Application of ZmHSP22 protein and its coding gene in regulating corn tolerance to low temperature stress

Abstract

The present application relates to the field of genetic engineering, in particular to the application of ZmHSP22 protein and its coding gene in regulating the low temperature stress tolerance of plants. The nucleotide sequence of ZmHSP22 gene is shown as SEQ ID No. 1; the protein amino acid sequence is shown as SEQ ID No. 3. After knocking out the ZmHSP22 gene, the cold tolerance of the transgenic plants is improved compared with the control group under low temperature stress treatment. The present application verifies that the ZmHSP22 protein has a regulating function on the low temperature stress tolerance of plants (especially corn), and the ZmHSP22 protein and its related biological materials can be applied to the regulation of the cold tolerance of plants, providing excellent candidate gene resources for the cultivation and improvement of new varieties of cold-tolerant plants. The method for breeding cold-tolerant plants provided has strong purpose compared with the traditional breeding method, significantly shortens the period of cold-tolerant breeding, improves the efficiency of cold-tolerant breeding, lays a theoretical foundation for the study of the mechanism of plant response to adverse signals and the molecular mechanism of tolerance to adverse environment, and has important theoretical and practical significance for promoting the process of corn breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the HSP22 protein and its encoding gene in regulating plant 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, as one of the world's three major food crops, is also 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 and survival, as well as the grain-filling process during maturity. Cold stress affects the plasticity of plant enzymes and membranes, alters physiology and metabolism, and sometimes causes water shortage and drought, creating stress conditions for plants and adversely affecting their growth, development, and yield. Low temperature is also associated with protein dysfunction and denaturation, thereby inducing the accumulation of heat shock proteins. In Arabidopsis thaliana, tobacco, maize, rapeseed, chicory, poplar, wheat, and barley, many heat shock proteins respond to and are upregulated in response to cold stress. Under low-temperature stress, heat shock proteins are induced and transported to various organelles to protect cells from the stress.

[0003] Therefore, studying the effects of low-temperature stress on maize is of great significance to maize production. Traditional breeding techniques are relatively difficult to cultivate and improve stress tolerance traits, and cannot quickly and efficiently obtain superior cold-resistant varieties. However, 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 transgenic and other genetic engineering methods has become one of the new approaches to improving plant stress resistance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of the ZmHSP22 protein and its encoding gene in regulating plant tolerance to low temperature stress. By constructing CRISPR / Cas9 materials, the target gene ZmHSP22 was rapidly knocked out in wild-type plants, and the results showed that the transgenic plants exhibited improved tolerance to low temperature stress compared to wild-type plants.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] The use of maize ZMHSP22 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects:

[0007] (1) Improve the cold resistance of corn;

[0008] (2) Breeding transgenic maize with improved resistance to low-temperature stress;

[0009] (3) Improve cold-resistant maize germplasm resources.

[0010] Preferred,

[0011] The cold tolerance of maize can be improved by inhibiting or reducing the expression level and / or activity of ZMHSP22 protein in maize.

[0012] Preferred,

[0013] The amino acid sequence of the corn ZMHSP22 protein is any one of the following:

[0014] (A1) The amino acid sequence shown in SEQ ID No. 3;

[0015] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 3.

[0016] Preferred,

[0017] The nucleotide sequence of the gene encoding the maize ZMHSP22 protein is any one of the following:

[0018] (B1) The nucleotide sequence shown in SEQ ID No. 1;

[0019] (B2) The nucleotide sequence shown in SEQ ID No. 2;

[0020] (B3) A nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0021] (B4) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No.2.

[0022] Preferred,

[0023] The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

[0024] A method for breeding cold-resistant maize.

[0025] Plants with improved cold tolerance were obtained by inhibiting the expression and / or activity of the ZMHSP22 protein; the amino acid sequence of the ZMHSP22 protein is any of the following:

[0026] (A1) The amino acid sequence shown in SEQ ID No. 3;

[0027] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 3.

[0028] The method for breeding cold-resistant maize provided by this invention involves knocking out the ZMHSP22 gene in the recipient plant using CRISPR / Cas9 technology, or introducing a recombinant expression vector containing the target gene encoding the protein into the recipient B73-329 maize to obtain a transgenic plant; the cold resistance of the transgenic maize plant is improved.

[0029] The recombinant expression vector can be constructed using existing plant expression vectors, including binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or other derived plant expression vectors.

