Application of rice Lhca4 gene and its encoded protein in improving plant resistance to salt stress

Abstract

The invention discloses rice Lhca4 The invention discloses a gene and its encoded protein for improving plant resistance to salt stress; the ... Lhca4 The transgenic rice obtained by overexpressing the gene in rice and the wild-type rice were overexpressed after salt stress treatment. Lhca4 The chlorophyll content and plant height of transgenic rice lines with the gene were significantly higher than those of wild type. Lhca4 The gene was knocked out using gene editing technology to obtain transgenic rice. After salt stress treatment, the knockout Lhca4 The chlorophyll content, plant height and root length of the transgenic rice lines with the gene were significantly lower than those of the wild-type rice, thus confirming that Lhca4 The gene or its encoded protein has the function of regulating the resistance of plants to abiotic stress. The invention has application prospects in enhancing the stress resistance of rice, improving the resistance of plants to abiotic stress or cultivating plant varieties with resistance to abiotic stress.

Description

Technical Field

[0001] The present invention relates to Lhca4 New uses of genes and their encoded proteins, especially in rice Lhca4 New use of genes and their encoded proteins in improving plant resistance to abiotic stress, belonging to Lhca4 New areas of use for genes and their encoded proteins. Background Art

[0002] Rice ( Oryza sativa L.) is one of the three major staple crops in the world. In the process of rice production, salt stress is one of the most serious adversities affecting its yield. Its effects mainly include osmotic stress, ion toxicity and secondary oxidative stress (Zhou, H., Shi, H., Yang, Y., Feng, X., Chen, X., Xiao, F., Lin, H. and Guo,Y. (2024)Insights into plant salt stress signaling and tolerance. Journal of Genetics and Genomics 51, 16-34.), due to the high concentration of soluble salt in the soil, crop growth and development are poor, dry matter accumulation is reduced, and crop yields decrease. According to statistics, the global saline-alkali land area exceeds 800 million hectares, of which more than 100 million hectares of agricultural land in China are affected by salinization to varying degrees, seriously endangering food security. Rice salt tolerance is a quantitative trait controlled by multiple genes and involves many complex physiological processes (Hamam, AM, Coskun, D., Britto, DT, Plett, D. and Kronzucker, HJ (2019) Plasma-membrane electrical responses to salt and osmotic gradients contradict radiotracer kinetics, and reveal Na + -transport dynamics in rice ( Oryza sativa L.). Planta 249, 1037-1051.), At present, the main problem in the identification and gene utilization of rice salt-tolerant germplasm resources is the limited number of major salt-tolerant genes. How to accurately discover new salt-tolerant genes is an important issue facing the current research on rice germplasm resources.

[0003] Conventional breeding cycles are long, and their selection efficiency is difficult to meet the requirements of salt-tolerant breeding practice. With the development of modern biotechnology, gene editing and molecular marker-assisted selection technology have gradually become common technical means for rice salt tolerance mechanism research and breeding. Using SNP markers to construct high-precision genetic maps and locate target QTL / genes through association or linkage analysis has greatly promoted the efficiency of mining salt-tolerant candidate genes (Qian, G., Wang, M., Wang, X., Liu, K., Li, Y.,Bu, Y. and Li, L. (2023) Integrated transcriptome and metabolome analysis of rice leaves response to high saline-alkali stress. International Journal Molecular Science 24, 4062.). By using genetic populations or germplasm resources such as near-isogenic lines and combining SNPs, salt stress phenotype identification, salt tolerance candidate QTLs were located, and some salt tolerance QTLs or candidate genes were screened by combining transcriptome analysis and bioinformatics analysis, such as SKC1、OsSOS1 Ren, Wang, and others have shown that overexpression of S KC1 (Wang,H., Zhang, M., Guo, R., Shi, D., Liu, B., Lin, X. and Yang, C. (2012) Effects of salt stress on ion balance and nitrogen metabolism of old and youngleaves in rice ( Oryza sativa L.). Bmc Plant Biology 12,194.) and OsSOS1 (Ren, ZH, Gao, JP, Li, LG, Cai, XL, Huang, W., Chao, DY, Zhu, MZ, Wang, ZY, Luan, S. and Lin, HX (2005) A rice quantitative trait locus for salttolerance encodes a sodium transporter. Nature Genetics37,1141-6.) can improve salt tolerance, and their mechanism of action is to regulate the balance of potassium and sodium ions in rice, reduce ion toxicity, and thus enhance rice salt tolerance. Based on the study of these candidate genes, a group of excellent salt-tolerant germplasm resources have been cultivated. These research results also show that the strategy of using gene editing technology to study the salt tolerance mechanism of rice is worth further exploration.

