Application of overexpression of rice P3A gene in improving plant resistance to abiotic stress

Abstract

This invention discloses the use of the rice P3A gene and its encoded protein in improving plant resistance to abiotic stress and yield. The invention also discloses a method for improving plant resistance to abiotic stress, comprising: overexpressing the rice P3A gene in plants to obtain transgenic plants. The transgenic plants obtained show significantly enhanced resistance to low-temperature stress and a significant increase in yield. This invention has promising applications in improving and enhancing rice stress resistance and accelerating the process of stress-resistant molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of overexpression of the rice P3A gene in improving plant resistance to abiotic stress. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, providing staple food for more than half of the world's population. Originating in tropical and subtropical regions, rice is a warm-loving crop and is highly sensitive to low temperatures. Chill damage not only limits the geographical distribution of rice but also severely affects its growth and development. As rice cultivation expands from tropical and subtropical areas to higher altitudes, the probability of rice suffering chill damage at various stages of growth increases. Chill damage during the seedling stage can cause poor seedling development, yellowing or wilting of leaves, and reduced tillering, ultimately leading to a decline in rice yield and quality. Therefore, identifying key genes involved in regulating cold tolerance in rice seedlings and elucidating their mechanisms of action is of great significance for the prevention and control of chill damage in rice and the breeding of cold-resistant rice varieties.

[0003] The rice-specific P3 subfamily P3A (Ribosomal p3 protein) encodes an acidic ribosomal protein. Ribosomes, primarily composed of ribosomal RNA (rRNA) and ribosomal protein (RP), are the site of protein translation in organisms. Overexpression in Arabidopsis thaliana...

[0004] P3

[0005] Genes can significantly enhance cold tolerance in Arabidopsis and sweet potatoes. Studies have found that low temperatures not only affect the expression of ribosome-related proteins but also ribosome-mediated protein translation. Early research in tomatoes found that low temperatures can inhibit protein translation by affecting the function of ribosomes in plastids. More recent studies in Arabidopsis have shown that low temperatures can inhibit protein translation for a short period. These studies indicate a close relationship between ribosomes and plant cold tolerance. However, how ribosomes regulate plant cold tolerance remains unclear. Summary of the Invention

[0006] The main objective of this invention is to provide the use of overexpressing the rice P3A gene in improving plant resistance to abiotic stresses. Overexpression of P3A can also significantly increase rice seed setting rate and tiller number.

[0007] To achieve the above-mentioned objective, the first aspect of the present invention provides an isolated nucleic acid that encodes a rice P3A protein, the P3A protein comprising the amino acid sequence shown in SEQ ID NO: 2.

[0008] As a preferred embodiment of the present invention, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 1 or its degenerate sequence.

[0009] As a preferred embodiment of the present invention, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0010] A second aspect of the present invention provides a vector comprising the nucleic acid described in the first aspect of the present invention.

[0011] The recombinant plant expression vector is obtained by linking the coding gene of the rice P3A protein with an expression regulatory element. This vector can be composed of the rice P3A coding region. The promoter can be a constitutive promoter, an inducible promoter, an enhancing promoter, or a tissue- or organ-specific promoter. A suitable terminator sequence can be taken from the Ti-plasmid of *Agrobacterium tumefaciens*, such as the terminator regions of octopine synthase and carmine synthase. The recombinant plant expression vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance, hygromycin, and herbicide genes. Furthermore, the marker genes may also include phenotypic markers, such as green fluorescent protein.

[0012] The third aspect of this invention provides the application of rice P3A protein or its encoded nucleic acid in improving plant resistance to abiotic stresses and / or increasing plant yield.

[0013] As a preferred embodiment of the present invention, the application includes overexpressing the nucleic acid encoding the rice P3A protein in plants to obtain transgenic plants; preferably, the amino acid sequence of the P3A protein is as shown in SEQ ID NO: 2; more preferably, the nucleic acid encoding the P3A protein is as described in the first aspect of the present invention.

[0014] As a preferred embodiment of the present invention, the application specifically includes: (1) constructing a recombinant plant expression vector containing nucleic acid encoded by rice P3A protein; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) cultivating and screening to obtain transgenic plants with improved resistance to abiotic stress; preferably, the expression vector is as described in the second aspect of the present invention.

[0015] As a preferred embodiment of the present invention, the abiotic stress includes low temperature stress.

[0016] In a preferred embodiment of the present invention, the abiotic stress includes low-temperature stress. Preferably, the low temperature is below 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, or 0°C.

[0017] As a preferred embodiment of the present invention, the yield indicators include the seed setting rate, the number of effective tillers, and the yield per plant.