[0030] The recombinant expression vector described above amplifies the target sequence of SEQ ID No. 4 into the intermediate vector pCBC-MT1T2 using a double primer amplification method, and then ligates it into the pBUE411 vector via the Bsa1 restriction site. When constructing a recombinant expression vector using genes, 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 promoter (pUbi), or the stress-inducible promoter rd29A, etc., which can be used alone or in combination with other plant promoters; the translation control signal and start codon 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 that produce color changes 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.

[0031] In this invention, the promoter that initiates the transcription of the protein-coding gene in the recombinant expression vector is the rice U6 promoter.

[0032] Preferably, the method includes the following steps:

[0033] The gene encoding the ZMHSP22 protein was ligated into the pBCXUN vector to obtain the recombinant vector pBCXUN-ZMHSP22 containing the ZMHSP22 coding sequence. This recombinant vector was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 containing the recombinant vector pBCXUN-ZMHSP22. The recombinant Agrobacterium EHA105 / pBCXUN-ZMHSP22 was then used to infect maize and introduced into recipient plants to obtain transgenic plants with increased cold tolerance.

[0034] The amino acid sequence of the ZMHSP22 protein is any of the following:

[0035] (A1) The amino acid sequence shown in SEQ ID No. 2;

[0036] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.

[0037] Preferred,

[0038] The primers for detecting the ZMHSP22 gene are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0039] Preferred,

[0040] This includes using DNA homologous recombination technology, Cre / Loxp technology, and Crispr / Gas9 technology to silence or reduce the expression level of the maize ZMHSP22 gene, thereby obtaining transgenic plant lines.

[0041] The recombinant expression vector carrying the single target sequence of the ZmHSP22 gene is introduced into the recipient plant, including methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, and Agrobacterium-mediated transformation to introduce the recombinant expression vector containing the maize ZMHSP22 gene into maize, thereby obtaining transgenic maize lines.

[0042] The use of a protein consisting of the amino acid sequence shown in SEQ ID No. 3 or its encoding gene in the following (a), (b), or (c) is also within the scope of protection of this invention:

[0043] (a) A homozygous knockout line of the ZmHSP22 gene was obtained;

[0044] (b) Knockout lines of the ZmHSP22 gene can increase the ion leakage rate of maize under low temperature treatment.

[0045] (c) Knockout lines of the ZmHSP22 gene can increase the osmotic potential of maize under low temperature treatment.

[0046] (d) Knockout lines of the ZmHSP22 gene can improve cold tolerance in maize seedlings;

[0047] In the above applications or methods, the plant can be either a dicotyledonous plant or a monocotyledonous plant.

[0048] The beneficial effects of this invention are:

[0049] This invention verifies that the ZmHSP22 protein has a regulatory function on the low-temperature stress tolerance of plants (especially maize). Homozygous knockout lines of the ZmHSP22 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; it provides excellent candidate gene resources for breeding and improving new cold-tolerant plant varieties; and the method for breeding cold-tolerant plants provided is more targeted than traditional breeding methods, significantly shortening the cold-tolerant breeding cycle and improving the efficiency of cold-tolerant breeding. It lays a theoretical foundation for studying the mechanisms of plant responses to stress signals and the molecular mechanisms of tolerance to adverse environments, and has important theoretical and practical significance for promoting the progress of maize breeding. Attached Figure Description

[0050] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0051] Figure 1 This is a schematic diagram illustrating the detection of homozygous knockout lines of the ZmHSP22 gene.

[0052] Figure 2 This study investigated the 4℃ low-temperature treatment of the ZmHSP22 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.

[0053] Figure 3 This section describes the response of the ZmHSP22 mutant line and the wild-type control line in the ion permeation experiment after 4℃ low-temperature treatment, as well as the ion leakage rate after 4℃ treatment. * indicates extremely significant difference compared with the Col-0 group (P<0.05).

[0054] Figure 4 The results show the response of the ZmHSP22 mutant line and the wild-type control line in the ion permeation experiment after 4℃ low-temperature treatment, as well as the freezing point osmotic pressure after 4℃ treatment. * indicates extremely significant difference compared with the Col-0 group (P<0.05). Detailed Implementation

[0055] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] 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.

[0059] 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. BMCPlantBiol 14, 327.). The upstream promoter in the pBUE411 vector is the rice U6-26p promoter. Wild-type maize (B73-329) was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0060] 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.).

[0061] Escherichia coli strain DH5α(DE3) competent cells: a product of TransGen Biotech Ltd.

