CN119685390B - Use of FSLE gene or coded protein thereof in regulating and controlling flooding resistance of plants - Google Patents

Abstract

The present invention discloses the use of FSLE1 gene or its encoded protein in regulating the ability of plant to withstand flooding. The present invention obtains fsm1 mutants in a large-capacity rice mutant library, which show abnormal air cavity development. Experiments show that fsm1 mutants are flood-sensitive mutants; through map-based cloning, it is found that fsm1 mutants are mutants obtained by the deletion of FSLE1 gene. By complementing FSLE1 gene into fsm1 mutants, it is found that the strains complemented with FSLE1 gene restore the phenotype of wild-type rice. The present invention further constructs strains that overexpress FSLE1 gene, and simulates flooding treatment of wild-type rice WT and strains that overexpress FSLE1 gene at the tillering stage, and finds that compared with wild-type rice WT, strains that overexpress FSLE1 gene have significantly reduced yield reduction. The present invention has application prospects in improving the ability of plants to withstand flooding, cultivating rice varieties with strong flooding resistance, etc.

Description

Use of FSLE gene or coded protein thereof in regulating and controlling flooding resistance of plants

Technical Field

The invention relates to a gene related to flooding resistance separated from rice and application thereof, in particular to application of FSLE gene or coded protein thereof separated from rice in regulating and controlling the flooding resistance of the rice, belonging to the FSLE gene and application field thereof.

Background

Flooding disasters are one of the common abiotic stresses in rice production, and 25-30% of the world rice planting areas are easy to be subjected to flooding stress. When the plants are flooded for a long time, the plants can be subjected to oxidative stress to generate excessive Reactive Oxygen Species (ROS) to damage cells, a large amount of mineral elements and main intermediate metabolites in plant root systems are dissolved and lost, meanwhile, anaerobic respiration can generate toxic secondary metabolites such as ethanol, acetaldehyde and the like which are unfavorable for plant growth, and finally, the plant roots, even the whole plant cells, are lack of oxygen, and the plants die when serious.

In recent years, some attention has been paid to the waterlogging-tolerant molecular mechanism of rice. The SUB1A is the earliest discovered main QTL locus for controlling the flooding resistance of rice, the rice flooding-resistant variety containing the SUB1A gene can induce the expression of fermentation genes such as alcohol dehydrogenase and the like under the flooding condition, so that the growth of plants under water is inhibited, the flooding-resistant capability of the plants and the survival capability of the plants after flooding is improved （Xu, K., Xu, X., Fukao, T., et al. (2006). Sub1A is an ethylene-response-factor-likegene that confers submergence tolerance to rice,Nature, 442: 705-8）.Yoko Hattori and the like are utilized to utilize a strain C9285 surviving in deep water, the SK1 gene and the SK2 gene are positioned by map cloning, the expression products of the SK1 gene and the SK2 gene cause remarkable elongation among joints through GA, the flooding-resistant rice phenomenon （Hattori, Y., Nagai, K., Furukawa, S., et al. (2009). The ethylene response factorsand allow rice to adapt to deep water,Nature, 460: 1026-U116）. protein kinase CIPK15 with the internode elongation exposing the water surface is generated to regulate and control the plant energy and the pressure sensor SnRK1A, and the hypoxia signal is connected with the sugar sensing cascade reaction depending on SNRK1, thereby regulating the energy generation and promoting the continuous growth of the rice under the deep water environment （Lee, K. W., Chen, P. W., Lu, C. A., et al. (2009). Coordinated Responses to Oxygenand Sugar Deficiency Allow Rice Seedlings to Tolerate Flooding,Science Signaling, 2）.

The aeration tissue (AERENCHYMA) is an interconnected and continuous gas space formed by some cavities in the plant parenchyma, and the formation of the aeration tissue is also an adaptive way for flooding stress, which is beneficial to the transportation of upper oxygen to lower parts. Classical wisdom holds that aeration tissue provides a diffusion pathway for plants to transport oxygen from the aerial parts of the plant to the hypoxic or anoxic root system to ensure the normal metabolic needs of the root. The rice leaves are the main sites for photosynthesis, and the rice leaves continuously transmit oxygen generated by photosynthesis to other tissue cells, especially the roots of the rice, through ventilation tissues (air cavities) of midribs, so that the plants of the rice can grow normally under the flooding adversity.

At present, the research on the rice aeration tissue mainly focuses on the formation of root-soluble aeration tissue, and the molecular mechanism of the formation of the rice leaf aeration tissue is not clear, so that the gene for regulating and controlling the development of the rice leaf aeration tissue is excavated, and the method has important application value in the cultivation of new varieties of water-logging-resistant rice.

Disclosure of Invention

The main purpose of the invention is to apply FSLE gene, FSLE1 protein, expression cassette containing FSLE gene or recombinant plant expression vector containing FSLE gene to regulate and control the flooding resistance of plants or to cultivate flooding-resistant plant varieties.

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises:

One aspect of the invention is the application of FSLE gene, FSLE protein, expression cassette containing FSLE gene or recombinant plant expression vector containing FSLE1 gene to regulate and control the flooding resistance of plants.

In a preferred embodiment of the invention, the plant is a gramineous plant.

