small amount of the bacterial liquid into the culture medium and spread it evenly with a spreading stick. Label the carrier, place it upside down, and incubate it in a 28°C incubator overnight.

[0101] Without activation treatment, the transformed colonies can be directly cultured for 4-5 days. Before shaking the culture, use a sterilized spoon to evenly spread all the colonies, then scrape an appropriate amount of bacteria and shake the culture. Bacteria stored at 4℃ must be activated.

[0102] (4) OD value adjustment: Scrape off the Agrobacterium with a key, place the bacteria in AAM liquid culture medium, and culture at 200 rpm at 28°C for 2 h. Then adjust the OD value of the bacterial solution to 0.12-0.15 using AAM liquid culture medium.

[0103] (5) Infection and co-cultivation: Collect about 100 embryonic calli into a 100 mL Erlenmeyer flask; Pour the adjusted concentration of Agrobacterium into the conical flask for infection, and shake it for 20 min on a shaker at 100 g. After shaking, pour out the infection solution, dry the callus with filter paper, move the infected callus to the co-cultivation medium, and cover it with sterile filter paper to ensure that all callus tissues are in contact with the filter paper surface. Culture in the dark at 22°C for 4 days.

[0104] (6) Resistance screening of transformed callus: Collect the callus tissue that has completed the co-culture stage into a 50 mL sterile centrifuge tube, rinse the callus tissue 10 times with sterile water until the washing liquid is clear. Then pour into the suspension culture medium, add 1 mL of Tim washing liquid with a concentration of 200 mg / mL, and shake it on a shaker at 100 g for 1 h. After shaking, pour out the filtrate and absorb the water with filter paper. Transfer the callus tissue to the selection culture medium and use tweezers to evenly place the callus particles to prevent contact inhibition and large-area contamination. 2-3 dishes per carrier can be screened. Culture in the dark at 28℃ for 2 weeks. This process is called the first screening. During this period, pay attention to observe whether there is any contamination. After two weeks, subculture once on the same culture medium, and double the number of screening culture dishes, so that each carrier can have 4-6 dishes. The selection lasts for about 4 weeks in total. This process is called the second screening. When the callus tissue has obvious yellow round particles the size of millet grains falling off, the next stage can be carried out.

[0105] (7) Differentiation and rooting: Select the white and dense callus and transfer it to the differentiation medium. Before using the differentiation medium, make sure to dry the water vapor completely. 20 pieces can be inoculated in each petri dish. Pay attention not to place them on the edge of the petri dish as much as possible, as it is easy to come into contact with water. Incubate under light at 28 °C for 3 - 4 weeks. Note that when placing the materials, they must be placed in layers. Prevent high temperature from burning the callus due to the heat generated by the light under the materials, which may affect the differentiation ability. It is best to place them on the bottom layer of the tissue culture room shelf to prevent the generation of water vapor and affect the differentiation of the callus. Then, subculture once on the same medium. Handle it gently to prevent the water droplets on the lid from dripping onto the callus. The callus that comes into contact with water will no longer differentiate. The callus just subcultured onto the differentiation medium needs to be placed for two days or covered with a black plastic bag for shading to prevent the callus from overheating and browning.

[0106] (8) Seedling strengthening: If relatively strong seedlings appear, transfer them to the 1 / 2 MS seedling strengthening medium. Incubate under light at 28 °C for 2 - 3 weeks. The seedlings just transferred to the seedling strengthening medium should be placed for two days before light treatment.

[0107] (9) Transplanting of tissue culture seedlings: Wash the residual medium on the roots, transfer the seedlings with good root systems to the greenhouse, and keep the soil moist in the first few days.

[0108] (10) Complementation FSLE1 Gene strain T 1 Creation of generation lines: Transfer the FSLE1 gene overexpression vector constructed into the fsm1 mutant through Agrobacterium tumefaciens, and identify multiple positive complementary transgenic lines. Self-cross the T 0 generation plants to obtain the T 1 generation complementary transgenic lines, randomly select CP1 and CP2 for subsequent analysis.

