Use of overexpression of rice snrk1a gene in improving plant resistance to abiotic stress and yield

Abstract

The present application belongs to the field of agricultural biotechnology, and discloses the use of overexpression of rice SnRK1 alpha gene in improving plant resistance to abiotic stress and yield. The present application also discloses the use of rice SnRK1 alpha protein or its coding nucleic acid in improving plant resistance to abiotic stress and / or improving plant yield, comprising: constructing a recombinant plant expression vector containing the coding nucleic acid of rice SnRK1 alpha protein; transforming the constructed recombinant plant expression vector into plant tissue or plant cells; cultivating and screening the transgenic plants with improved resistance to abiotic stress or improved yield. The present application has very important theoretical and practical significance for improving and enhancing the stress resistance of rice, cultivating high-yield and stress-tolerant varieties, and accelerating the process of stress-resistant molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the use of overexpression of the rice SnRK1α gene in improving plant resistance to abiotic stress and yield. Background Technology

[0002] Rice, as my country's most important food crop, plays a crucial role in ensuring national food security and economic development through stable yield growth. Originating in tropical and subtropical regions, rice is highly sensitive to cold climates; its growth is severely affected when temperatures drop below 15℃. In my country's double-cropping rice areas in South China and the middle and lower reaches of the Yangtze River, as well as single-cropping rice areas in Northeast China, rice often encounters temperatures below 15℃ during its growth. In recent years, due to the large-scale promotion of direct-seeded rice and the gradual expansion of rice cultivation areas to higher altitudes and latitudes, rice has been increasingly affected by low-temperature disasters, becoming a major obstacle to rice production in my country. Breeding and utilizing cold-resistant rice varieties is the most practical and economical method to combat low-temperature disasters. Therefore, identifying and studying key genes regulating rice's cold tolerance is of great significance for preventing and controlling low-temperature disasters in rice.

[0003] Cold resistance and yield in rice are complex traits controlled by multiple genes. Currently, we have identified only a small number of genes related to both cold resistance and yield in rice, and genes that simultaneously enhance both are extremely rare. Therefore, utilizing modern molecular breeding techniques has become a quick and effective method for improving cold resistance and increasing yield in rice.

[0004] Sucrose non-fermenting related kinase 1 (SnRK1) is an important signaling molecule that plays a crucial role in maintaining energy balance, regulating carbon metabolism, and influencing growth and development in plant cells. SnRK1 typically exists as a heterotrimer, with its α-subunit possessing conserved kinase domains and other core sequences. Studies have shown that SnRK1 kinase can sense intracellular energy states, particularly at low energy levels, and regulate metabolic pathways by phosphorylating substrates, ensuring that plants can adapt to different environmental conditions.

[0005] However, to date, there have been no reports of SnRK1 protein or its encoding gene improving plant cold stress tolerance. Summary of the Invention

[0006] The main objective of this invention is to provide the use of overexpressing the rice SnRK1 gene in improving plant resistance to abiotic stresses. At the same time, overexpressing rice SnRK1 can also significantly increase the seed setting rate and tiller number of rice.

[0007] To achieve the above-mentioned objective, the first aspect of the present invention provides an isolated nucleic acid that encodes a rice SnRK1 protein, the SnRK1 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] Using the above technical solution, the present invention provides a novel rice stress-resistance related SnRK1α gene, which is a DNA fragment of a complete coding region isolated and cloned from rice. Analysis of the protein sequence encoded by this gene shows that it has a plant-specific, highly conserved Ser / Thr kinase domain, which can respond to abiotic stress, preferably low temperature stress, by phosphorylating downstream substrates.

[0011] A second aspect of the present invention provides a vector comprising the nucleic acid described in the first aspect of the present invention.

[0012] Using the above technical solution, a recombinant plant expression vector is obtained by linking the coding gene of the rice SnRK1 protein with an expression regulatory element. This recombinant plant expression vector can be composed of the rice SnRK1 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 also include phenotypic markers, such as green fluorescent protein. Preferably, the construction of the recombinant plant expression vector includes inserting the coding gene of the rice SnRK1α protein before the GFP of the plant expression vector p1300s.

[0013] The third aspect of this invention provides the application of rice SnRK1 protein or its encoded nucleic acid in improving plant resistance to abiotic stresses and / or increasing plant yield.