[0004] In existing research, rice salt tolerance has been widely studied, and many salt-tolerant germplasms have been identified. However, the agronomic traits of most salt-tolerant germplasms are poor, and the number of major salt-tolerant genes identified is relatively small. Therefore, systematic evaluation of the salt tolerance of rice germplasm resources, discovery of excellent new salt-tolerant genes from rice itself, and analysis of their salt-tolerant genetic mechanisms are important issues facing current rice germplasm resource research. This is of great significance for increasing rice yields, expanding the scope of rice cultivation, and ensuring grain production in saline-alkali rice-producing areas. Summary of the invention

[0005] The main purpose of the present invention is to Lhca4 The gene or its encoded protein is used to regulate plant resistance to abiotic stress and other aspects.

[0006] The present invention finds that the Lhca4 The transgenic rice obtained by overexpressing the gene in rice and the wild-type rice were treated with salt stress. Lhca4 The chlorophyll content and plant height of transgenic rice lines with the gene were significantly higher than those of wild type. Lhca4 The gene was knocked out using gene editing technology to obtain transgenic rice and wild-type rice, which were treated with salt stress. Lhca4 The chlorophyll content, plant height and root length of the transgenic rice lines with the gene were significantly lower than those of the wild-type rice, which ultimately determined that the rice Lhca4 The gene or its encoded protein has the function of regulating the resistance of plants to abiotic stress, thereby completing the present invention.

[0007] The present invention mainly provides Lhca4 Use of a gene or its encoded protein (Lhca4 protein) in regulating plant resistance to abiotic stress;

[0008] As a preferred specific embodiment of the present invention, the regulation of plant resistance to abiotic stress is to improve the resistance of plants to abiotic stress; wherein, the improvement of plant resistance to abiotic stress includes improving plant resistance to salt stress or improving plant resistance to drought stress; more preferably, the improvement of plant resistance to salt stress includes promoting plant growth under salt stress, for example: increasing the chlorophyll content of plants, increasing plant height or root length, etc.

[0009] As a reference implementation scheme, Lhca4 Overexpression of genes in plants can improve the resistance of plants to salt stress and promote the growth and development of plants under salt stress.

[0010] As a preferred embodiment, the present invention provides a method for improving plant resistance to salt stress, comprising: overexpressing in a plant Lhca4 Gene, Lhca4 The expression of the gene is increased or the function or activity of the Lhca4 protein is enhanced; for example, Lhca4 The gene is operably connected to an expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into a plant, so that Lhca4 The gene is overexpressed in plants, and the resulting transgenic plants have enhanced resistance to salt stress.

[0011] The base described in the present invention Lhca4 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) to (e) below:

[0012] (a) the polynucleotide sequence shown in SEQ ID No. 1;

[0013] (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2;

[0014] (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress;

[0015] (d) a polynucleotide sequence that is at least 75% or more identical to the polynucleotide sequence shown in any one of (a)-(c), and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress; preferably, a polynucleotide sequence that is at least 85% or more identical to the polynucleotide sequence shown in any one of (a)-(c), and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress; more preferably, a polynucleotide sequence that is at least 90% or more identical to the polynucleotide sequence shown in any one of (a)-(c), and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress; most preferably, a polynucleotide sequence that is at least 95% or more identical to the polynucleotide sequence shown in any one of (a)-(c), and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress;

[0016] (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), and the polynucleotide sequence still has the function or activity of regulating plant resistance to abiotic stress.

[0017] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0018] In addition, those skilled in the art can also optimize the nucleotide sequence shown in SEQ ID NO. 1 to enhance the expression efficiency in plants.

[0019] The present invention can also be used to obtain mutants by deleting one or more amino acid residue codons in the DNA sequence shown in SEQ ID No. 1, and / or performing missense mutations of one or more base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to the 5' end and / or 3' end of the amino acid sequence of the protein encoded by its gene.

[0020] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to Lhca4 The nucleotide sequence of the gene is mutated. Lhca4 Nucleotides with 75% or higher identity to the nucleotide sequence of a 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 the function of regulating plant physical dormancy.

[0021] 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.

