Application of ZmNCED4 protein and coding gene thereof in regulating and controlling low-temperature stress resistance of plants

Abstract

The invention relates to application of a ZmNCED4 protein and a coding gene thereof in regulating and controlling low-temperature stress resistance of plants. The ZmNCED4 protein and the coding gene thereof have obvious influence on low-temperature stress resistance of plants (especially corn), and the low-temperature stress resistance of the corn can be improved by inhibiting expression of the coding gene of the ZmNCED4 protein in the corn. The application provides a good germplasm resource for cultivating new varieties of low-temperature-resistant plants, and lays a theoretical foundation for researching a mechanism of responding to stress signals and a molecular mechanism of tolerating unfavorable environments of the plants.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to application of a ZmNCED4 protein and a coding gene thereof in regulating plant resistance to low temperature stress. Background Art

[0002] Plants that grow in fixed places in nature are inevitably affected by various adverse stresses. Low temperature stress, as one of the main external environmental factors, not only affects the growth and development of plants and their geographical distribution, but also seriously affects the yield of crops. Therefore, studying the physiological and biochemical changes and molecular mechanisms of plant responses to low temperature stress and finding key genes that regulate plant responses to low temperature stress can provide valuable theoretical support for the cultivation of cold-resistant crop varieties to increase crop yields. Corn (Zea mays) belongs to the genus Zea of ​​the Poaceae family and is an important food crop and feed crop. Low temperature stress after corn sowing will reduce the germination rate of corn and delay emergence, thereby affecting the uniformity of emergence and the vitality of seedlings during the seedling stage. Low temperature restricts the growth and development of seedlings, affects the intensity of photosynthesis, and thus affects the yield of corn. Improving corn cold stress tolerance through genetic engineering methods is of great significance for maintaining and increasing corn yields.

[0003] The biosynthesis of ABA is an important pathway that affects its content. The biosynthesis of ABA can be divided into two pathways: one is the terpenoid pathway, also known as the C15 direct pathway, in which farnesyl pyrophosphate (FPP) is directly formed into 15-carbon ABA through cyclization and oxidation. The other is the carotenoid pathway, also known as the C40 indirect pathway, in which β-carotene polymerized from mevalonic acid (MVA) is catalyzed by zeaxanthin epoxidase (ZEP) encoded by AtABA1 / ZEP to form zeaxanthin, which is then oxidized to form flavonoids by 9-cis-epoxycarotenoid dioxygenases (NCEDs). NCEDs are considered to be the key enzymes for ABA synthesis. Subsequently, flavonoids are converted into ABA in the cytoplasm through the catalysis of short-chain alcohol dehydrogenase (encoded by AtABA2) and aldehyde oxidase (AAO3) and the activation of molybdenum cofactor sulfurase (MoCo) encoded by AtABA3. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide an application of ZmNCED4 protein and its encoding gene in regulating plant resistance to low temperature stress. The present invention can improve the resistance to low temperature stress of corn by inhibiting the expression of the encoding gene of ZmNCED4 protein in corn.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] Application of ZmNCED4 protein, or its encoding gene, or its inhibitor, or biological material containing its encoding gene or inhibitor in any of the following aspects:

[0007] (1) Regulate plant tolerance to low temperature stress;

[0008] (2) Breeding plants with improved tolerance to low temperature stress;

[0009] The amino acid sequence of the ZmNCED4 protein is:

[0010] (1) the amino acid sequence shown in SEQ ID NO.1; or,

[0011] (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0012] It should be understood that, considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0013] The nucleotide sequence of the ZmNCED4 protein encoding gene is:

[0014] (1) the nucleotide sequence shown in SEQ ID NO.2, which encodes the ZmNCED4 protein shown in SEQ ID NO.1; or

[0015] (2) a nucleotide sequence encoding a protein having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; or,

[0016] (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0017] Among them, the maize ZmNCED4 gene is numbered Zm00001d007876 in the maize genome database, consisting of 1986 bases, and the reading frame of the gene is from the 151st base to the 1878th base from the 5' end. The gene reading frame consists of one exon.

[0018] In some embodiments, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

[0019] Preferably, the plant is a monocotyledonous plant; more preferably, it is corn.

[0020] On the basis of the above scheme,

[0021] The application improves the low temperature stress tolerance of plants by inhibiting the expression of the coding gene of the ZmNCED4 protein, and is applied to the breeding of low temperature stress resistant plants.

[0022] On the basis of the above scheme,

[0023] The application improves the low temperature sensitivity of plants by promoting the expression of the coding gene of the ZmNCED4 protein, and is applied to the breeding of low temperature sensitive plants.