In a preferred embodiment of the invention, the regulation of the flooding resistance of the plant is to improve the flooding resistance of the plant, wherein the improvement of the flooding resistance of the plant is to promote the development of aeration tissues of the plant or reduce the yield reduction amplitude under flooding stress.

For reference, the present invention provides one embodiment. Namely, through over-expression of the coding gene of FSLE protein related to the flooding resistance of the plant, the expression quantity or activity of FSLE protein related to the flooding resistance of the plant is improved, so that the flooding resistance of the plant is improved, the development of ventilation tissues of the plant is promoted, or the yield reduction amplitude is reduced under flooding stress.

A preferred embodiment of the invention provides a method for cultivating a flooding-tolerant plant comprising overexpressing the FSLE gene in the plant, enhancing the expression level of the FSLE gene or enhancing the function or activity of the FSLE protein, e.g., by ligating the FSLE gene derived from rice with an expression regulatory element to obtain a recombinant plant expression vector expressing the gene in the plant, transforming the plant with the recombinant plant expression vector, and overexpressing the FSLE1 gene in the plant to obtain a transgenic plant having improved flooding-tolerant capability.

The invention provides a FSLE gene plant recombinant expression vector, which comprises a FSLE gene derived from rice and an expression regulatory element to obtain the recombinant plant expression vector, wherein the recombinant plant expression vector can consist of a 5 'end non-coding region, a nucleotide shown as SEQ ID NO.2 and a 3' non-coding region, wherein the 5 'end non-coding region can comprise a promoter sequence, an enhancer sequence or/and a translation enhancing sequence, the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter, and the 3' non-coding region can comprise a terminator sequence, an mRNA cutting sequence and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells, for selection of transformed cells or tissues. The marker gene includes a gene encoding antibiotic resistance, a gene conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also includes phenotypic markers such as beta-galactosidase and fluorescent protein.

Transformation protocols and protocols for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, high velocity ballistic bombardment, and the like. In certain embodiments, the FSLE gene of rice can be provided to plants using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al PLANT CELL reports 1986.5:81-84).

The invention further provides application of FSLE genes, FSLE1 proteins, expression cassettes containing FSLE genes or recombinant plant expression vectors containing FSLE1 genes in regulating and controlling rice tillering numbers, plant heights, leaf lengths or widths, wherein the regulating and controlling rice tillering numbers are increasing rice tillering numbers, the regulating and controlling rice plant heights are reducing plant heights, the regulating and controlling rice leaf lengths or widths are reducing rice leaf lengths or widths, and the method comprises the steps of mutating FSLE genes in rice to reduce the expression quantity of FSLE genes or cause defects of normal functions of FSLE proteins.

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

The FSLE gene in the plant can be knocked out or mutated by a conventional method such as a conventional gene knockout or gene editing technology, for example, a FSLE1 gene knockout vector is constructed or a FSLE1 gene CRISPR/Cas9 gene editing vector is constructed by a gene editing technology, and FSLE gene in the plant is knocked out or mutated by a conventional method, and all the methods are well known to those skilled in the art.

It is known to those skilled in the art that the main principle of CRISPR/Cas gene editing systems or gene editing methods is to find the location where gene editing is to be performed, i.e. to target DNA sequences, in the host genome by means of a nucleic acid fragment called guide-RNA (gRNA), and then cleave the DNA by means of Cas proteins. In the present application, the Cas protein includes, but is not limited to, cas9, cas12a, cas12j, cas12e, cas13, and/or Cas14, among others.

The interference FSLE gene or the normal function can be realized by adopting RNA interference technology (RNAi) to interfere the normal expression of the FSLE protein coding gene or the promoter thereof or cause the normal function to be defective, and the RNA interference technology is conventional technology in the field, and the RNA interference technology specifically combines the 21-23bp short-chain double-stranded RNA (siRNA) or long-chain double-stranded RNA (dsRNA; double-STRAND RNA) with the mRNA homologous region expressed by the target gene to degrade the mRNA and inhibit the gene expression.

Another aspect of the invention provides FSLE genes or encoded proteins thereof from rice which are capable of regulating the flooding tolerance of plants.

(A) A polynucleotide sequence shown in SEQ ID NO. 2;

(b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1;

(c) A polynucleotide sequence capable of hybridizing to the polynucleotide sequence of (a) or (b) under stringent hybridization conditions, the polynucleotide sequence still having the function of regulating the flooding-resistance of a plant;

(d) A polynucleotide sequence having at least 95% or more identity to any one of the polynucleotide sequences shown in (a) - (c), which polynucleotide sequence still has the function of regulating the flooding-resistance of a plant;

(e) A polynucleotide sequence complementary to the polynucleotide sequence of any one of (a) - (d), which polynucleotide sequence still has the function of regulating the flooding-resistance of a plant.

The percentage of sequence identity described in the present invention may be obtained by well known bioinformatics algorithms, including Myers and Miller algorithms, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul algorithms, as is well known to those skilled in the art.

In addition, the nucleotide shown in SEQ ID NO.2 can be optimized by a person skilled in the art to enhance the expression efficiency in plants.

