Application of ZmCIPK4 protein and its encoding gene in regulating maize's tolerance to low temperature stress

Abstract

This invention relates to the application of the ZmCIPK4 protein and its encoding gene in regulating maize's tolerance to low-temperature stress. This invention improves the low-temperature stress tolerance of maize plants by knocking out the ZmCIPK4 gene. Maize plants with the ZmCIPK4 gene knocked out showed significantly higher tolerance to low-temperature stress compared to wild-type controls. This invention is of great significance for understanding the molecular mechanisms of cold tolerance in maize; furthermore, this gene plays an important role in breeding cold-resistant maize varieties, thus providing important possibilities for breeding new stress-resistant crop varieties, which is of great significance to agricultural production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the ZmCIPK4 protein and its encoding gene in regulating maize's tolerance to low-temperature stress. Background Technology

[0002] With the gradual decrease in arable land and the increase in the world's population, the demand for food is growing. Maize, one of the world's three major food crops, originated from the Mexican sorghum in its wild ancestry. It was first domesticated by humans more than 9,000 years ago and first appeared in the southwestern United States more than 4,000 years ago. Subsequently, maize cultivation spread to various parts of the world, with its growing environment gradually shifting from tropical to subtropical and temperate regions. This process was accompanied by a gradual decrease in the ambient temperature for maize growth. Furthermore, maize is a crop that is relatively sensitive to temperature. Among all biological and environmental stresses, low-temperature stress is one of the main limiting factors affecting crop survival and yield. Low-temperature stress has a significant impact on maize seedling germination, seedling survival, and grain filling during maturity. Therefore, studying the effects of low-temperature stress on maize is of great significance to maize production. Traditional breeding techniques for cultivating and improving stress-tolerant traits are relatively difficult and cannot quickly and efficiently yield superior cold-resistant varieties. With the development of molecular biology techniques, in-depth research into the molecular mechanisms of plant stress resistance, and significant progress in genetic engineering research, introducing exogenous genes for stress resistance into plants using genetic engineering methods such as transgenics has become one of the new approaches to improve plant stress resistance.

[0003] Ca 2+ Calcineurin B-like proteins (CBLs) and their target proteins, CBL-interacting protein kinases (CIPKs), play a crucial role as second messengers in intracellular signal transduction induced by developmental and environmental signals. They have become part of a stress response signaling network mediated by plant calcineurin.

[0004] CBLs are calcium-binding proteins containing an EF-hand domain, which serves as the structural basis for Ca2+ binding and interacts with Ca2+. 2+ In signal transduction, a group of protein kinases known as CBL-interacting protein kinases (CIPKs) engage in specific interactions. CIPKs are Ca2+-dependent serine / threonine kinases with highly conserved SNF-like kinase domains and NAF amino acid motifs, belonging to the plant SnRK3 (SNF-1-associated protein kinase 3) protein family.

[0005] The CBL-CIPKS complex is a complex calcium signaling system that responds to various stresses in plants. The ZmCIPK gene is upregulated under salt stress, drought stress, heat stress, and cold stress. The expression patterns of ZmCIPKs differ between the cold-tolerant inbred line B73 and the cold-sensitive inbred line Mo17 under cold stress. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of the ZmCIPK4 protein and its encoding gene in regulating maize's tolerance to low-temperature stress. Knocking out the ZmCIPK4 gene can enhance maize's resistance to low temperatures (ion permeability, osmotic potential, etc.), offering significant possibilities for breeding new stress-resistant (cold-tolerant) maize varieties and holding great importance for agricultural production.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The application of ZmCIPK4 protein in regulating maize's tolerance to low-temperature stress is characterized by the following application:

[0009] (1) Improve the ability of corn to tolerate low temperature stress;

[0010] (2) Select and breed maize varieties resistant to low temperature stress;

[0011] The amino acid sequence of the ZmCIPK4 protein is shown in SEQ ID NO.3.

[0012] The application of the ZmCIPK4 gene in regulating maize's tolerance to low-temperature stress is characterized by the following:

[0013] (1) Improve the ability of corn to tolerate low temperature stress;

[0014] (2) Select and breed maize varieties resistant to low temperature stress;

[0015] The ZmCIPK4 gene is used to encode the ZmCIPK4 protein as described in claim 1;

[0016] The specific measures of corn's ability to withstand low-temperature stress are: increased ion permeability, increased osmotic potential, and increased cold tolerance during the seedling stage.

[0017] Based on the above scheme, the application improves the ability of maize to tolerate low temperature stress by knocking out the ZmCIPK4 gene in maize plants, or by breeding maize varieties that are tolerant to low temperature stress.

[0018] In the above application, a recombinant expression vector containing the target gene encoding the protein is introduced into the receptor B73-329 corn using CRISPR / Cas9 technology.