[0024] In actual implementation, the expression or overexpression method can be transgenic, hybridization, backcrossing, selfing or asexual reproduction, wherein the transgenic includes using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, conductivity, Agrobacterium-mediated method to introduce a recombinant expression vector containing the gene encoding the ZmNCED4 protein into corn to obtain a transgenic corn strain.

[0025] The target sequence for editing the above-mentioned ZmNCED4 protein encoding gene is characterized in that the target sequence is shown in SEQ ID NO.3.

[0026] The primers used to amplify the above ZmNCED4 protein encoding gene are characterized by:

[0027] The nucleotide sequences of the primers are shown in SEQ ID NO.4-5.

[0028] A mutant of a gene encoding a ZmNCED4 protein, characterized in that:

[0029] The nucleotide sequence of the mutant is shown in SEQ ID NO.6-7.

[0030] The application of the ZmNCED4 protein and its encoding gene in regulating plant tolerance to low temperature stress has the following beneficial effects:

[0031] ZmNCED4 protein and its encoding gene have a significant effect on the tolerance of plants (especially corn) to low temperature stress. Specifically, by inhibiting the expression of the encoding gene of ZmNCED4 protein in corn, the tolerance of corn to low temperature stress can be improved. This application provides good germplasm resources for breeding new varieties of low temperature tolerant plants and lays a theoretical foundation for studying the mechanism of plant response to adverse signals and the molecular mechanism of tolerance to adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention has the following accompanying drawings:

[0033] Figure 1The seed germination conditions of the mutant strains crispr1-2300 and crispr2-2500 under normal temperature and low temperature treatment in Example 1 of the present invention; wherein CK represents the wild-type corn strain, Cr1 and Cr2 represent the mutant strains crispr1-2300 and crispr2-2500, respectively.

[0034] Figure 2 This is a statistical chart of seed germination rates of the mutant strains crispr1-2300 and crispr2-2500 at room temperature in Example 2 of the present invention; wherein CK represents wild-type corn plants, Cr1 and Cr2 represent the mutant strains crispr1-2300 and crispr2-2500, respectively.

[0035] Figure 3 This is a statistical graph of seed germination rates of the mutant strains crispr1-2300 and crispr2-2500 under low temperature treatment in Example 2 of the present invention; wherein CK represents wild-type corn plants, Cr1 and Cr2 represent the mutant strains crispr1-2300 and crispr2-2500, respectively.

[0036] Figure 4 This is the specific sequencing comparison result of the strain with ZmNCED4 gene knocked out in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0038] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0039] In the following examples, the pBSK vector is a commonly used cloning vector and is commercially available; the pBCXUN vector (the pBCXUN vector uses the commercial vector pCAMBIA1300 as the backbone, and the hygromycin resistance gene hpt therein is replaced with the herbicide resistance gene barM; at the same time, the promoter of the maize ubiquitin gene Ubi is cloned into the vector by enzyme ligation to drive the transcription of downstream overexpressed genes), using the Ubi promoter to drive the expression of the NCED4 gene; the Agrobacterium EHA105 strain was purchased from Shanghai Weidi Biotechnology Co., Ltd. (Cat. No.: AC1010S).

[0040] The main reagents in the following examples are: various restriction endonucleases, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, KOD purchased from NEB, Toyobo and other biological companies; dNTPs purchased from Genestar; plasmid mini-extraction kit and agarose gel recovery kit purchased from Shanghai Jierui Bioengineering Co., Ltd.; agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampicin (Rif) and other antibiotics and glucose, BSA, LB Medium, etc. purchased from Sigma, Bio-Rad and other companies; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and various other chemical reagents used in the examples were imported or domestic analytical reagents.

[0041] The primers used in the examples were synthesized by Liuhe BGI, and related sequencing was performed.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field, such as Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21, or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased through regular channels.

[0043] Example 1 Phenotypic Verification of Maize ZmNCED4 Mutant

[0044] I. Construction of ZmNCED4 mutants

[0045] 1. Construction of recombinant expression vector

[0046] gRNA sequence: TCCGTGAACCGGCACGCGT (Seq ID No.3)

[0047] The designed target site was synthesized from a single-stranded form to a double-stranded form by touch down PCR and the connecting fragment was connected to the pBUE411 vector.

[0048] The pBUE411 vector obtained above was digested with restriction endonuclease Bsa1, and then ligated with Soluton1 ligase to obtain a recombinant plasmid. The recombinant plasmid was sent for sequencing, and the recombinant plasmid between the restriction site Bsa1 of the pBUE411 vector indicated by sequencing was named pBUE411-gnced4.