The invention may also be carried out by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID NO.2 and/or by making one or more base pair missense mutations.

The nucleotide sequence of the FSLE gene can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of FSLE gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, as long as the encoded protein has a function of regulating the flooding resistance of plants.

In addition, the nucleotide sequence described in the present invention may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

The amino acid sequence of FSLE protein in the present invention is selected from the amino acid sequences shown in any one of the following (a) - (d):

(a) An amino acid sequence shown in SEQ ID NO. 1;

(b) A protein variant is obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and the protein variant still has the function or activity of regulating the flooding resistance of plants;

(c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.1, wherein the protein variant still has the function or activity of regulating the flooding resistance of plants;

(d) A protein with 80% or more than 80% identity with the amino acid sequence shown in SEQ ID No.1, which still has the function or activity of regulating the flooding resistance of plants.

The invention obtains the fsm1 mutant in a large-capacity rice mutant library, which is expressed as abnormal development of an air cavity, tests find that the fsm1 mutant is a flooding sensitive mutant, and further discovers that the fsm1 mutant is a FSLE gene deletion mutant through map-based cloning, and the phenotype of the fsm1 mutant is reduced in plant height, reduced in leaf length and leaf width, increased in tiller number, increased in parenchyma cells, reduced in relative air cavity area and the like. By supplementing the FSLE gene back into the fsm1 mutant, the strain of the restoring FSLE gene was found to restore the phenotype of wild rice. The invention further constructs an over-expression FSLE gene line, simulates a wild rice WT and an over-expression FSLE gene line in a water flooding treatment tillering stage, and discovers that the over-expression FSLE gene line has obviously reduced yield reduction amplitude compared with the wild rice WT after the water flooding treatment. The invention has application prospect in improving the flooding resistance of plants, and can be used for cultivating rice varieties with strong flooding resistance and applying the rice varieties to actual production processes.

Definition of terms in connection with the present invention

Unless defined otherwise, 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.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoroamidites, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.

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

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a 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-integrating vector, such as a plasmid, or may integrate 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.

The term "operably connected" refers to a functional connection between two or more elements and the elements operably connected may be contiguous or non-contiguous.

The term "recombinant plant expression vector" means one or more DNA vectors for effecting plant transformation, which vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

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

The term "expression" refers to the transcription and/or translation of an endogenous gene or transgene in a plant cell.

Drawings

FIG. 1 is a graph showing the phenotype observation results of wild type rice WT and fsm1 mutants, wherein FIG. 1-A is a wild type rice WT and fsm1 mutant mature period plant type graph, FIG. 1-B is a wild type rice WT and fsm1 mutant mature period flag leaf shape graph, FIG. 1-C is a freehand slice cross-section graph of wild type rice WT and fsm1 mutant young seedling period leaves, asterisks show air cavities and arrows show thin wall cells, FIG. 1-D is a paraffin slice cross-section graph of wild type rice WT mature period flag leaves, which is formed by combining a plurality of high-magnification photomicrographs due to a large midrib area, FIG. 1-E is a fsm1 mutant mature period flag leaf paraffin slice cross-section graph due to a large midrib area, which is formed by combining a plurality of high-magnification photomicrographs due to a large midrib area, and FIG. 1-F is a wild type rice WT and fsm1 mutant leaf curl graph.

FIG. 2 is a schematic diagram of gene cloning of FSLE gene, wherein FIG. 2-A is a schematic diagram of map-site cloning of FSLE gene, and FIG. 2-B is a schematic diagram of FSLE gene structure and mutation position.

FIG. 3 is a diagram showing the identification result of the strain of the gene of FIG. FSLE, wherein FIG. 3-A is a schematic diagram showing the structure of the vector pCam A-FSLE1, FIG. 3-B is a diagram showing the identification result of the strain of the gene of FIG. FSLE by using the primers pCam A-F and FSLE1-R, and FIG. 3-C is a diagram showing the amplification result of the strain of the gene of FIG. FSLE by using the primers RICE-ACTIN-F and RICE-ACTIN-R of the reference gene ACTIN.

FIG. 4 is a statistical result of phenotype observation of a strain of the feedback FSLE1 gene, wherein FIG. 4-A is a cross-sectional view of a free hand slice of seedling leaves of wild-type rice WT, fsm1 mutant, CP1 and CP2, FIG. 4-B is a statistical analysis result of leaf length of wild-type rice WT, fsm1 mutant, CP1 and CP2, FIG. 4-C is a statistical analysis result of leaf width of wild-type rice WT, fsm1 mutant, CP1 and CP2, FIG. 4-D is a statistical analysis result of relative air cavity areas of wild-type rice WT, fsm1 mutant, CP1 and CP2, FIG. 4-E is a statistical analysis result of plant heights of wild-type rice WT, fsm1 mutant, CP1 and CP2, FIG. 4-F is a statistical analysis result of leaf curliness of wild-type rice WT, fsm1 mutant, CP1 and CP2, and tillering number of wild-type rice, fsm1 mutant, CP1 and CP 2.