[0109] (11) The formula of the medium used is as follows:

[0110] N6 medium: The solutes are potassium nitrate 2830 g / L, ammonium sulfate 463 g / L, calcium chloride (CaCl 2 ·2H 2 O) 166 g / L, magnesium sulfate (MgSO 4 ·7H 2 O) 185 g / L, potassium dihydrogen phosphate 400 g / L, ferrous sulfate (FeSO 4 ·7H 2 O) 27.8 g / L, manganese sulfate (MnSO 4 ·H 2 O) 4.4 g / L, zinc sulfate (ZnSO 4 ·7H 2O) 1.6 g / L, boric acid 0.8 g / L, potassium iodide 1.6 g / L, vitamin B1 (thiamine hydrochloride) 1.0 g / L, vitamin B6 (pyridoxine hydrochloride) 0.5 g / L, nicotinic acid 0.5 g / L, glycine 2.0 g / L, and the solvent is deionized water.

[0111] Induction medium: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, casein hydrolysate 0.8 g / L, proline 0.3 g / L, sucrose 30 g / L, and phytagel 3 g / L.

[0112] Suspension medium: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, casein hydrolysate 0.8 g / L, proline 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, and acetosyringone 100 μM.

[0113] Co-culture medium: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, proline 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, acetosyringone 100 μM, and agar powder 8 g / L.

[0114] Selection medium: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, proline 0.3 g / L, G418 50 mg / L, ticarcillin 200 mg / L, sucrose 30 g / L, and agar powder 8 g / L.

[0115] MS medium: The solutes are CaCl 2 ·2H 2 O 440 mg / L, KH 2 PO 4 170 mg / L, MgSO 4 ·7H 2 O 370 mg / L, NH 4 NO 3 1650 mg / L, KNO 3 1900 mg / L, KI 0.83 mg / L, CoCl 2 ·6H 2 O 0.025 mg / L, H 3 BO 4 6.2mg / L, Na 2 MoO 4 ·7H 2 O 0.25 mg / L, MnSO 4 ·4H 2O 22.3 mg / L, CuSO 4 ·5H 2 O 0.025 mg / L, ZnSO 4 ·7H 2 O 8.6 mg / L, FeSO 4 ·7H 2 O 27.8 mg / L, Na 2 EDTA 37.3 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, inositol 100 mg / L, glycine 2.0 mg / L, sucrose 30000 mg / L, agar powder 7000 mg / L, with the balance being deionized water.

[0116] 1 / 2 MS medium: The final concentration of the solutes in MS is halved.

[0117] Differentiation medium: On the basis of MS medium, add 2 mg / L KT, 0.2 mg / L NAA, 2 mg / L 6 - BA, 0.2 mg / L IAA, 0.8 g / L casein hydrolysate, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel, with the balance being sterile water.

[0118] 4 complementation FSLE1 Positive identification and phenotypic analysis of the complemented gene lines

[0119] For the complemented FSLE1 gene lines, use DNAMAN software to design a pair of primers for identifying the complemented FSLE1 gene lines. To distinguish the FSLE1 gene of rice itself, the forward primer in this primer pair is located on the vector backbone, and the reverse primer is located on the FSLE1 gene. The primer sequences of the rice internal reference gene ACTIN RICE - actin - F and RICE - actin - R are as follows:

[0120] pCam23A - F: CCCAAAGTGCTATCCACGATCCAT (SEQ ID NO.27);

[0121] FSLE1 - R: CCACGAAGCTGCATTACTTGG (SEQ ID NO.28).

[0122] RICE - actin - F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO.29);

[0123] RICE-actin-R: GATGGGCCAGACTCGTCGTAC (SEQ ID NO.30).

[0124] The identification results are as shown in Figure 3 -B Figure 3 -C. The plants corresponding to the band numbers that are present in both Figure 3 -B Figure 3 -C are the positive plants in which the pCam23A-FSLE1 complementary vector has been successfully transferred into the fsm1 mutant. Among them, the positive plants all exhibit wild-type phenotypes, indicating the success of the complementation experiment. The T 0 positive plants obtained by self-crossing the T 1 positive plants were randomly selected as CP1 and CP2 for subsequent analysis.