[0022] The amino acid sequence of the Lhca4 protein described in the present invention is selected from the amino acid sequences shown in any one of the following (a)-(d):

[0023] (a) the amino acid sequence shown in SEQ ID NO.2;

[0024] (b) a protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant resistance to abiotic stress;

[0025] (c) a protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant resistance to abiotic stress;

[0026] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 1, wherein the protein still has the function or activity of regulating plant resistance to abiotic stress.

[0027] In order to facilitate the purification or detection of the Lhca4 protein, a tag can be connected to the amino terminus or and / or carboxyl terminus of the protein. As an example, the present invention provides some specific tags that can be connected to the amino terminus or carboxyl terminus of the protein. The specific amino acid sequences of these tags can be shown in Table 1, such as Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc, etc.

[0028] The Lhca4 protein of the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0029] Another aspect of the present invention is to further provide a Lhca4 An expression cassette, a recombinant vector or a recombinant host cell of a gene; preferably, the expression cassette, the recombinant vector or the recombinant host cell is a recombinant eukaryotic expression cassette, a recombinant eukaryotic expression vector or a recombinant plant cell; more preferably, the recombinant eukaryotic expression cassette or the recombinant eukaryotic expression vector is a recombinant plant expression cassette or a recombinant plant expression vector.

[0030] For reference, the present invention provides a Lhca4 Plant recombinant expression vector, including:

[0031] Will Lhca4 The gene is operably connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.1 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0032] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0033] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell to be transformed. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods may be used to transform Medicago truncatula. MtMET1 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0034] Another aspect of the present invention is to provide Lhca4 The use of a gene or a protein encoded by the gene in reducing the resistance of plants to abiotic stress comprises: Lhca4 Gene mutation Lhca4 The reduced expression of the gene or the defect in the normal function of the Lhca4 protein will cause the plant to have phenotypes such as growth stunting or delay under salt stress, such as decreased chlorophyll content in leaves, delayed plant height and root length development, etc.

[0035] The mutations include Lhca4 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Specifically, 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 includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes, but is not limited to, ZFN, TALE and / or CRISPR / Cas, etc.

[0036] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. Lhca4 Gene knockout mutation, such as constructing Lhca4 Gene knockout vectors or constructed using gene editing technology Lhca4 CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants LAP1 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0037] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, to target the DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0038] The interference Lhca4 The normal expression or normal function of the gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the LAP1 protein or its promoter or to cause defects in its normal function. RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short-chain double-stranded RNA (siRNA) or a long-chain double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0039] The present invention can be used to transform any plant species, including but not limited to monocotyledonous plants or dicotyledonous plants; more preferably, the plants include crops, vegetables or ornamental plants, fruit trees, etc., for example, they can be rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, kidney bean, peanut or sugarcane, etc., preferably rice.

[0040] Definitions of terms used in this invention

[0041] 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.

[0042] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0043] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as 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 via covalent peptide bonds.

[0044] The "stringent hybridization conditions" described herein refer to conditions of low ionic strength and high temperature as known in the art. Generally, under stringent conditions, the detectable degree of hybridization of the probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times above background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be lower than the thermal melting point (T) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. T m The temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T mStringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60° C. for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42° C.; or 5×SSC, 1% SDS, incubated at 65° C., washed in 0.2×SSC and washed in 0.1% SDS at 65° C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer.

[0045] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating 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.

[0046] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0047] The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. 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, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0048] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0049] 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

[0050] Figure 1 The aboveground parts of the salt-tolerant rice variety MH63 and the salt-sensitive rice variety 02428 after salt stress Lhca4 Changes in gene expression levels.

[0051] Figure 2 This is the map of the pYLCRISPR / Cas9Pubi-H vector.

[0052] Figure 3 for Lhca4 - KO Sequencing analysis of gene targets in transgenic rice.

[0053] Figure 4 for Lhca4 - KO Identification of salt tolerance phenotype in transgenic rice.

[0054] Figure 5 This is a map of the overexpression vector pMDC43.

[0055] Figure 6 for Lhca4 Gene in Lhca4 - OE Expression analysis in transgenic lines.