FIG. 5 is a graph showing the results of a flooding test of mutants of wild-type rice WT and FSLE gene 1, wherein FIG. 5-A is a graph showing seedlings of wild-type rice WT and fsm1 grown for 7 days without the flooding test, FIG. 5-B is a graph showing seedlings of wild-type rice WT and fsm1 mutant in 7 days with the flooding test, FIG. 5-C is a statistical analysis of the length of seedlings after the flooding test of wild-type rice WT and fsm1 mutant, FIG. 5-D is a statistical analysis of the length of roots after the flooding test of wild-type rice WT and fsm1 mutant, FIG. 5-E is a result of detecting the relative expression level of FSLE1 gene in wild-type rice WT and over-expressed FSLE gene strain, and FIG. 5-F is a statistical analysis of the reduction of the yield of individual plants after the flooding test of wild-type rice WT and over-expressed FSLE gene strain.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.

Carrier, strain and test material

PCam23A vector is stored by the institute of biotechnology of China academy of agricultural sciences, agrobacterium AGL1 is purchased from Beijing African biological gene technology Co., ltd., product number ZK296, rice wild type is Nipponbare (Oryza satival. Ssp. JaponicarrieNipponbare; hereinafter also referred to as wild type rice WT), and is stored by the institute of biological technology of China academy of agricultural sciences crop high light efficiency functional genome team.

Test reagent

Primers used for PCR were synthesized by Beijing and sequencing were all done by Beijing, inc. of Biotech Co. The restriction enzymes SmaI and XbaI, infusion recombinase, high-fidelity enzyme are all purchased from Beijing Liujingjing trade company (TaKaRa), the antibiotics are purchased from American SIGMA, and the rest reagents are all of domestic analytical purity.

The reagent formulation used in the test procedure was as follows:

2,4-D (2 mg/mL) is prepared by heating and dissolving 2,4-D in 5-10: 10 mL of 1: 1N KOH, and adding ultrapure water to desired volume. Preserving at room temperature.

6-BA (3 mg/mL) 150 mg of 6-BA was weighed, dissolved in 5 mL of 1N KOH and then fixed to a volume of 50 mL with sterile water. And (5) filtering and sterilizing.

Tim (200 mg/mL) of Tim, 2 g Tim, was dissolved in sterile water to a volume of 10 mL, and filter sterilized.

G418 (150 mg/mL) and sterilizing by filtration with sterile water.

Rif (25 mg/mL) 0.5 g Rif was dissolved in 1. 1N NaOH and then fixed to 10 mL with methanol or directly fixed to 10 mL with DMSO and filter sterilized. Stored at-20℃and working concentration 25. Mu.g/mL.

Kanamycin (50 mg/mL) kanamycin sulfate powder 0.5 g was weighed, dissolved in 10mL ultrapure water, filtered and sterilized, and the working concentration was 50. Mu.g/mL.

Data processing

The following test examples were run on data using GRAPHPAD PRISM statistical software, and the significance analysis of a set of data was tested using the Students t test, with P <0.05 (x) indicating significant differences and P <0.01 (x) indicating very significant differences.

Test example 1 phenotypic analysis of fsm1 mutant and cloning test of regulatory Gene

Phenotypic and genetic analysis of 1 fsm1 mutants

The fsm1 mutant (flooding sensitive mutant) is obtained by using the large-capacity rice mutant library (Wan et al, 2008) created in the early stage of the laboratory, and the phenotypes such as abnormal development of the air cavity are expressed, so that the mutant is analyzed from the aspects of phenotypic characteristics and genetics.

The mutant is stably inherited through planting mutation phenotype for a plurality of years, compared with wild type Nippon rice, the plant height of the fsm1 mutant is obviously reduced (figure 1-A, figure 4-E), the leaf length is obviously reduced (figure 1-B, figure 4-B), the leaf width is obviously reduced (figure 1-B, figure 4-C), the leaves of wild type seedlings and fsm1 mutant seedlings are respectively provided with 2 air cavities (shown by asterisks in figure 1-C), the area of the relative air cavities is obviously reduced (figure 1-C, figure 4-D), meanwhile, the number of parenchyma cells (cells of the part shown by arrows in figure 1-C) of the fsm1 mutant is obviously increased compared with that of wild type seedlings, the flag leaves of the wild type mature period are provided with 4 air cavities, the fsm1 mutant is only provided with 2 air cavities, the area is obviously reduced, the parenchyma cells are obviously increased (figure 1-D, figure 1-E), and the leaf rolling degree is obviously increased (figure 1-F, figure 4-F) and the tillering number is obviously increased (figure 1-A, figure 4-G).

Forward and reverse crossing of the fsm1 mutant with wild type japonica rice, the F ₁ generation plants showed a wild type phenotype, and the phenotype was presumed to be recessive gene control. F ₂ generation is generated by selfing the F ₁ generation plants, phenotypic analysis and observation are carried out on the F ₂ generation population, chi square detection is carried out on the segregation ratio of F ₂ generation plants, and the ratio of the number of plants showing the phenotype of wild type rice to the number of plants showing the phenotype of mutant is found to accord with the Mendelian segregation law (table 1), so that the phenotype of the mutant shown by the fsm1 mutant is controlled by a pair of single recessive nuclear genes.

TABLE 1F ₂ generation of segregating population statistics

Note that χ ² _0.05 (1) = 3.841.