[0014] As a preferred embodiment of the present invention, the application includes overexpressing the nucleic acid encoding the rice SnRK1 protein in plants to obtain transgenic plants; preferably, the amino acid sequence of the SnRK1 protein is as shown in SEQ ID NO: 2; more preferably, the nucleic acid encoding the SnRK1 protein is as described in the first aspect of the present invention.

[0015] As a preferred embodiment of the present invention, the application specifically includes: (1) constructing a recombinant plant expression vector containing rice SnRK1 protein-encoded nucleic acid; (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 and / or increased yield; preferably, the expression vector is as described in the second aspect of the present invention.

[0016] As a preferred embodiment of the present invention, the abiotic stress includes low temperature stress.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Compared with the prior art, the present invention has the following technical advantages:

[0021] This invention, through protein-protein interaction and in vivo and in vitro phosphorylation experiments, discovered that SnRK1α can interact with and phosphorylate the ABA core component ABI5 (ABA insensitive 5). Furthermore, this invention investigated phenotypic changes in rice plants overexpressed and knocked out under extreme low temperatures of 6°C and relative low temperatures of 20°C, performing gene cloning and functional analysis. The relationship between candidate genes and abiotic stress responses during the rice seedling and booting stages was analyzed. Results showed that overexpression of the SnRK1α gene in rice significantly improved the rice's ability to withstand low-temperature stress, and overexpressed SnRK1α plants exhibited increased seed setting rate and yield. This invention has significant theoretical and practical implications for improving and enhancing rice stress resistance, cultivating high-yielding and stress-tolerant varieties, and accelerating the process of stress-resistant molecular breeding. Attached Figure Description

[0022] Figure 1 This study compares different mutations in the rice SnRK1α gene with the wild type.

[0023] Figure 2PCR positive identification of SnRK1α overexpression and knockout transgenic plants was performed. Lanes 1-3 were used for overexpression material identification, lanes 4-7 were used for knockout mutant primer identification, lane 8 was used for negative control, and lane 9 was used for negative control with added double-distilled water.

[0024] Figure 3 The expression levels of the SnRK1α gene overexpression material are shown in various lines. The expression levels of each overexpression line are 4-6 times higher than those of the wild type.

[0025] Figure 4 This study illustrates the cold tolerance phenotypes of SnRK1α-overexpressing, CRISPR knockout transgenic plants and wild-type plants at 6°C during the seedling stage. A shows the phenotypes of the snrk1α-1 and snrk1α-2 mutants after 2 days of treatment at 6°C and 7 days of recovery. B shows the survival rate statistics of the snrk1α-1 and snrk1α-2 mutants before and after 6°C treatment. C shows the phenotypes of the snrk1α-3 and snrk1α-4 mutants after 2 days of treatment at 6°C and 7 days of recovery. D shows the survival rate statistics of the snrk1α-3 and snrk1α-4 mutants before and after 6°C treatment. E shows the phenotypes of SnRK1α-overexpressing plants after 3 days of treatment at 6°C and 7 days of recovery. F shows the survival rate statistics of SnRK1α-overexpressing plants before and after 6°C treatment.

[0026] Figure 5 This study statistically analyzed the agronomic traits of SnRK1α-overexpressing, CRISPR knockout transgenic plants and wild-type plants. AD represents the statistical results of plant height (A), seed setting rate (B), number of effective tillers (C), and yield per plant (D) after 20℃ chilling injury. Detailed Implementation

[0027] 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.

[0028] Definitions of terms involved in this invention

[0029] 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.

[0030] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either 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 refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, specific nucleic acid sequences implicitly encompass variants of their 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.

[0031] 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.

[0032] 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.

[0033] The term "off-target" refers to the phenomenon where there are numerous base segments in the genome sequence that are very close to a specific nucleotide sequence. During gene editing, these segments, which are very close to the target of the guide RNA, may also bind to and be knocked out. Off-target effects can be ruled out by verifying whether the phenotypes of mutants produced by different targets are consistent.

[0034] In this invention, the term "transformation" refers to the genetic transformation of polynucleotides or polypeptides into plants by introducing the gene encoding the rice SnRK1α 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.

[0035] 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.

[0036] Example 1: Construction of SnRK1α gene overexpression vector

[0037] Primers were designed based on the full-length homologous sequence from Nipponbare, and the full-length coding region of the SnRK1α protein was amplified using Nipponbare cDNA as a template. Sequencing confirmed its sequence as SEQ ID NO: 3.