[0056] Figure 7 for Lhca4 - OE Identification of salt tolerance phenotype in transgenic rice. DETAILED DESCRIPTION

[0057] 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 proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0058] Test Example 1: Rice plants after salt stress Lhca4 Changes in gene expression

[0059] 1. Salt stress treatment of rice and total RNA extraction from rice plants

[0060] Seeds of the salt-tolerant rice variety MH63 and the salt-sensitive rice variety 02428 were sown in hydroponic boxes, and treatment groups and control groups were set up, with 3 biological replicates in each group, and cultured in an artificial climate chamber. The culture conditions were 14 hours of light / 10 hours of darkness, and the temperature was constant at 28°C. Distilled water (pH 5.5) was used for cultivation for 6 days, and then Yoshida nutrient solution was used to continue cultivation in the artificial climate chamber. The nutrient solution was changed every 6 days, and salt stress treatment was started at the rice three-leaf one-heart stage. At 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after salt stress treatment, the aboveground leaves were cut and quickly frozen in liquid nitrogen. Three biological replicates were taken for each sample. All samples were frozen in liquid nitrogen and stored at -80°C.

[0061] After extracting sample RNA using an RNA extraction kit (Tiangen Company), the RNA concentration was determined (Thermo NANODROP), and the integrity of the extracted RNA was detected by 1.0% agarose gel electrophoresis. RNA samples with better integrity were selected for reverse transcription and cDNA synthesis.

[0062] 2.qRT-PCR detection Lhca4 Gene expression

[0063] Obtained from the Rice Genome Annotation Project database (http: / / rice.uga.edu / index.shtml / ) Lhca4 Gene (MSU Locus: LOC_Os0g33820 ;RAP Locus: Os08g0435900 ) sequence, and designed its specific primers using Primer5, the sequence is as follows:

[0064] Lhca4-RT-F: 5′-CTTCCTCCTCTCTAAGCC-3′ (SEQ ID No. 3);

[0065] Lhca4-RT-R: 5′-AGGTAGACTTGAGCGACGC-3′ (SEQ ID No. 4).

[0066] Using cDNA as template, UBQ As the internal reference gene, PCR was used to detect the quality of cDNA of each sample and to normalize the cDNA of each sample. Take a 0.2ml micro-PCR tube, prepare 25 μL reaction system according to Table 1, mix gently, and centrifuge briefly.

[0067] Table 1 Fluorescence quantitative PCR reaction system (25 μL)

[0068]

[0069] The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0070] Rice UBQ Primer sequences:

[0071] UBQ-F: 5'- GCTCCGTGGCGGTATCAT -3' (SEQ ID No. 5);

[0072] UBQ-R: 5'-CGGCAGTTGACAGCCCTAG-3' (SEQ ID No. 6).

[0073] Real-time PCR was performed according to the instructions of the Tiangen kit (SYBR Green). The qRT-PCR analysis results showed that the melting curves were all single-peaked, the amplification products had good specificity, and the fluorescence curves could well reflect the amplification results. According to the amplification results, Lhca4 The gene is induced by salt stress, and Lhca4 The expression of the gene was significantly upregulated after 6 h of salt stress, and Lhca4 The gene expression level in MH63 was significantly higher than that in 02428 ( Figure 1 ).

[0074] Test Example 2 Lhca4 Construction of gene editing knockout vector and acquisition and identification of transgenic plants

[0075] 1. Lhca4 Gene editing knockout target primer design and CRISPR / Cas vector construction

[0076] According to the principle of gene editing, the target of gRNA is designed through online Blast homology analysis. The target is located on the exon of the gene and as close as possible to the front end of the protein or the important functional domain area to ensure the specificity of the target. The knockout vector is pYLCRISPR / Cas9Pubi-H. Figure 2 This is the map of the pYLCRISPR / Cas9Pubi-H vector; the process of constructing the CRISPR / Cas vector (numbered pYLCRISPR / Cas9Pubi-H) is as follows:

[0077] (1) Ligation reaction system

[0078] Table 2 Ligation reaction system

[0079]

[0080] (2) Reaction conditions

[0081] React at 25℃ for 1 h. (Can be left overnight at 4℃)

[0082] 2. Lhca4 Obtaining KO transgenic rice

[0083] Take 5 μL of the above reaction solution, add 20 μL of competent cells (DH5α), mix and place on ice for 30 min (do not shake); gently take out, heat shock at 42℃ for 35 s, and immediately place on ice for 2 min; add 100 μL LB, shake and culture at 37℃ for 1 h; take 60 μL of bacterial solution and spread on LB plate containing kanamycin, invert and culture at 37℃ overnight. Select single clones and shake bacteria, extract plasmids for sequencing.