[0062] Example 1: Obtaining and Identifying ZmHSP22 Transgenic Plants

[0063] The ZmHSP22 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. This gene consists of 1270 nucleotides, of which positions 438-568 are intron sequences. The cDNA sequence of the ZmHSP22 gene is shown in SEQ ID No. 2, which consists of 1139 nucleotides, of which positions 185-841 are coding sequences (ORF). Both SEQ ID No. 1 and SEQ ID No. 2 encode the protein shown in SEQ ID No. 3, which consists of 219 amino acid residues. SEQ ID No. 10 (TAGCGTCCGCGAGTACGAT) is the gRNA sequence located on the first exon.

[0064] 1. Construction of the recombinant expression vector pBUE411-pCBC-MTaTb

[0065] Ta for Figure 1 The sequence shown was used to construct a single Ta target amplification on pCBC-T1T2 using pCBC-T1T2 plasmid as a template and double primer amplification. This ligation fragment was then ligated into the pBUE411 vector to obtain the pBUE411-pCBC-MT1T2 vector.

[0066] BsF: 5'-ATATATGGTCTCGATTGATCGTACTCGCGGACGCTA-3' (SEQ ID No. 4)

[0067] F0:5'-TGATCGTACTCGCGGACGCTAGTTTTAGAGCTAGAAATAGC-3'(SEQ ID No.5)

[0068] (Large font indicates the identification site of Bsa1, and bold font indicates the Ta sequence, which is the reverse complementary sequence of positions 370-388 of SEQ ID No. 2);

[0069] R0:5'-AACTAGCGTCCGCGAGTACGATCAATCTCTTAGTCGACTCTAC-3'(SEQ ID No.6)

[0070] BsR:5'-ATTATTGGTCTCGAAACTAGCGTCCCGCGAGTACGATC-3'

[0071] (SEQ ID No.7)

[0072] (Large font indicates the identification site of Bsa1, and bold font indicates the Ta sequence, which is the sequence of positions 370-388 of SEQ ID No. 2).

[0073] The pBUE411 vector, prepared by digestion with restriction endonuclease Bsa1, was amplified using four primers with pCBC-MT1T2 as a template. The F0 and R0 primers were diluted tenfold from their working concentrations. PCR amplification was then performed, and the ligation fragment pCBC-MTaTb was recovered from the gel. This fragment was then ligated using Soluton 1 ligase to obtain the recombinant plasmid. The recombinant plasmid was sequenced, and the plasmid containing the pCBC-MTaTb DNA fragment inserted between the Bsa1 restriction sites of the pBUE411 vector was named pBUE411-pCBC-MTaTb.

[0074] 2. Obtaining and identifying ZmHSP22 gene knockout maize

[0075] 1) Recombinant Agrobacterium was prepared by introducing the recombinant expression vector pBUE411-pCBC-MTaTb into competent Agrobacterium GV3101 cells via a freeze-thaw method into ZmHSP22 transgenic maize and maize plants transformed with the empty vector pBUE411-pCBC-MTaTb. The transformed recombinant Agrobacterium was identified by PCR using primer pairs consisting of primers F0 and R0. Agrobacterium GV3101 cells that were identified as containing pCBC-MTaTb (the PCR target band size was approximately 1000 bp) were named pBUE411-pCBC-MTaTb-1.

[0076] The recombinant Agrobacterium pBUE411-pCBC-MTaTb-1 obtained above was transformed into wild-type maize (ecotype B73-329) 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 was performed on maize callus tissue (screening for bar resistance was conducted by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University).

[0077] 2) Identification of CRISPR knockout lines of ZmHSP22 gene

[0078] 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.

[0079] 3. Identification of ZmHSP22 gene CRISPR line by sequencing

[0080] Total DNA was extracted from wild-type maize (B73-329 ecotype) and knockout plants (I1 and D4), and the DNA sequence differences of the ZmHSP22 gene in each material were detected by PCR.

[0081] Specifically as follows:

[0082] The PCR amplification method is as follows:

[0083] The primer sequences for amplifying the ZmHSP22 gene are as follows:

[0084] HSP22RT-F1: 5'-GCAGGATCTTCGTGAACCTT-3' (SEQ ID No. 8) (bits 2-21 of SEQ ID No. 2);

[0085] HSP22RT-R1: 5'-CTTGTCCATCCTGTAAACCTCTG-3' (SEQ ID No. 9) (the inverse complementary sequence of positions 726-748 of SEQ ID No. 2).