[0019] The recombinant expression vector can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBD121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or other derived plant expression vectors. The recombinant expression vector amplifies the target sequence described in sequence 4 into the intermediate vector pCBC-MT1T2 using a double-primer amplification method, and then ligates it into the pBUE411 vector via a Bsa1 restriction site. When constructing a recombinant expression vector using the aforementioned gene, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), the stress-inducible promoter rd29A, etc. These can be used alone or in combination with other plant promoters. The translation control signal and start codon are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants, antibiotic resistance markers, or chemical reagent resistance marker genes. Alternatively, no selective marker genes can be added, and transformed plants can be directly screened for stress.

[0020] In this invention, the promoter that initiates the transcription of the protein-coding gene in the recombinant expression vector is the rice U6 promoter.

[0021] The ZmCIPK4 gene is characterized by the following nucleotide sequence:

[0022] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0023] (2) The nucleotide sequence shown in SEQ ID NO.2.

[0024] The target sequence of the ZmCIPK4 gene described above is characterized in that the target sequence of the ZmCIPK4 gene is as shown in SEQ ID NO.4. This target sequence is a gRNA sequence located on an exon.

[0025] More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the ZmCIPK4 gene target sequence (as shown in SEQ ID NO.4) between the multiple cloning sites Bsa1 of the pBUE411 vector.

[0026] Furthermore, the recombinant expression vector carrying the single target sequence of the ZmCIPK4 gene is introduced into the recipient plant. Specifically, this can be done by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrocoagulation, or Agrobacterium-mediated transformation, and then cultivating the transformed plant tissues into plants.

[0027] The beneficial effects of the ZmCIPK4 protein and its encoding gene described in this invention in regulating maize's tolerance to low-temperature stress are as follows:

[0028] Homozygous knockout lines of the ZmCIPK4 gene showed significantly improved tolerance to low-temperature stress compared to wild-type control plants. This invention is of great significance for the study of the molecular mechanism of cold tolerance in maize; furthermore, this gene plays an important role in breeding cold-resistant maize varieties, thus providing important possibilities for breeding new stress-resistant crop varieties, which is of great significance to agricultural production. Attached Figure Description

[0029] The present invention includes the following figures:

[0030] Figure 1 This is a schematic diagram illustrating the detection of homozygous knockout lines of the ZmCIPK4 gene.

[0031] Figure 2 This study investigated the 4℃ low-temperature treatment of the ZmCIPK4 mutant line and the wild-type control from the same batch. Maize seedlings were grown at 23℃ for 13 days, treated at 4℃ for 3 days, and then recovered at 23℃ for 24 hours.

[0032] Figure 3 The reaction of the ZmCIPK4 mutant line and the wild-type control line in the same batch after 4℃ low-temperature treatment in the ion permeation experiment. * indicates that the difference was extremely significant compared with the Col-0 group (P<0.05).

[0033] Figure 4 The osmotic pressure of the ZmCIPK4 mutant line and the wild-type control line after 4℃ low-temperature treatment is shown. * indicates extremely significant difference compared with the Col-0 group (P<0.05). Detailed Implementation

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Unless otherwise specified, all percentages in the following examples refer to mass percentages. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0037] The intermediate vector pCBC-MT1T2 of the pBUE411 vector was provided by China Agricultural University (reference: Xing, HL#, Dong, L.#, Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, and Chen, QJ* (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327.). The upstream promoter in the pBUE411 vector is the rice U6-26p promoter. The wild-type maize vector B73-329 was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0038] Agrobacterium tumefaciens: Agrobacterium tumefaciens strain GV3101, provided by our laboratory (reference: R. Berres, L. Otten, B. Tinland et al. Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T_6bgene. Plant Cell Reports, 1992(11):192-195.).

[0039] Escherichia coli strain DH5α(DE3) competent cells: a product of TransGen Biotech Ltd.

[0040] Example 1: Obtaining and Identifying ZmCIPK4 Transgenic Plants

[0041] The ZmCIPK4 gene involved in this embodiment is derived from maize (Zea may), and its sequence in the maize genome is shown in SEQ ID NO.1 of the sequence listing. This sequence consists of 1800 nucleotides and is the sequence of the ZmCIPK4 gene in the maize genome. The cDNA sequence of the ZmCIPK4 gene is shown in SEQ ID NO.2 of the sequence listing. This sequence consists of 1800 nucleotides and is the cDNA sequence of the ZmCIPK4 gene, wherein positions 165-1451 are the coding sequence (ORF). Both SEQ ID NO.1 and SEQ ID NO.2 encode the protein shown in SEQ ID NO.3 of the sequence listing. SEQ ID NO.3 consists of 429 amino acid residues.