[0084] The indica rice variety MH63 was transformed by Agrobacterium-mediated method. The mature seeds of MH63 were taken and mechanically shelled. The plump, smooth and sterile seeds were selected and sterilized and inoculated on the induction medium for induction culture. The rice callus with good appearance and good growth was selected as the recipient material. The pYLCRISPR / Cas9Pubi-H was transferred into the rice callus by Agrobacterium-mediated method. The callus was transformed with a culture solution containing 100 μM acetosyringone and Agrobacterium with an OD value of 0.3-0.5. The callus soaked in the transformation solution was placed on the co-culture medium for co-culture, and co-cultured at 28℃ in the dark for 50-55 hours. The callus with no obvious Agrobacterium on the surface was selected and transferred to the N6 antibacterial medium containing 2.0 mg / L 2,4-D and 500 mg / L cephalosporin, and cultured in the dark at 28℃ for 3-4 days. The callus was transferred to the screening medium for culture for 30 days, and subcultured every 10 days. Take fresh hygromycin-resistant callus, inoculate it into the pre-culture medium, culture it in the dark at 28℃ for 7 days, then place it in the light culture room (12 h light / 12 h dark) for another 7 days, then transfer it to the regeneration medium (250 mL tissue culture bottle) and continue to culture it in the light until regenerated plants grow.

[0085] The culture medium formula involved in this test example is shown in Table 3.

[0086] Table 3 Culture medium and its formula used for genetic transformation

[0087]

[0088] 3. Lhca4 Identification of -KO transgenic positive lines

[0089] To obtain T1 generation Lhca4 -KO positive transgenic lines, all transformant lines were PCR amplified and sequenced using the gene editing target information.

[0090] Gene editing target specific primers are:

[0091] Lhca4-F: 5′- TACACCCGCTTCGCTTCC -3′ (SEQ ID No. 7);

[0092] Lhca4-R: 5′-CGCCCAATCATCGGTTTC-3′ (SEQ ID No. 8).

[0093] The sequencing results showed that two target site sequence homozygous mutants were obtained in the transformant strains, namely KO-1 with a T inserted at the target site and KO-2 with a G replaced by a T at the target site ( Figure 3 ), indicating that Lhca4 The gene has been knocked out in the MH63 background.

[0094] 4.Lhca4 Identification of salt tolerance phenotype of -KO transgenic lines

[0095] The wild type MH63, two T4 Lhca4 -KO transgenic rice (KO-1 and KO-2) seeds were sown in hydroponic boxes, MH63 and knockout materials were planted in half plates, treatment groups and control groups were set up, and 3 biological replicates were set up in an artificial climate chamber. The culture conditions were 14 hours of light / 10 hours of darkness, and the temperature was constant at 28°C. Distilled water (pH 5.5) was used for 6 days, and then Yoshida nutrient solution was used to continue culturing in the artificial climate chamber. The nutrient solution was changed every 6 days. Salt stress treatment was started at the three-leaf and one-heart stage of rice. The salt solution was Yoshida nutrient solution with 140 mM NaCl. The nutrient solution was changed every 5 days. After 8 days of salt stress, chlorophyll, root length, and plant height were measured.

[0096] The phenotypic survey results showed that after 8 days of salt treatment, Lhca4 The chlorophyll content, plant height, and root length of the -KO transgenic line were significantly lower than those of the wild type, with an average decrease of 44.8%, 21.3%, and 29.6%, respectively ( Figure 4 ).

[0097] Test Example 3 Lhca4 Construction of gene overexpression vector and acquisition and identification of transgenic plants

[0098] 1.Lhca4 Construction of gene overexpression vector

[0099] The scheme for constructing the overexpression vector is to first add BsaI restriction sites at both ends of the synthetic gene sequence, then connect the vector used and the synthetic overexpression gene sequence with T4 ligase after BsaI digestion to construct the overexpression vector pMDC43, and finally transform the competent Escherichia coli. Figure 5This is a map of the overexpression vector pMDC43.

[0100] The specific construction process is as follows:

[0101] (1) Enzyme digestion

[0102] The enzyme digestion reaction system is shown in Table 4.

[0103] Table 4 Enzyme digestion reaction system

[0104]

[0105] Reaction conditions: 37 °C, 30 min.

[0106] (2) Connection

[0107] Add the following ingredients directly to the 5 uL after enzyme digestion for ligation:

[0108] The connection system is shown in Table 5.

[0109] Table 5 Connection system

[0110]

[0111] The reaction conditions are 25℃ and ligation for 30 min. If the target fragment contains a BsaI restriction site, ligation should be performed at 25℃ for 1-2h and then at 16℃ overnight.