2. Map-based cloning of phenotype genes for regulating and controlling fsm1 mutants

The fsm1 mutant is hybridized with indica rice, F ₁ generation is selfed to obtain F ₂ generation, and a single plant with phenotype in F ₂ generation segregating population is selected for gene localization. And (3) carrying out PCR amplification and electrophoresis on 6 single plants showing mutant phenotype in the F ₂ population by adopting more than 150 pairs of preliminary positioning molecular markers which are uniformly distributed in the whole genome stored in a laboratory, and screening to find that the target gene has linkage relationship with the 4-10 markers and the 4-11 markers on chromosome 4. Subsequently, the number of phenotypic individuals was expanded for further linkage analysis, and the analysis result confirmed that these molecular markers were linked to the target gene, and the target gene was located in the region of about 10.5 cM between chromosome 4-10 marker and chromosome 4-11 marker (FIG. 2-A).

A plurality of molecular markers are designed between the 4-10 markers and the 4-11 markers, the molecular marker primer list is shown in table 2, the F ₂ generation population is utilized to carry out fine localization on the phenotypic individual, and finally the target gene is localized in the interval of about 0.2 cM. By sequencing all genes in this region, a 72bp deletion was found in FSLE gene exons, i.e., the 105 th to 176 th bases of CDS (FIG. 2-B), which resulted in a deletion of the function of the encoded protein, and thus the FSLE gene was a candidate gene for regulating the fsm1 mutant phenotype.

The amino acid sequence of FSLE protein is shown as SEQ ID NO.1 ：MDRLNAKLYLQNCYIMKENERLRKKALLLNQENQALLTELKQRLAKTKAAAAAAAATKANGNGNMPAGGGRASLPDLNSAPPAHGHDKAVPKSKKTAAK* （SEQ ID NO.1）.

The nucleotide sequence of FSLE gene is shown as SEQ ID NO.2 ：ATGGACAGGCTGAACGCGAAGCTGTACCTGCAGAACTGCTACATCATGAAGGAGAACGAGCGGCTGCGCAAGAAGGCGCTGCTGCTGAACCAGGAGAACCAGGCCTTGCTCACCGAGCTCAAGCAGCGGCTCGCCAAGACGAAGGCGGCGGCGGCGGCGGCGGCCGCGACCAAGGCTAACGGCAACGGCAACATGCCCGCCGGCGGCGGCCGCGCGTCCCTCCCCGACCTCAACTCGGCTCCGCCGGCGCACGGCCATGACAAGGCCGTGCCCAAGTCCAAGAAGACGGCCGCCAAGTAA （SEQ ID NO.2）.

TABLE 2 molecular marker primer sequences

Test example 2 test for the anaplerosis and phenotyping of FSLE1 Gene

1. Rice RNA extraction and reverse transcription

RNA extraction Total RNA of rice was extracted using RNA prep pure plant kit of Tiangen Biochemical technology (Beijing) Co., ltd., product No. DP 432. The tissue sample for extracting RNA is rapidly put into tinfoil paper after being sampled in a field, and is brought back to Beijing laboratory after being frozen by liquid nitrogen for RNA extraction. For specific steps of RNA extraction, see the instructions.

RNA reverse transcription was performed using FASTKING CDNA first Strand Synthesis kit (Degenome) from Tiangen Biochemical technologies (Beijing) Co., ltd., product No. KR116 according to the instructions. The specific method comprises the following steps:

A reaction system of 20. Mu.L can be established by taking 50-ng-2. Mu.g total RNA, and the template RNA is thawed on ice, and 5 XgDNA Buffer, FQ-RT Primer Mix, 10X King RT Bufer and RNase-Free ddH ₂ O are thawed at room temperature and rapidly placed on ice after thawing. Before use, each solution was vortexed and mixed well and centrifuged briefly to collect the liquid remaining on the tube wall. The following steps are carried out on ice.

The gDNA removal reaction system was 5 XgDNA Buffer 2. Mu.L, RNA 50 ng-2. Mu.g, RNase-Free ddH ₂ O to a total of 10. Mu.L. Incubate 3 min at 42 ℃ and then place on ice.

The reverse transcription reaction system was 10X King RT Bufer. Mu.L, FASTKING RT Enzyme Mix 1. Mu.L, FQ-RTPrimer Mix. Mu.L, RNase-Free ddH ₂ O to 10. Mu.L.

Mix in the reverse transcription reaction was added to the reaction solution in the gDNA removal step, and mixed well. Incubation at 42 ℃ was 15 min. After incubation at 95℃and 3min, the resulting cDNA was placed on ice and used for subsequent experiments or for cryopreservation.

Construction of 2 FSLE1 Gene repair vector

Primers FSLE-23 AF and FSLE-23 AR were designed using the primer design software DNAMAN, the nucleotide sequences of which are shown below:

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

FSLE1-23AR:GCATGCCTGCAGGTCGACTCTAGACCACGAAGCTGCATTACTTGG (SEQ ID NO.26)。

PCR amplification was performed using the cDNA obtained as described above as a template, FSLE-23 AF and FSLE-23 AR as primers, and high-fidelity enzyme PRIMESTAR HS DNA Polymerase with GC Buffer (TaKaRa: R044A) purchased from Peking Liuzhitong Summit Co., ltd.