[0018] As a preferred embodiment of the present invention, the plants include, but are not limited to, monocotyledons or dicotyledons; more preferably, the plants include crops, vegetables or ornamental plants, fruit trees, etc., such as rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, sugarcane or Arabidopsis thaliana, etc., preferably rice.

[0019] Compared with the prior art, the present invention has the following technical advantages:

[0020] This invention utilizes the cloning of Arabidopsis thaliana RPP3 Homologous genes in rice P3A Furthermore, phenotypic changes in rice plants overexpressing and knocked out under extreme low temperatures of 6℃ and relative low temperatures of 20℃ were investigated to perform gene cloning and functional analysis. The relationship between candidate genes and abiotic stress responses during the rice seedling and booting stages was analyzed. The results showed that overexpression in rice... P3A The gene can significantly improve the ability of rice to resist low temperature stress, and overexpression of it can also improve the resistance of rice to low temperature stress. P3A The invention increases the seed setting rate and yield of rice. It has significant theoretical and practical implications for improving and enhancing the stress resistance of rice, cultivating high-yielding and stress-resistant varieties, and accelerating the process of stress-resistant molecular breeding. Attached Figure Description

[0021] Figure 1 For rice P3A Knockout status of each mutant line. Figure 1 A: Mutation type 1 ( p3a-1 Mutation type 2 involves the deletion of 25 bp (from 336 bp to 360 bp) in the coding region of P3A, causing frameshift during translation and ultimately leading to premature termination of translation. p3a-2 The coding region of P3A has 135 bp (from 201 bp to 335 bp) deleted, resulting in the loss of 45 amino acids during the translation of the P3A protein; Figure 1 B, using RT-PCR to p3a-1 and p3a-2 The expression level of mutant mRNA was measured. P3A in... p3a-1 The mutant produced new transcripts, and their expression levels were similar to those in the wild type; p3a-2 Transcriptional instability occurred in the mutant, and expression was undetectable. (Explanation) p3a-1 and p3a-2 These are two different types of loss-of-function mutants.

[0022] Figure 2 PCR-positive identification of hygromycin in transgenic plants of overexpression and knockout materials. Lanes 1-6 were used for... p3a Mutant, lanes 7-12 are P3A Overexpression was indicated by lane 13 as a negative control and lane 14 as a negative control with added double-distilled water.

[0023] Figure 3 For rice P3A Expression levels of the gene overexpression material in various lines. The expression levels of each overexpression line were 15-20 times higher than those of the wild type.

[0024] Figure 4 for P3A Cold tolerance phenotypes of overexpression and CRISPR knockout transgenic plants and wild-type plants at 6℃ during the seedling stage. A shows the phenotypes of the mutants after 3 days of treatment at 6℃ and 7 days of recovery. B shows the statistical results of the survival rate of the mutants before and after treatment at 6℃. C shows the overexpression... P3A Phenotypic images after 3 days of treatment at 6 ℃ and 7 days of recovery. D indicates overexpression. P3A Survival rate statistics before and after treatment at 6 ℃. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0026] 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 pertains.

[0027] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, 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 a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0028] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terminology applies 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 terminology covers amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0029] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, an engineered selection marker for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0030] In this invention, the term "transformation" refers to the genetic transformation of polynucleotides or polypeptides into plants by introducing the gene encoding the rice P3A protein into plant cells. Methods for introducing such polynucleotides or polypeptides into plants are well known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation. "Stable transformation" refers to the integration of the introduced polynucleotide construct into the genome of the plant cell and its inheritance through its progeny; "transient transformation" refers to the introduction of a polynucleotide into a plant but its temporary expression or presence in the plant.

[0031] In this invention, the term "effective tiller number" refers to the tillers in rice that are ultimately able to produce grains. "Grain setting rate" refers to the proportion of full grains to the total number of grains.

[0032] Example 1 P3A Construction of gene overexpression vectors

[0033] Cloning the homolog of Arabidopsis thaliana RPP3 in Japanese rice P3A Sequencing confirmed its sequence as SEQ ID NO: 3.

[0034] Using two restriction endonucleases KpnI and SacI The pCAMBIA1300s vector (addgene, #KM1033157) was digested with enzymes to linearize it and then recovered.

[0035] According to Japan's Haruyoku Gene P3A Primers were designed based on the full-length sequence (SEQ ID NO: 3). Using Nipponbare cDNA as a template, the full-length coding region of the P3A protein (SEQ ID NO: 1) was amplified. The stop codon (TGA) was removed, and a linearized adapter of the pCAMBIA1300s vector was added to the 5′ and 3′ ends. The amplification primers are shown in Table 1.

[0036] Table 1 P3A Amplification primers

[0037]

[0038] The lowercase part is the connector sequence.