[0125] Phenotypic observations were made on wild-type rice WT fsm1 mutants, CP1, and CP2 lines. It can be seen that the leaf air cavity area ( Figure 4 -A Figure 4 -D), leaf length ( Figure 4 -B), leaf width ( Figure 4 -C), plant height ( Figure 4 -E), leaf curl degree ( Figure 4 -F), and tiller number ( Figure 4 -G) of the CP1 and CP2 complementation lines have all been restored to wild-type levels. The above results prove that the FSLE1 gene mutation is the cause of the fsm1 mutant phenotype.

[0126] Experimental Example 3 FSLE1 Overexpression and Functional Analysis Experiment of the Gene

[0127] 1 Overexpression FSLE1 Preparation of Gene Lines

[0128] The pCam23A-FSLE1 vector prepared in Experimental Example 2 was transferred into wild-type Nipponbare rice using the method in Experimental Example 2 to prepare overexpression FSLE1 gene lines. Two overexpression FSLE1 gene lines, OE5 and OE12, were randomly selected for identification. The results showed that the FSLE1 gene expression levels in OE5 and OE12 were significantly increased ( Figure 5 -E), indicating the successful construction of the overexpression FSLE1 gene lines. The T 0 positive plants obtained by self-crossing the T 1 positive plants were used for subsequent analysis.

[0129] 2 FSLE1Experiment on application of gene regulation in rice's ability to tolerate flooding

[0130] Rice is planted in paddy fields, and its roots are in an oxygen-deficient environment. The required oxygen is transported from the above-ground part to the underwater roots through aerenchyma, which directly affects the normal growth of rice under flooded and oxygen-deficient conditions. fsm1 The aerenchyma area of ​​the mutant was significantly reduced. FSLE1 The application of genes in regulating rice waterlogging tolerance has an impact on wild-type rice WT and fsm1 The results showed that the mutants were subjected to germination waterlogging experiments. fsm1 Compared with wild-type rice WT, the mutant had significantly lower seedling height ( Figure 5 -A, Figure 5 -B, Figure 5 -C), while the root length was significantly shortened ( Figure 5 -A Figure 5 -B, Figure 5 -D), indicating FSLE1 Loss of gene function fsm1 The mutant is a flooding-sensitive mutant.

[0131] For research FSLE1 Whether the gene has the ability to regulate rice flooding tolerance, simulate the flooding environment of sudden flood disasters, and compare the effects of wild-type rice WT and overexpression FSLE1 Plants of the genetic lines OE5 and OE12 were flooded for 5 days at the tillering stage, and then restored to normal growth conditions. Seeds were harvested and statistical analysis of yield was performed. Figure 5 -F, the yield reduction ratio of WT, OE5 and OE12 plants after flooding was calculated and statistical analysis was performed. It was found that overexpression FSLE1 The average yield per plant of the gene plants OE5 and OE12 decreased by about 8%, and the average yield per plant of the wild-type rice WT decreased by about 22%. FSLE1 The yield reduction of the genetically modified plants was significantly reduced, indicating that FSLE1 The gene has the ability to regulate rice's tolerance to flooding.

Claims

1. FSLE1 Gene, FSLE1 protein, containing FSLE1 Gene expression cassette or containing FSLE1 Use of a recombinant plant expression vector of a gene in improving the waterlogging resistance of a plant; include ,Will FSLE1 Overexpression of genes in plants increases FSLE1 The expression amount or expression level of the gene; or enhance the function or activity of FSLE1 protein; the plant is rice; the FSLE1 The nucleotide sequence of the CDS of the gene is the polynucleotide sequence shown in SEQ ID NO.2; The amino acid sequence of the FSLE1 protein is the amino acid sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The improvement of the waterlogging resistance of plants is to promote the development of rice aerenchyma or reduce the yield reduction under waterlogging stress.

3. A method for cultivating a flood-resistant plant variety, characterized in that: include: Build contains FSLE1 Gene overexpression recombinant plant expression vector; transforming the overexpression recombinant plant expression vector into plants, so that FSLE1 The gene is overexpressed in the plant, and the resulting transgenic plant has improved waterlogging tolerance or reduced yield reduction under waterlogging stress; The FSLE1 The nucleotide sequence of the CDS of the gene is the polynucleotide sequence shown in SEQ ID NO.2; The plant is rice.

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