Application of ZmNCED4 protein and its coding gene in regulating plant tolerance to low temperature stress

Abstract

The application relates to application of ZmNCED4 protein and a coding gene thereof in regulating low-temperature stress tolerance of plants. The ZmNCED4 protein and the coding gene thereof have a significant influence on low-temperature stress tolerance of plants (especially corn), and the low-temperature stress tolerance of corn can be improved by inhibiting expression of the coding gene of the ZmNCED4 protein. The application provides a good germplasm resource for breeding new low-temperature-resistant plant varieties, and lays a theoretical foundation for studying a mechanism of plant response to adverse environment signals and a molecular mechanism of tolerance to adverse environments.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the ZmNCED4 protein and its encoding gene in regulating plant tolerance to low-temperature stress. Background Technology

[0002] Plants that grow sessilely in nature inevitably suffer from various abiotic stresses. Low-temperature stress, as one of the major external environmental factors, not only affects plant growth and development and geographical distribution but also severely impacts crop yield. Therefore, studying the physiological and biochemical changes and molecular mechanisms of plant responses to low-temperature stress, and identifying key genes regulating these responses, can provide valuable theoretical support for breeding cold-resistant crop varieties to improve crop yield. Maize (Zea mays), belonging to the genus Zea in the Poaceae family, is an important food and forage crop. Low-temperature stress after sowing reduces germination rate and delays emergence, thus affecting seedling uniformity and viability. Low temperatures restrict seedling growth and development, affecting photosynthetic intensity and consequently impacting yield. Improving maize's tolerance to low-temperature stress through genetic engineering is of great significance for maintaining and increasing maize yield.

[0003] Biosynthesis of ABA is a crucial pathway affecting its content. ABA biosynthesis can be divided into two pathways: one is the terpene pathway, also known as the C15 direct pathway, where farnesyl pyrophosphate (FPP) directly forms 15-carbon ABA through cyclization and oxidation. The other is the carotenoid pathway, also known as the C40 indirect pathway, where β-carotene polymerized from mevalonic acid (MVA) is catalyzed by zeaxanthin cyclooxygenase (ZEP) encoded by AtABA1 / ZEP to form zeaxanthin, which is then oxidized by 9-cis-epoxycarotenoid dioxygenases (NCEDs) to form xanthoaldehyde. NCEDs are considered key enzymes in ABA synthesis. Subsequently, xanthoaldehyde is converted into ABA in the cytoplasm through the catalysis of short-chain alcohol dehydrogenases (encoded by AtABA2) and aldehyde oxidases (AAO3), and activation by molybdenum cofactor sulfonase (MoCo) encoded by AtABA3. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of the ZmNCED4 protein and its encoding gene in regulating plant tolerance to low-temperature stress. This invention improves the low-temperature stress tolerance of maize by inhibiting the expression of the ZmNCED4 protein encoding gene in maize.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Applications of ZmNCED4 protein, or its encoding gene, or its repressor, or biological materials containing its encoding gene or repressor in any of the following aspects:

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

[0008] (2) Select and breed plants with improved tolerance to low temperature stress;

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

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

[0011] (2) 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.1.

[0012] It should be understood that, taking into account the degeneracy of codons and the codon preferences of different species, those skilled in the art may use codons suitable for the expression of a particular species as needed.

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

[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 with the same function obtained by substituting, deleting, or inserting one or more nucleotides into 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 strict conditions.

[0017] The maize ZmNCED4 gene, numbered Zm00001d007876 in the maize genome database, consists of 1986 bases. Its reading frame (RF) extends from position 151 to position 1878 at the 5' end. This RRF consists of a single exon.

[0018] In some implementations, the biomaterial is an expression cassette, vector, host cell, or recombinant bacteria.

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

[0020] Based on the above plan,

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

[0022] Based on the above plan,

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

[0024] In practice, the expression or overexpression methods can be transgenic, hybrid, backcross, self-pollination, or asexual reproduction. The transgenic method includes introducing a recombinant expression vector containing the coding gene of ZmNCED4 protein into maize using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrocoagulation, or Agrobacterium-mediated methods to obtain transgenic maize lines.

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

[0026] Primers used to amplify the ZmNCED4 protein-encoding gene described above are characterized by:

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

[0028] A mutant of the ZmNCED4 protein-encoding gene, characterized by:

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

[0030] The beneficial effects of the ZmNCED4 protein and its encoding gene described in this invention in regulating plant tolerance to low-temperature stress are as follows:

[0031] The ZmNCED4 protein and its encoding gene have a significant impact on the low-temperature stress tolerance of plants, especially maize. Specifically, inhibiting the expression of the ZmNCED4 protein encoding gene in maize can improve its low-temperature stress tolerance. This application provides excellent germplasm resources for breeding new low-temperature tolerant plant varieties and lays a theoretical foundation for studying the mechanisms of plant responses to adverse signals and the molecular mechanisms of tolerance to adverse environments. Attached Figure Description

[0032] The present invention includes the following figures:

[0033] Figure 1The image shows the seed germination of mutant lines cristr1-2300 and cristr2-2500 in Example 1 of this invention under ambient temperature and low temperature treatment; where CK represents wild-type maize lines, and Cr1 and Cr2 represent mutant lines cristr1-2300 and cristr2-2500, respectively.