[0039] The target DNA fragment (360 bp) was amplified and recovered by PCR. Homologous recombination of the target fragment with the linearized vector was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, Code no: C115-01). Positive clone plasmids were verified by PCR and sequencing. Sequencing results showed that the DNA fragment shown in SEQ ID NO.1 was inserted between the two restriction sites of the p1300S vector. P3A The gene fragment was used to obtain a recombinant vector named p1300S- P3A (The overexpression strain is named) P3A -OE).

[0040] Example 2 P3A Construction of CRISPR gene knockout vector

[0041] according to P3A The target sites 1 and 2 for gene cDNA sequence knockout were designed (Table 2), and the website used was: http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html;

[0042] Table 2 Knockout Target Sites

[0043]

[0044] Primers P3A target1-BsF, P3A target1-F0, P3A target2-R0, and P3A target2-BsR (Table 3) were designed based on the target sites. Four-primer PCR amplification was performed using a 100-fold diluted pCBC-MT1T2 plasmid as a template. The PCR products were purified and recovered, and the final vector was constructed using an enzyme digestion-ligation system. The vector was named CRISPR-P3A.

[0045] Table 3 Gene knockout primers

[0046]

[0047] Figure 1 A shows rice P3A Knockout details for each mutant lineage. Mutation type 1 ( p3a-1 Mutation type 2 involves the deletion of 25 bp (from 336 bp to 360 bp) in the coding region of P3A, causing frameshift during translation and ultimately leading to premature termination of translation. p3a-2 The coding region of P3A has 135 bp (from 201 bp to 335 bp) missing, resulting in the loss of 45 amino acids during the translation of the P3A protein.

[0048] The expression vector p1300S-P3A and the knockout vector CRISPR-P3A were transformed into Agrobacterium EHA105 competent cells using the freeze-thaw method (competent cells were purchased from Shanghai Sangon Biotech Co., Ltd.). The specific experimental method was in accordance with the experimental guidelines for molecular cloning.

[0049] 1) Sterilization: Remove the shells from healthy, plump Nipponbare seeds, soak them in 70% ethanol for 1-2 minutes, add 50% bleach and place on a shaker (200 rpm) for about 1-1.5 hours. Rinse with sterile water 4-6 times, place the seeds on sterile filter paper to absorb excess moisture, and then evenly place them in NBD medium and incubate in the dark at 28°C. All the above steps are performed in a laminar flow hood.

[0050] 2) Subculture: After about 10-15 days of dark culture, the rice seed buds are separated and transferred to NBD subculture medium for continued dark culture at 28°C. After 10 days, the seeds are separated from the callus and the callus is transferred to a new NBD subculture medium. After about 4-5 days of dark culture at 28°C, Agrobacterium transformation can be carried out.

[0051] 3) During this period, Agrobacterium containing plasmids (p1300S-P3A, CRISPR-P3A) was streaked on LB medium containing kanamycin and rifampin. After 2 days, single clones were picked, streaked again, and cultured for 1 day.

[0052] 4) Collect the bacterial cells from the culture medium and vortex them in NBC1 medium containing acetylsuccinone (AS), adjusting the OD to approximately 0.1-0.2.

[0053] 5) Transformation: Select healthy callus tissue into a sterile Erlenmeyer flask, add the above-prepared suspension, gently shake at room temperature for about 10 minutes, discard the bacterial solution, place the callus tissue on sterile filter paper, absorb the excess bacterial solution, and place it in a laminar flow hood to blow air until the callus tissue turns slightly white. Then transfer the callus tissue to NBC2 medium with a layer of sterile filter paper, and co-culture at 22 ℃ in the dark for 2 days.

[0054] 6) Screening: Transfer the co-cultured callus to NBS1 medium containing the corresponding antibiotic, and culture in the dark at 28 ℃ for 10-12 days. Then transfer it to NBS2 medium and continue to culture in the dark at 28 ℃ for 10-12 days.

[0055] 7) Differentiation: After transferring the callus to NBR1 medium, culture it in the dark at 28 ℃ for 6 days, then transfer it to an artificial climate incubator with 15 h light / 9 h darkness and culture it at 28 ℃ for 15-20 days. During this period, callus tissue with green spots is transferred to NBR2 medium and cultured until it differentiates into seedlings.

[0056] 8) Cut the roots and leaves of the transgenic seedlings that are about 5 cm tall, transfer them to the rooting medium, and culture them at 28 ℃ in an artificial climate incubator with 12 h light / 12 h darkness.

[0057] 9) Hardening off seedlings: Once the root system of the transgenic seedlings is sufficiently developed, open the culture bottle for about 2 days, wash off the culture medium, place the seedlings in water for 1 week, and then transfer them to the soil for planting.