[0049] 2. Obtaining and identifying ZmNCED4 gene knockout maize

[0050] (1) Obtaining ZmNCED4 transgenic maize and maize plants transformed with pBUE411-gnced4

[0051] The recombinant expression vector pBUE411-gnced4 constructed in 1 was introduced into Agrobacterium GV3101 competent cells by freeze-thaw method. The recombinant Agrobacterium pBUE411-gnced4 obtained above was transformed into wild type maize (LH244 ecotype) by 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.). Maize callus tissue was transformed and infected (screened by bar resistance, the process was completed by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University)

[0052] (2) Identification of CRISPR / Cas9 knockout strains of ZmNCED4

[0053] The pBUE411 vector can produce homozygous stable knockout strains in the T0 generation, and then the homozygous knockout strains with the CRISPR / Cas9 background removed can be obtained by selfing the F2 generation.

[0054] 3. Sequencing and identification of ZmNCED4 CRISPR strains

[0055] Total DNA was extracted from wild-type maize (ecotype LH244) and knockout plants (2300 and 2500), and PCR was used to detect the DNA sequence differences of the ZmNCED4 gene in the materials. The details are as follows:

[0056] The PCR amplification method is as follows:

[0057] Among them, the primer sequence for amplifying the ZmNCED4 gene is:

[0058] ZmNCED4RT-F1:5'-ATGGCGTCGTCCTCCATCTC-3'(Seq ID No.4)

[0059] ZmNCED4RT-R1: 5'-GAGGTCGTCCTCCGACATGGCGA-3' (Seq ID No. 5)

[0060] The reaction conditions of the above primers are as follows:

[0061] (1) Establishment of reaction system:

[0062] The PCR reaction system is shown in Table 1 below:

[0063] Table 1

[0064]

[0065] (2) Repeat three times, shake gently to mix, and perform the experiment using a Bio-Rad PCR instrument.

[0066] (3) Setting of reaction program:

[0067] The PCR reaction program is shown in Table 2 below:

[0068] Table 2

[0069]

[0070]

[0071] The band size was about 720 bp using 1% agarose gel electrophoresis at 200 V, and was sent to a sequencing company for sequencing.

[0072] The results of ZmNCED4 PCR detection and sequencing are as follows Figure 4 As shown in Figure 1, there are two different knockout forms of the ZmNCED4 gene: crispr1-2300 (SEQ ID NO.6) inserts a T after the 490bp of the CDS sequence of ZmNCED4 (SEQ ID NO.8) in the target site; crispr2-2700 (SEQ ID NO.7) is a deletion of 16 bases from the 477bp to the 492bp of the CDS sequence of ZmNCED4 (SEQ ID NO.8). Both knockout forms lead to frameshift mutations. The specific sequencing comparison results are shown in Figure 1. Figure 4 shown.

[0073] Example 2 Detection of the effect of knocking out the ZmNCED4 gene on the low temperature coping ability of plants

[0074] The dried seeds were soaked in water at 25°C for 12 h to allow them to swell, and then transferred to a constant temperature incubator at 10°C and 25°C for dark culture. The germination rates of the CK and ZmNCED4 mutant strains were counted every 24 h. Figure 2 and 3 As shown in the germination rate graph, under low temperature conditions, the germination rate of Cr1 and Cr2 seeds was much higher than that of the control group seeds.

[0075] Compared with the wild type, two independent mutant lines with different knockout forms obtained by CRISPR / Cas9 gene editing technology showed obvious cold-resistant phenotypes after low-temperature treatment, proving that the ZmNCED4 mutation is beneficial to improving corn's tolerance to low temperatures.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0077] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Claims

1. Application of ZmNCED4 protein or its encoding gene in any of the following aspects: (1) Regulate plant tolerance to low temperature stress; (2) Breeding plants with improved tolerance to low temperature stress; The amino acid sequence of the ZmNCED4 protein is: (1) the amino acid sequence shown in SEQ ID NO.1; or (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 is obtained by replacing, deleting or inserting one or more amino acid residues, and has the same functional protein. The nucleotide sequence of the ZmNCED4 protein encoding gene is: (1) the nucleotide sequence shown in SEQ ID NO.2, which encodes the ZmNCED4 protein shown in SEQ ID NO.1; or (2) a nucleotide sequence encoding a protein having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; or, (3) a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The application improves the low temperature stress tolerance of plants by inhibiting the expression of the coding gene of the ZmNCED4 protein, and is applied to the breeding of low temperature stress resistant plants.

3. A target sequence for editing the ZmNCED4 protein encoding gene according to claim 1, characterized in that: The target sequence is shown in SEQ ID NO.

3.

4. Primers for amplifying the ZmNCED4 protein encoding gene according to claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO.4-5.

5. A mutant of a gene encoding a ZmNCED4 protein, characterized in that: The nucleotide sequence of the mutant is shown in SEQ ID NO.6-7.

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