[0034] Figure 2 This is a statistical chart showing the seed germination rate of the mutant lines cristr1-2300 and cristr2-2500 in Example 2 of the present invention at room temperature; where CK represents wild-type maize plants, and Cr1 and Cr2 represent the mutant lines cristr1-2300 and cristr2-2500, respectively.

[0035] Figure 3 This is a statistical chart showing the seed germination rate of the mutant lines cristr1-2300 and cristr2-2500 under low-temperature treatment in Example 2 of the present invention; where CK represents wild-type maize plants, and Cr1 and Cr2 represent the mutant lines cristr1-2300 and cristr2-2500, respectively.

[0036] Figure 4 The specific sequencing comparison results are shown for the ZmNCED4 gene knockout strain in Example 1 of this invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings.

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

[0039] In the following examples, the pBSK vector is a commonly used cloning vector and is commercially available; the pBCXUN vector (which uses the commercial vector pCAMBIA1300 as its backbone, replacing the hygromycin resistance gene hpt with the herbicide resistance gene barM; and simultaneously cloning the promoter of the maize ubiquitin gene Ubi into the vector via enzyme digestion and ligation to drive the transcription of downstream overexpressed genes) uses the Ubi promoter to drive the expression of the NCED4 gene; the Agrobacterium EHA105 strain was purchased from Shanghai Weidi Biotechnology Co., Ltd. (product number: AC1010S).

[0040] The main reagents used in the following examples were: various restriction endonucleases, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, and KOD were purchased from NEB, Toyobo, and other biotechnology companies; dNTPs were purchased from Genestar; plasmid miniprep kits and agarose gel extraction kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampin (Rif), and other antibiotics, as well as glucose, BSA, LBMedium, etc., were purchased from Sigma, Bio-Rad, and other companies; reagents used for real-time quantitative PCR were purchased from TaKaRa; and all other chemical reagents used in the examples were imported or domestically produced analytical grade reagents.

[0041] The primers used in the examples were synthesized by Huada Biotechnology Co., Ltd. and subjected to relevant sequencing.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21), or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] Example 1: Phenotypic Verification of the Maize ZmNCED4 Mutant

[0044] I. Construction of ZmNCED4 mutant

[0045] 1. Construction of recombinant expression vectors

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

[0047] The designed target was synthesized from single-stranded to double-stranded form using touch-down PCR, and this linker fragment was then ligated into the pBUE411 vector.

[0048] The obtained pBUE411 vector was digested with the restriction endonuclease Bsa1, and then ligated using Soluton 1 ligase to obtain a recombinant plasmid. The recombinant plasmid was then sequenced, and the recombinant plasmid that was sequenced to be located between the Bsa1 restriction sites in the pBUE411 vector 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 step 1 was introduced into Agrobacterium GV3101 competent cells via freeze-thaw method. The obtained recombinant Agrobacterium pBUE411-gnced4 was transformed into wild-type maize (LH244 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 (screened for bar resistance, the process was completed by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University) was also performed.

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

[0053] 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.

[0054] 3. Identification of ZmNCED4 CRISPR line by sequencing

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

[0056] The PCR amplification method is as follows:

[0057] The primer sequences for amplifying the ZmNCED4 gene are as follows:

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

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

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

[0061] (1) Establishment of the reaction system:

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

[0063] Table 1

[0064]

[0065] (2) Three replicates were made, and the mixture was shaken to mix. The experiment was performed using a Bio-Rad PCR instrument.

[0066] (3) Setting the reaction procedure:

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

[0068] Table 2

[0069]

[0070]

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

[0072] ZmNCED4 PCR detection sequencing results are as follows Figure 4 As shown, the ZmNCED4 gene has two different knockout forms: crispr1-2300 (SEQ ID NO.6) is obtained by inserting a T after the 490bp of the CDS sequence (SEQ ID NO.8) of ZmNCED4 at the target site; crispr2-2700 (SEQ ID NO.7) is obtained by deleting 16 bases from the 477bp to the 492bp of the CDS sequence (SEQ ID NO.8) of ZmNCED4. Both knockout forms result in frameshift mutations. Specific sequencing alignment results are shown below. Figure 4 As shown.

[0073] Example 2: Detection of the effect of ZmNCED4 gene knockout on plant low-temperature coping ability

[0074] The dried seeds were soaked in water at 25℃ for 12 hours to absorb moisture, and then transferred to constant temperature incubators at 10℃ and 25℃ respectively for dark culture. The germination rate of the CK and ZmNCED4 mutant lines was counted every 24 hours. Figure 2 and 3 As shown in the germination rate diagram, 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 through CRISPR / Cas9 gene editing technology showed obvious low-temperature tolerance phenotypes after low-temperature treatment, demonstrating that the ZmNCED4 mutation is beneficial to improving the low-temperature tolerance of maize.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0077] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. Applications of ZmNCED4 protein or its encoding gene in any of the following aspects: (1) Regulating the germination rate of corn at low temperatures; (2) Selecting and breeding maize varieties with improved germination rate at low temperatures; The application improves the low-temperature germination rate of maize by inhibiting the expression of the gene encoding the ZmNCED4 protein, and is applied to breeding maize with improved low-temperature germination rate. The amino acid sequence of the ZmNCED4 protein is the amino acid sequence shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the ZmNCED4 protein is the nucleotide sequence shown in SEQ ID NO.2, which encodes the ZmNCED4 protein shown in SEQ ID NO.1.

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