[0042] I. Construction of the recombinant expression vector pBUE411-pCBC-MT1T2

[0043] Ta is the sequence shown in SEQ ID NO.4. Using the pCBC-T1T2 sequence as a template, the Ta single target was amplified on pCBC-T1T2 through double primer amplification, and this ligation fragment was then ligated into the pBUE411 vector.

[0044] BsF: 5'-ATATATGGTCTCGATTGAGACAGCGCGCGTGGT ATA-3' (SEQ ID NO.5)

[0045] F0: 5'-TGAGACAGCGCGCGTGGTATAGTTTTAGAGCTAG AAATAGC-3' (SEQ ID NO.6)

[0046] The 7th-12th bp of the sequence shown in SEQ ID NO.5 above is the recognition site of Bsa1, and the 18th-36th bp of the sequence shown in SEQ ID NO.5 and the 3rd-21st bp of the sequence shown in SEQ ID NO.6 are the reverse complementary sequences of the 259th-277th bp of the sequence shown in SEQ ID NO.2;

[0047] R0:5'-AACTATACCACGCGCGCTGTCTCAATCTCTTAGTCGACTCTAC-3'(SEQ ID NO.7)

[0048] BsR:5'-ATTATTGGTCTCGAAACTATACCACGCGCGCTGTCTC-3'(SEQ ID NO.8)

[0049] The 7th-12th bp of the sequence shown in SEQ ID NO.8 is the recognition site of Bsa1. The 4th-22th bp of the sequence shown in SEQ ID NO.7 and the 18th-36th bp of the sequence shown in SEQ ID NO.8 are both Ta sequences, which are the 259th-277th bp of the sequence shown in SEQ ID NO.2.

[0050] The pBUE411 vector obtained above was digested with restriction endonuclease Bsa1. Using the two pairs of primers described above and pCBC-MT1T2 as a template, four-primer amplification was performed. The F0 and R0 primer concentrations were diluted tenfold from the working concentration. PCR amplification was then performed, and the ligation fragment was recovered from the gel. The fragment was then ligated using Soluton 1 ligase to obtain the recombinant plasmid. The recombinant plasmid was sent for sequencing. The recombinant plasmid showing that a DNA fragment of pCBC-MTaTb was inserted between the Bsa1 restriction sites of the pBUE411 vector was named pBUE411-pCBC-MTaTb.

[0051] II. Obtaining and Identifying ZmCIPK4 Gene Knockout Maize

[0052] 1. Obtaining ZmCIPK4 transgenic Arabidopsis and maize plants transformed with the empty vector pBUE411-pCBC-MTaTb: The recombinant expression vector pBUE411-pCBC-MTaTb constructed in step one was introduced into Agrobacterium GV3101 competent cells using the freeze-thaw method. The transformed recombinant Agrobacterium was identified by PCR using primer pairs consisting of primers F0 and R0. The Agrobacterium GV3101 cells that were identified as containing pCBC-MTaTb (the target PCR band size was approximately 1000 bp) were named pBUE411-pCBC-MTaTb-1.

[0053] The recombinant Agrobacterium pBUE411-pCBC-MTaTb-1 obtained above was transformed into maize wild-type (B73-329 ecotype) using the Agrobacterium inflorescence infection method (SJ Clough, AF Bent. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 1998, 16(6):735-743.). Transformation of maize callus tissue (screening for bar resistance was performed; the process was completed by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University) was also performed.

[0054] 2. Identification of ZmCIPK4 CRISPR knockout lines

[0055] The pBUE411 vector can produce homozygous stable knockout lines in the T0 generation, and then homozygous knockout lines with CRISPR / Cas9 background removed can be obtained through self-pollination in the F2 generation.

[0056] III. ZmCIPK4 CRISPR line sequencing and identification

[0057] Total DNA was extracted from wild-type maize (B73-329 ecotype) and knockout plants (D1 and D2), and the DNA sequence differences of the ZmCIPK4 gene in the materials were detected by PCR. Details are as follows:

[0058] The PCR amplification method is as follows:

[0059] The primer sequences for amplifying the CIPK4 gene are as follows:

[0060] CIPK4RT-F1: 5'-GGCAGCACCCTGTAAACTAA-3' (SEQ ID NO.9, bits 88-107 of SEQ ID NO.2);

[0061] CIPK4RT-R1: 5'-AGCTCCATCACGAGGTACA-3' (the inverse complementary sequence of positions 436-454 of SEQ ID NO.10 and SEQ ID NO.2).

[0062] The reaction conditions for the above primers are as follows:

[0063] (1) Establishment of the reaction system

[0064] PCR reaction system

[0065]

[0066]

[0067] (2) Three replicates were made, and the mixture was shaken to mix. The experiment was performed using a Bio-Rad PCR instrument.