[0038] The pCAMBIA1300s vector (addgene, #KM1033157) was linearized and recovered by digesting it with two restriction endonucleases, KpnI and SacI.

[0039] Primers were designed based on the full-length SnRK1α sequence of Nipponbare (SEQ ID NO: 3). Using Nipponbare's cDNA as a template, the full-length coding region of the SnRK1α 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.

[0040] Table 1 SnRK1α amplification primers (5'-3')

[0041]

[0042] The lowercase part is the connector sequence.

[0043] The target DNA fragment (1530 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 SnRK1α gene fragment shown in SEQ ID NO.1 was inserted between the two restriction sites of the p1300S vector, and the recombinant vector was named p1300S-SnRK1α.

[0044] Example 2: Construction of SnRK1α gene CRISPR knockout vector

[0045] Knockout target sites 1-1, 1-2, 2-1, and 2-2 were designed based on the SnRK1α gene cDNA sequence (Table 2). The website used was: http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html.

[0046] Table 2 Knockout target sites (5'-3')

[0047] Target site 1-2 GAGCGTTGGGTATCCTCAG(SEQ ID NO:7) Target site 2-1 CGTCGCCAAATGAGAAATA(SEQ ID NO:8) Target site 2-1 TAAATTATATGCTGGACCC(SEQ ID NO:9)

[0048] Primers SnRK1αtarget1-1-BsF, SnRK1αtarget1-1-F0, SnRK1αtarget1-2-R0, SnRK1αtarget1-2-BsR, SnRK1αtarget2-1-BsF, SnRK1αtarget2-1-F0, SnRK1αtarget2-2-R0, and SnRK1αtarget2-2-BsR were designed based on the target site. Four-primer PCR amplification was performed using a 100-fold diluted pCBC-MT1T2 plasmid as a template. The PCR product was purified and recovered, and the final vector was constructed using an enzyme digestion-ligation system. The vector was named CRISPR-SnRK1α.

[0049] Table 3 Gene knockout primers (5'-3')

[0050]

[0051] Figure 1Mutation type 1 (snrk1α-1) involves a 5bp deletion in the Target 1 sequence at exon 7, leading to a frameshift mutation at amino acid 300, prematurely terminating translation at amino acid 303. Mutation type 2 (snrk1α-2) involves a 1bp deletion in the Target 2 sequence at exon 10, leading to a frameshift mutation at amino acid 436, prematurely terminating translation at amino acid 440. Mutation type 3 (snrk1α-3) involves a 676bp deletion between the Target 1 sequence at exon 2 and the Target 2 sequence at exon 5, leading to a frameshift mutation at amino acid 48, prematurely terminating translation at amino acid 67. Mutation type 4 (snrk1α-4) involves a 1bp deletion in the Target 1 sequence at exon 2, leading to a frameshift mutation at amino acid 54, prematurely terminating translation at amino acid 69.

[0052] Example 3 Agrobacterium transformation

[0053] The expression vector p1300S-SnRK1α and the knockout vector CRISPR-SnRK1α 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.

[0054] Example 4 Genetic Transformation

[0055] 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.

[0056] 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.

[0057] 3) During this period, Agrobacterium containing plasmids (p1300S-SnRK1α, CRISPR-SnRK1α) was streaked on LB medium containing kanamycin and rifampin. After 2 days, single clones were picked, streaked again, and cultured for 1 day.

[0058] 4) Collect the bacterial cells from the culture medium and vortex them in NBC1 medium containing acetylsuccinone (AS), adjusting the OD to about 0.1-0.2.

[0059] 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 lined with a layer of sterile filter paper and co-culture at 22°C in the dark for 2 days.

[0060] 6) Screening: Transfer the co-cultured callus to NBS1 medium containing the corresponding antibiotic, incubate in the dark at 28°C for 10-12 days, then transfer it to NBS2 medium and continue incubation in the dark at 28°C for 10-12 days.

[0061] 7) Differentiation: After transferring the callus to NBR1 medium, culture it in the dark at 28°C for 6 days, then transfer it to an artificial climate incubator with 15h light / 9h darkness and culture it at 28°C for 15-20 days. During this period, callus tissue with green spots is transferred to NBR2 medium and cultured until it differentiates into seedlings.