And (3) carrying out agarose gel electrophoresis separation, gel cutting and gel recovery on the obtained PCR product, and recombining the obtained recovered product with pCam A vector subjected to SmaI and XbaI digestion by utilizing recombinase In-Fusion Snap Assembly Master Mix (TaKaRa product number: 638948). The recombinant vector pCam A-FSLE1 was obtained by sequencing the SmaI and XbaI sites of pCam A vector with the nucleotide of CDS sequence of FSLE1 gene (FIG. 3-A), and the arrow in the figure shows FSLE gene sequence.

3. Preparation of the Rev FSLE Gene Strain

The recombinant vector pCam A-FSLE1 prepared above is introduced into agrobacterium AGL1 to obtain recombinant strain AGL1/pCam A-FSLE1, and the positive recombinant strain is obtained through enzyme digestion verification.

The recombinant strain AGL1/pCam A-FSLE1 is transformed into the fsm1 mutant by adopting rice genetic transformation to obtain a T ₀ generation strain of the anaplerotic FSLE1 gene. The specific operation steps are as follows:

(1) In the seed sterilization and callus induction stage, mature seeds are dehulled, and 300 full dehulled rice grains are selected and placed into a 50mL sterilization centrifuge tube, and the seeds are washed three times by sterilized ultrapure water. Then using 40mL 75% ethanol to sterilize 5min, using 40mL 50% sodium hypochlorite to sterilize 5min, repeating the sterilization, finally using sterile water to wash 10 times until the water is clarified, using sterile filter paper to suck the sterilized seeds dry, placing the seeds on an induction culture medium, culturing the seeds on a 20-grain/dish under the dark culture condition at 28 ℃ for 28 days until the callus with the size of the fallen grains grows out.

(2) And the stage of subculturing and preculturing the callus, namely transferring embryogenic callus with good state to MS culture medium. Each dish can be placed with about 100 grains. Culturing for 7 days under the dark condition at 28 ℃. The picked callus can be put back for continuous culture, a batch of induction can be picked for 3-4 times, and meanwhile, a large piece of non-shedding callus can be put on a new MS culture medium, and then shed again.

(3) The preparation of agrobacterium is to add YEP culture medium (or LB culture medium) with corresponding screening resistance, kanamycin (50 mg/mL) and rifampicin (25 mg/mL) 1 day in advance, plate-coat the agrobacterium on the culture medium, scrape the agro-rod colony on the original culture dish with the coating rod, uniformly coat on the new culture medium, if the agro-rod colony is bacterial liquid, pour the bacterial liquid into the culture medium, a small amount of bacterial liquid, uniformly coat with the coating rod, make carrier label, invert and place, and culture box at 28 ℃ overnight.

The method comprises the steps of directly culturing the transformed colonies for 4-5 days without activating treatment, uniformly smearing all the colonies by using a sterilized spoon before shaking, and scraping a proper amount of bacteria for shaking. The bacteria preserved at 4 ℃ must be subjected to an activation treatment.

(4) OD value adjustment, namely scraping off agrobacterium with a key, placing the agrobacterium into AAM liquid culture medium, and culturing the agrobacterium with a shaking table at 28 ℃ for 2h turns at 200 turns. Then, the OD value of the bacterial liquid is adjusted to 0.12-0.15 by AAM liquid culture medium.

(5) Infection and co-cultivation, namely collecting about 100 embryogenic calli into a triangular flask of 100 mL, pouring the agrobacterium with the adjusted concentration into a conical flask for infection, and shaking 20min by using a shaking table of 100 g. Pouring out the soaking dye liquor after the vibration is finished, sucking the callus by using filter paper, transferring the infected callus to a co-culture medium, and covering sterile filter paper to ensure that all the callus contacts the surface of the filter paper on the filter paper. Dark culture at 22℃4 d.

(6) Resistance screening of transformed calli, namely collecting calli subjected to the co-culture stage into a 50 mL sterilization centrifuge tube, flushing the calli with sterile water for 10 times until the flushing liquid is clear. Then pouring the suspension medium, adding 1mL of Tim lotion with the concentration of 200 mg/mL, shaking the suspension medium by using a shaking table 100 g for 1h, pouring out filtrate after the shaking is finished, and sucking the water by using filter paper. Transferring the callus onto a selective culture medium, and uniformly placing callus particles by forceps to prevent contact inhibition and large-area bacteria infection. And (3) screening a carrier in a 2-3 dish. Dark culture at 28 ℃ for 2 weeks, the process became a sieve. During the period, the observation of pollution or not is carried out. And (3) subculturing on the same culture medium once after two weeks, and doubling the number of culture medium dishes for screening, wherein each carrier is 4-6 dishes. A total of about 4 weeks was selected. This process becomes a two-screen. And (5) the yellow round solid particles with obvious small rice size of the callus can fall off to carry out the next stage.