[0068] (3) Setting the reaction procedure:

[0069] PCR reaction procedure

[0070]

[0071] The band size was detected by electrophoresis at 157V using a 1% agarose gel, and was found to be approximately 360bp. The sample was then sent to a sequencing company for sequencing.

[0072] ZmCIPK4 PCR detection and sequencing results are as follows Figure 1The diagram illustrates two different knockout patterns of the ZmCIPK4 gene: D1, which involves the deletion of one base at position 268 (as shown in SEQ ID NO. 2) at the target site (as shown in SEQ ID NO. 4); and D2, which involves the deletion of two bases at positions 272-273 (as shown in SEQ ID NO. 2). Both knockout patterns result in frameshift mutations.

[0073] Example 2: Low-temperature treatment experiment of ZmCIPK4 gene knockout lines

[0074] Low temperature stress can cause leaf damage. It can also lead to leaf dehydration and wrinkling, such as... Figure 1 As shown, 14-day-old maize seedlings (including those 3-4 days after germination) grown under normal light conditions at 23℃ were placed in a 4℃ incubator for 3 days. The treated ZmCIPK4CRISPR / Cas9 knockout lines D1 and D2, along with the wild-type (WT, B73-329 ecotype) from the same batch, were then placed at 23℃ for 24 hours to recover before phenotypic observation and image acquisition. It can be seen that compared to wild-type maize plants, the tolerance of D1 and D2 to low-temperature stress was significantly improved after low-temperature treatment. Figure 2 Knocking out the ZmCIPK4 gene significantly improves maize's tolerance to low-temperature stress (this was verified by more than three low-temperature treatments).

[0075] I. Plant Ion Leakage Measurement Experiment

[0076] When plants are subjected to low-temperature stress, low temperatures can damage plant cells, causing the cell membrane to lose or partially lose its selective permeability to ions. Therefore, measuring ion leakage can reflect the plant's tolerance to various abiotic stresses such as low-temperature stress (Verslues PE1, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J. 2006 Feb;45(4):523-39.). The aboveground parts of maize seedlings were placed in 15ml centrifuge tubes, 10ml of deionized water was added, and the plants were completely submerged below the water surface. The mixture was shaken at 23℃ for 120rpm for 1h, and the ion leakage was measured using a particle leakage detector and recorded as S0. Then all samples were placed in a 100℃ water bath for 1h and shaken at 23℃ for 120rpm. After 1 hour, the sample was measured again and recorded as S1. (Note: Before measuring each sample, the instrument probe must be rinsed and the value of the rinsing solution (deionized water) measured and recorded as S2 and S3. The final particle leakage rate is calculated using the following formula:)

[0077] Ion leakage(%)=S0-S2 / S1-S3

[0078] This experiment measured the average value of at least three maize seedlings from the same lineage (three experiments, * indicates significant difference, P<0.05).

[0079] like Figure 3 The significantly lower leakage rates of D1 and D2 particles compared to the wild type (WT, B73-329 ecotype) demonstrate that their tolerance to low-temperature stress under low-temperature stress treatment is significantly improved compared to the wild type.

[0080] III. Osmotic Measurement Experiment

[0081] Maintaining osmotic pressure is crucial for plants to maintain homeostasis. When plants are subjected to external stress, they accumulate protective substances within their cells, thus increasing their freezing point osmotic pressure. After treating 14-day-old maize seedlings at 4°C for 12 hours, the above-ground parts of the seedlings were squeezed using a syringe, and the sap was collected in 1.5 ml centrifuge tubes. The centrifuge volume was increased by 12000 rpm for 5 minutes, the supernatant was discarded, and the osmotic pressure was measured using a freezing point osmotic pressure meter. Figure 4As shown, the freezing point osmotic pressure of D1 and D2 is higher than that of the wild type (WT, B73-329 ecotype), indicating that their tolerance to low-temperature stress is significantly improved compared to the wild type. All of the above experiments demonstrate that knocking out the ZmCIPK4 gene can improve the cold tolerance of maize.

[0082] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. ZmCIPK4 The application of genes in regulating maize's tolerance to low-temperature stress is characterized by, The application is as follows: By knocking out ZmCIPK4 Genes enhance maize's tolerance to low-temperature stress; The ZmCIPK4 The gene, as shown in SEQ ID NO.1, encodes the ZmCIPK4 protein shown in SEQ ID NO.

3.

2. The application as described in claim 1, characterized in that, Knockout in corn ZmCIPK4 The gene is specifically: as shown in SEQ ID NO.4 ZmCIPK4 The target sequence of the gene is knocked out by one base, which is located at the 268th bp of the sequence shown in SEQ ID NO.2.

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