[0112] (3) Transformation of Escherichia coli

[0113] Take 5 μL of the above reaction solution, add 20 μL of competent cells (DH5α), mix and place on ice for 30 min (do not shake); gently take out, heat shock at 42℃ for 35 s, and immediately place on ice for 2 min; add 100 μL LB, shake and culture at 37℃ for 1 h; take 60 μL of bacterial solution and spread it on LB plate containing kanamycin, and culture it upside down at 37℃ overnight.

[0114] 2.Lhca4 Gene overexpression transgenic plants

[0115] The overexpression vector pMDC43 obtained above was transformed into Agrobacterium EHA105 by freeze-thaw method. The indica rice variety MH63 was transformed by Agrobacterium-mediated method to construct Lhca4 Overexpression of transgenic rice (abbreviated as Lhca4 - OE ).

[0116] The specific operation of rice genetic transformation refers to step 2 of Experimental Example 2.

[0117] 3.Lhca4 Molecular identification of gene overexpression transgenic plants

[0118] Using marker genes hyg For the obtained Lhca4 - OE The transgenic lines were tested by PCR, 1.0% agarose gel electrophoresis, and observed under UV light.

[0119] The marker gene specific primers are:

[0120] Hyg-F: 5′-TTGGCGACCTCGTATTGGGAA-3′ (SEQ ID No. 9);

[0121] Hyg-R: 5′-CAAAGATCGTTATGTTTATCGGCACT-3′ (SEQ ID No. 10).

[0122] Select 2 Lhca4 - OE Transgenic lines (named OE-1 and OE-2 ), qRT-PCR was used to analyze the Lhca4 The changes in expression levels were used to identify the overexpression efficiency of the transgenic lines.

[0123] Test results show OE-1 and OE-2 middle Lhca4 The expression levels of MH63 were significantly upregulated compared with wild-type MH63 ( Figure 6 ).

[0124] 4.Lhca4 Identification of salt tolerance phenotypes of gene overexpression transgenic lines

[0125] The wild-type MH63, Lhca4 - OE Transgenic rice lines OE-1 and OE-2 The seeds of MH63 and overexpression materials were sown in hydroponic boxes, and MH63 and overexpression materials were planted in half plates. Treatment groups and control groups were set up, with 3 biological replicates in each group. They were cultured in an artificial climate chamber with 14 hours of light / 10 hours of darkness and a constant temperature of 28°C. Distilled water (pH 5.5) was used for 6 days, and then Yoshida nutrient solution was used to continue the culture in the artificial climate chamber. The nutrient solution was changed every 6 days. Salt stress treatment was started at the rice three-leaf and one-heart stage. The salt solution was Yoshida nutrient solution with 140 mM NaCl. The nutrient solution was changed every 5 days. After 14 days of salt stress, chlorophyll, root length, and plant height were measured.

[0126] The phenotypic survey results showed that after 14 days of salt treatment, Lhca4The chlorophyll content and plant height of the -OE transgenic strain were significantly higher than those of the wild type, increasing by 57.6% and 13.8% respectively on average compared with the wild type ( Figure 7 ).

Claims

1. Lhca4 gene, Lhca4 protein, or Lhca4 Use of gene expression cassette, recombinant plant expression vector or recombinant host cell in regulating rice resistance to salt stress; Lhca4 The nucleotide sequence of the CDS of the gene is selected from the nucleotide sequence described in (a) or (b) below: (a) the nucleotide sequence shown in SEQ ID No. 1; (b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; The amino acid sequence of the Lhca4 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The regulation of rice resistance to salt stress is to improve the resistance of rice to salt stress.

3. The use according to claim 2, characterized in that: The method of improving the resistance of rice to salt stress includes: improving the chlorophyll content of rice, increasing the plant height or root length of rice.

4. The use according to claim 1 or 2, characterized in that: include: Will Lhca4 Gene overexpression in rice improves Lhca4 The expression amount or expression level of the gene may enhance the function or activity of the Lhca4 protein, thereby improving the resistance of rice to salt stress.

5. The use according to claim 1, characterized in that: The regulation of rice resistance to salt stress is to reduce the resistance of rice to salt stress.

6. The use according to claim 5, characterized in that include: The rice Lhca4 Gene mutation Lhca4 Reduced gene expression may cause defects in the normal function of Lhca4 protein.

7. A method for cultivating rice varieties resistant to salt stress, characterized in that: include: (1) Constructing a method comprising the method according to claim 1 Lhca4 Recombinant plant expression vector of gene; (2) transforming the constructed recombinant plant expression vector into recipient rice tissues or rice cells; and (3) cultivating and screening transgenic rice with improved resistance to salt stress.

Images