(7) Differentiation and rooting, namely transferring the white and compact callus onto a differentiation culture medium, and taking care of airing water vapor before the differentiation culture medium is used, wherein each dish can be connected with 20 grains, taking care of not being placed at the edge of the culture dish as much as possible, and being easy to contact with water. Light culture at 28℃for 3-4 weeks. Care must be taken to keep the material in place. The lower layer of the material has the capability of preventing the differentiation of the callus caused by high-temperature burn generated by lamplight heat. Preferably placed at the lowest layer of the tissue culture chamber shelf to prevent water vapor from being generated and influence the differentiation of callus. Then the culture medium is used for subculture once again, the user takes the culture medium lightly and releases the culture medium lightly, so that the water drops on the cover are prevented from dripping on the callus, and the callus which is in contact with water is not differentiated any more. The callus on the differentiation medium just after the subculture needs to be placed for two days or is subjected to shading treatment by a black plastic bag, so that the overheat browning of the callus is prevented.

(8) Strong seedlings, if stronger seedlings appear, the seedlings are connected to 1/2 MS strong seedling culture medium. Light culture at 28℃for 2-3 weeks. The young seedlings which are just connected to the strong seedling culture medium are placed for two days and then are subjected to light treatment.

(9) Transplanting tissue culture seedlings, namely washing off residual culture medium on roots, transferring seedlings with good root systems into a greenhouse, and keeping soil moist for the first few days.

(10) And (3) creating a mending FSLE gene line T ₁ generation line, namely transferring the constructed FSLE gene over-expression vector into an fsm1 mutant through agrobacterium, and identifying and obtaining a plurality of positive complementary transgenic lines. The T ₀ generation plants were selfed to obtain T ₁ generation complementary transgenic lines, and CP1 and CP2 were randomly selected and used for subsequent analysis.

(11) The medium formulation used was as follows:

The solute of the N6 culture medium is potassium nitrate 2830 g/L, ammonium sulfate 463 g/L, calcium chloride (CaCl ₂·2H₂ O) 166. 166 g/L, magnesium sulfate (MgSO ₄·7H₂ O) 185 g/L, monopotassium phosphate 400 g/L, ferrous sulfate (FeSO ₄·7H₂ O) 27.8 g/L, manganese sulfate (MnSO ₄·H₂ O) 4.4 g/L, zinc sulfate (ZnSO ₄·7H₂ O) 1.6g/L, boric acid 0.8 g/L, potassium iodide 1.6g/L, vitamin B1 (thiamine hydrochloride) 1.0 g/L, vitamin B6 (pyridoxine hydrochloride) 0.5g/L, nicotinic acid 0.5g/L, glycine 2.0 g/L, and the solvent is deionized water.

Induction medium 2,4-D,0.8 g/L hydrolyzed casein, 0.3 g/L proline, 30 g/L sucrose and 3 g/L plant gel were added at a final concentration of 2.5 mg/L on the basis of N6 medium.

Suspension medium 2,4-D,0.8 g/L hydrolyzed casein, 0.3 g/L proline, 30 g/L sucrose, 10 g/L glucose and 100 μm acetosyringone were added to the N6 medium at a final concentration of 2.5 mg/L.

Co-culture medium 2,4-D with final concentration of 2.5 mg/L, proline with final concentration of 0.3 g/L, sucrose with final concentration of 30 g/L, glucose with final concentration of 10 g/L, acetosyringone with final concentration of 100 μm and agar powder with final concentration of 8 g/L are added on the basis of N6 culture medium.

The culture medium is selected, 2,4-D with the final concentration of 2.5 mg/L, proline with the final concentration of 0.3G/L, temetin with the final concentration of 50 mg/L and G418,200 mg/L, sucrose with the final concentration of 30G/L and agar powder with the final concentration of 8G/L are added on the basis of the N6 culture medium.

MS culture medium comprises CaCl₂·2H₂O 440 mg/L、KH₂PO₄170 mg/L、MgSO₄·7H₂O 370 mg/L、NH₄NO₃1650 mg/L、KNO₃1900 mg/L、KI 0.83 mg/L、CoCl₂·6H₂O 0.025 mg/L、H₃BO₄6.2 mg/L、Na₂MoO₄·7H₂O 0.25 mg/L、MnSO₄·4H₂O 22.3 mg/L、CuSO₄·5H₂O 0.025 mg/L、ZnSO₄·7H₂O 8.6 mg/L、FeSO₄·7H₂O 27.8 mg/L、Na₂EDTA 37.3 mg/L、 thiamine hydrochloride 0.1 mg/L solute, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, inositol 100 mg/L, glycine 2.0 mg/L, sucrose 30000mg/L, agar 7000 mg/L, and deionized water for the rest.

1/2MS culture medium, the final concentration of solute in MS is halved.

Differentiation medium MS medium was supplemented with 2 mg/L KT,0.2 mg/L NAA,2 mg/L6-BA, 0.2 mg/L IAA,0.8 g/L hydrolyzed casein, 0.3 g/L proline, 30 g/L sucrose and 3 g/L vegetable gel, the balance being sterile water.