[0058] The culture medium formulations used in the above genetic transformation process are shown in Table 4; the preparation of hormone and antibiotic stock solutions in the culture medium is shown in Table 5.

[0059] Table 4 Genetic Transformation Culture Media

[0060]

[0061] Table 5. Preparation of Hormone and Antibiotic Storage Solutions

[0062]

[0063] The T0 generation transgenic seedlings were verified by PCR and propagated. T1 generation transgenic seeds were harvested, and the corresponding homozygous mutant materials were obtained through positive verification and sequencing. The obtained homozygous mutant materials were propagated again, and the offspring were screened with hygromycin (50 mg / L) to obtain the corresponding Cas9-free homozygous mutant materials.

[0064] Table 6 shows the results of PCR positive identification of overexpression and knockout transgenic plants using primers for hygromycin.

[0065] Table 6 Hygromycin Primers

[0066]

[0067] Example 5: Molecular identification and stress resistance identification of transgenic plants

[0068] (1) Select T2 generation P3A Transgenic overexpression, T3 generation P3A Seeds of knockout mutants and wild-type Nipponbare.

[0069] (2) Rice cultivation in soil: After the newly harvested seeds break dormancy, soak them in a 28 ℃ incubator for 3 days until they germinate, and then sow them. Select seeds with uniform germination and sow them evenly in a mixture of nutrient soil and vermiculite in a ratio of 3:1. Cover the surface with a layer of vermiculite and then let them grow normally in a 28 ℃ incubator. Water them once every 2-3 days during this period.

[0070] (3) Rice hydroponics: Select seeds with consistent germination and sow them in a 96-well PCR plate with the bottom removed. Place the plate in a 28 ℃ incubator and grow. Change the water every 2-3 days. Add an appropriate amount of nutrient solution when the third leaf just emerges. Change the water to clean water after the third leaf has fully unfolded.

[0071] (4) After the disinfected rice seeds germinate at room temperature, they are sown. Each experimental group has at least 3 replicates. After two weeks of cultivation under 28 ℃ light conditions, the seeds are treated at 6 ℃ for 2-4 days (depending on the actual situation), and then transferred to 28 ℃ to recover growth for 1 week.

[0072] (5) Regarding survival rate, the criterion in this study was whether new leaves had grown. If new leaves were present, the plant was considered alive; otherwise, it was considered dead. According to... Figure 4 It is evident that overexpression of rice in rice P3A Genes can significantly improve rice's ability to resist low-temperature stress, by increasing the amount of rice... P3A Mutations or knockouts of genes can significantly reduce the ability of rice to resist low-temperature stress. Therefore, rice P3A protein, its encoding gene, and recombinant vectors can be used to enhance the crop's resistance to abiotic stresses.

[0073] (6) The expression level of transgenic rice at the RNA level was detected by quantitative real-time PCR (AceQ qPCR SYBR Green Master Mix (vazyme)) and sequencing (primers are shown in Table 7).

[0074] Figure 1 B is P3A gene knockout Expression levels in the strain. (From...) Figure 1 As can be seen from B, P3A is... p3a -1 The mutant produced new transcripts, and their expression levels were similar to those in the wild type; p3a-2 Transcriptional instability occurred in the mutant, and expression was undetectable. (Explanation) p3a -1 and p3a-2 These are two different types of loss-of-function mutants.

[0075] Figure 3 for P3A Expression levels of the gene overexpression material in various lines. From Figure 3 Compared to wild-type materials, P3A In gene overexpression materials P3A Gene expression levels were increased to varying degrees.

[0076] The following overexpression and mutant line primers are used for subsequent identification of homozygosity in materials:

[0077] Table 7 Primers for quantitative real-time PCR detection of RNA expression levels in transgenic rice

[0078]

[0079] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. Use of a rice P3A protein or a nucleic acid encoding the same for increasing the resistance of rice to low temperature stress, characterized in that, The application includes overexpressing the nucleic acid encoding the rice P3A protein in rice to obtain transgenic rice; The amino acid sequence of the P3A protein is shown in SEQ ID NO: 2, and the sequence encoding the nucleic acid is shown in SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The applications include, (1) Construct a recombinant expression vector containing nucleic acid encoded by rice P3A protein; (2) Transform the constructed recombinant plant expression vector into rice tissues or rice cells; (3) Cultivate and screen transgenic rice with improved resistance to low temperature stress.

3. Use according to claim 2, characterized in that, The low temperature mentioned refers to the seedling stage temperature. Temperature below 10℃.

4. The application according to claim 3, characterized in that, The low temperature mentioned refers to a temperature below 6℃ during the seedling stage.

5. The application according to claim 2, characterized in that, The low temperature mentioned refers to a temperature of 20℃ during the booting stage.

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