[0086] The reaction conditions for the above primers are as follows:

[0087] (1) Establishment of the reaction system

[0088] PCR reaction system

[0089]

[0090] (2) Three replicates were made, and the mixture was shaken to mix. The experiment was performed using a Bio-Rad PCR instrument.

[0091] (3) Setting the reaction procedure:

[0092] PCR reaction procedure

[0093]

[0094] The band size was detected by electrophoresis at 157V on a 1% agarose gel and was approximately 750bp. The sample was then sent to a sequencing company for sequencing.

[0095] The PCR detection and sequencing results of the ZmHSP22 gene are as follows: Figure 1 As shown, the ZmHSP22 gene has two different knockout forms: I1, which involves inserting a base after position 385 in the sequence shown in SEQ ID No. 2, and D4, which involves deletion at positions 383-386 in the sequence shown in SEQ ID No. 2. Both knockout forms result in frameshift mutations.

[0096] Example 2: Low-temperature treatment experiment on ZmHSP22 gene knockout lines

[0097] Low temperature stress can cause leaf damage, as well as leaf dehydration and wrinkling, such as... Figure 2 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 ZmHSP22 gene CRISPR / Cas9 knockout lines I1 and D4, along with the same batch of wild-type (B73-329 ecotype), were then placed at 23℃ for 24 hours to recover before phenotypic observation and image collection. It can be seen that compared to wild-type maize plants, I1 and D4 showed significantly improved tolerance to low-temperature stress after low-temperature treatment. Knocking out the ZmHSP22 gene significantly improved maize's tolerance to low-temperature stress (this experiment was verified through more than three low-temperature treatments).

[0098] 1) Plant ion leakage test

[0099] When plants are subjected to low-temperature stress, the low temperature 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. The aboveground parts of corn seedlings were placed in 15ml centrifuge tubes, and 10ml of deionized water was added to completely submerge the plants. The mixture was incubated at 23℃ and 120rpm for 1 hour using a particle leakage detector, and the result was recorded as S0. Then, all samples were placed in a 100℃ water bath for 1 hour, followed by an incubation at 23℃ and 120rpm for 1 hour. The results were then 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) must be recorded as S2 and S3.) The final particle leakage rate is calculated using the following formula:

[0100] Ion leakage(%)=S0-S2 / S1-S3

[0101] This experiment measured the average value of at least three maize seedlings from the same lineage (three experiments, * indicates significant difference, P<0.05).

[0102] like Figure 3 As shown, the leakage rates of I1 and D4 particles were significantly lower than those of the wild type (B73-329 ecotype), proving that their ability to tolerate low-temperature stress under low-temperature stress treatment was significantly improved compared to the wild type.

[0103] 2) Osmotic Measurement Experiment

[0104] 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 was performed at 12000 rpm for 5 minutes, the supernatant was discarded, and the osmotic pressure was measured using a freezing point osmotic pressure meter. Figure 4 As shown, the freezing point osmotic pressure of I1 and D4 is higher than that of the wild type (B73-329 ecotype), indicating that their tolerance to low temperature stress under low temperature stress treatment is significantly improved compared to the wild type.

[0105] The above experiments all demonstrate that knocking out the ZmHSP22 gene can improve the cold resistance of maize.

[0106] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0107] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of maize ZMHSP22 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects: (1) Improve the cold resistance of corn; (2) Breeding transgenic maize with improved resistance to low-temperature stress; (3) Improve cold-resistant maize germplasm resources; The cold tolerance of maize can be improved by inhibiting or reducing the expression level of ZMHSP22 protein in maize. The amino acid sequence of the corn ZMHSP22 protein is as follows: (A1) The amino acid sequence shown in SEQ ID No.

3.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the maize ZMHSP22 protein is any one of the following: (B1) The nucleotide sequence shown in SEQ ID No. 1; (B2) The nucleotide sequence shown in SEQ ID No.

2.

3. The application according to claim 1 or 2, characterized in that, The biomaterials include expression cassettes, vectors, and host cells.

4. A method for breeding cold-resistant maize, characterized in that, Plants with improved cold tolerance were obtained by inhibiting the expression of the ZMHSP22 protein; the amino acid sequence of the ZMHSP22 protein is as follows: (A1) The amino acid sequence shown in SEQ ID No.

3.

5. The breeding method for cold-resistant maize according to claim 4, characterized in that, This includes using DNA homologous recombination technology, Cre / Loxp technology, and CRISPR / Gas9 technology to improve corn... ZMHSP22 Gene silencing or reduced expression levels yield transgenic plant lines.

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