4. Positive identification and phenotypic analysis of the anaplerotic FSLE gene line

The strain of the anaplerotic FSLE gene was identified and a pair of primers designed using DNAMAN software to identify the strain of the anaplerotic FSLE gene was used. In order to distinguish FSLE genes of rice per se, a forward primer is positioned on a vector skeleton, and a reverse primer is positioned on FSLE genes. The primers of RICE reference gene ACTIN, RICE-ACTIN-F and RICE-ACTIN-R, the sequences of the primers are as follows:

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

FSLE1-R:CCACGAAGCTGCATTACTTGG (SEQ ID NO.28)。

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

RICE-actin-R: GATGGGCCAGACTCGTCGTAC (SEQ ID NO.30)。

The identification results are shown in the figure 3-B, the figure 3-C, and the plants corresponding to the numbers with the bands in the figure 3-B and the figure 3-C are positive plants obtained by successfully transferring pCam A-FSLE1 complementary vectors into the fsm1 mutant, wherein the positive plants are all expressed as wild type phenotypes, which indicates that the resupply experiment is successful. T ₀ generation positive plants were selfed to obtain T ₁ positive plants, and CP1 and CP2 were randomly selected for subsequent analysis.

Phenotypic observations of wild-type rice WT, fsm1 mutant, CP1 and CP2 strains revealed that the leaf cavity area (FIG. 4-A, FIG. 4-D), leaf length (FIG. 4-B), leaf width (FIG. 4-C), plant height (FIG. 4-E), leaf curliness (FIG. 4-F) and tillering number (FIG. 4-G) of the CP1 and CP2 complementation lines all restored to wild-type levels, which demonstrated that the FSLE gene mutation was responsible for the appearance of the fsm1 mutant phenotype.

Test example 3 overexpression and functional analysis of FSLE1 Gene

1. Preparation of an overexpressed FSLE Gene Strain

The pCam A-FSLE1 vector prepared in test example 2 was transferred into wild type Nippon Rice by the method of test example 2 to prepare an overexpressed FSLE gene strain. Two over-expressed FSLE1 gene lines OE5 and OE12 are randomly selected for identification, and the results show that the FSLE gene expression level in the OE5 and OE12 is obviously improved (fig. 5-E), which shows that the over-expressed FSLE1 gene line is successfully constructed, and T ₁ positive plants obtained by selfing T ₀ generation plants are used for subsequent analysis.

Application test of 2 FSLE1 gene in regulating and controlling flooding resistance of rice

The rice is planted in paddy fields, the roots are in an anoxic environment, and the needed oxygen is transported from the overground parts to the underwater roots by virtue of ventilation tissues, and the ventilation tissues directly influence the normal growth of the rice under the condition of flooding and anoxic. The ventilation tissue area of the fsm1 mutant is obviously reduced, and germination flooding experiments are carried out on wild rice WT and the fsm1 mutant in order to research the application of FSLE gene in regulating the flooding resistance of rice. The results show that the fsm1 mutant after seed germination has significantly reduced seedling height (FIG. 5-A, FIG. 5-B, FIG. 5-C) and significantly shortened root length (FIG. 5-A, FIG. 5-B, FIG. 5-D) compared with wild-type rice WT, indicating that the FSLE gene-function-deleted fsm1 mutant is a flooding-sensitive mutant.

In order to study whether FSLE gene has the capacity of regulating and controlling the flooding resistance of rice, simulating the flooding environment of sudden flood disasters, carrying out flooding treatment on wild rice WT and plants in the tillering stage of OE5 and OE12 of the over-expressed FSLE gene lines for 5 days, recovering normal growth conditions after the flooding treatment, harvesting seeds and carrying out statistical analysis on yield. The test results are shown in fig. 5-F, the individual yield reduction ratio of the WT, OE5 and OE12 plants after flooding treatment is calculated and statistical analysis is performed, and it is found that the individual yield of the over-expressed FSLE gene plants OE5 and OE12 is reduced by about 8% on average, and the wild rice WT is reduced by about 22% on average, that is, compared with the wild rice WT, the yield reduction amplitude of the over-expressed FSLE gene plants is significantly reduced, which indicates that the FSLE gene has the capacity of regulating and controlling rice flooding resistance.

Claims (3)

1. The application of FSLE1 gene, FSLE protein, expression cassette containing FSLE1 gene or recombinant plant expression vector containing FSLE1 gene in improving the flooding resistance of plant includes over-expressing FSLE1 gene in plant to raise the expression level or expression level of FSLE1 gene or enhancing FSLE1 protein function or activity, the plant is rice, the nucleotide sequence of CDS of FSLE gene is the polynucleotide sequence shown in SEQ ID No. 2;

   The amino acid sequence of FSLE protein is the amino acid sequence shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the improvement in the flooding tolerance of the plant is the promotion of the development of rice aeration tissue or the reduction of the yield under flooding stress.
3. 3. A method for cultivating a flooding-resistant plant variety is characterized by comprising the steps of constructing an over-expression recombinant plant expression vector containing FSLE gene, transforming a plant by using the over-expression recombinant plant expression vector, and enabling FSLE gene to be over-expressed in the plant, wherein the flooding-resistant capacity of the obtained transgenic plant is improved or the yield reduction amplitude of the obtained transgenic plant is reduced under flooding stress;

   the CDS nucleotide sequence of FSLE gene is the polynucleotide sequence shown in SEQ ID NO. 2;

   The plant is rice.