[0062] 8) Cut the roots and leaves of the transgenic seedlings that are about 5cm tall, transfer them to the rooting medium, and culture them at 28°C in an artificial climate incubator with 12h light / 12h darkness.

[0063] 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.

[0064] 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.

[0065] Table 4 Genetic Transformation Culture Media

[0066]

[0067]

[0068] Table 5 Preparation of Hormone and Antibiotic Storage Solutions

[0069] NAA 1 mg / ml (initial dissolved in ethanol) 4℃ IAA 1 mg / ml (initial dissolved in ethanol) -20℃ KT 1 mg / ml (initial dissolved NaOH) -20℃ 6-BA 2 mg / ml (initial dissolved in NaOH) -20℃ 2,4-D 2 mg / ml (initial dissolved in 95% ethanol) 4℃ Rifampicin 50 mg / ml (soluble in methanol) -20℃ Kanamycin 50mg / ml -20℃ Temetin 50mg / ml 4℃ Hygromycin 50mg / ml 4℃ Acetyleugenol 100 μM / L (DMSO) -20℃

[0070] Example 5 Molecular Identification of Transgenic Plants

[0071] The T0 generation transgenic seedlings produced in Example 3 were subjected to PCR verification and propagation. T1 generation transgenic seeds were harvested and, through positive verification and sequencing, were obtained. Figure 2The corresponding homozygous mutant materials snrk1α-1, snrk1α-2, snrk1α-3, and snrk1α-4 were obtained as shown. Simultaneously, the RNA expression levels in transgenic rice were detected using quantitative real-time PCR (AceQ qPCR SYBR Green Master Mix (vazyme)) and sequencing methods. Figure 3 As shown, compared with wild-type materials, the expression level of SnRK1α gene in SnRK1α overexpression materials OE-SnRK1α#1, OE-SnRK1α#2, and OE-SnRK1α#3 was increased to varying degrees.

[0072] The obtained materials were further planted and propagated, and the offspring were screened for seeds using hygromycin (50 mg / L) to obtain the corresponding Cas9free homozygous mutant materials.

[0073] The primers used for molecular identification of transgenic plants, including those for overexpression and mutant line identification, are shown in Table 6.

[0074] Table 6 Primers for molecular identification of transgenic plants (5'-3')

[0075]

[0076] Example 6: Identification of stress resistance in transgenic plants

[0077] (1) Seeds of T2 generation SnRK1α transgenic overexpression, T3 generation SnRK1α knockout mutant and wild-type Nipponbare were selected.

[0078] (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 prepared 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.

[0079] (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.

[0080] (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 according to the above conditions, they are treated at 6℃ for 2-4 days (depending on the actual situation), and then transferred to 28℃ to recover growth for 1 week.

[0081] (5) Regarding survival rate, the criterion is whether new leaves have grown. If new leaves are present, the plant is considered alive; otherwise, it is considered dead. Figure 4It is evident that overexpression of the rice SnRK1α gene in rice can significantly improve the rice's ability to resist low-temperature stress. Mutating or knocking out the SnRK1α gene in rice will significantly reduce the rice's ability to resist low-temperature stress. Therefore, the rice SnRK1α protein, its encoding gene, and recombinant vector can be used to enhance the crop's resistance to abiotic stress.

[0082] Example 7: Yield Identification of Transgenic Plants

[0083] The phenotypes of overexpression and knockout mutant plants subjected to a low temperature of 20℃ during the booting stage are shown in the figure. Figure 5 The results showed that overexpression of the rice SnRK1α gene in rice can significantly increase the number of effective tillers and yield per plant. Mutating or knocking out the SnRK1α gene in rice will significantly reduce the seed setting rate and number of tillers. Therefore, the rice SnRK1α protein, its encoding gene, and recombinant vector can be used to improve crop yield.

[0084] 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. The application of the nucleic acid encoding overexpression of rice SnRK1α protein in improving rice seedling resistance to low-temperature stress, characterized in that, The encoded nucleic acid is the nucleotide sequence shown in SEQ ID NO: 1, and the rice variety is Nipponbare.

2. The application according to claim 1, characterized in that, The applications include, (1) Construct a recombinant expression vector containing the coding nucleic acid of rice SnRK1α protein; (2) Transform the constructed recombinant plant expression vector into rice tissues or rice cells; (3) Transgenic rice with improved resistance to low temperature stress during the seedling stage was obtained through breeding and screening.

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