Corn ZmBARK1 sequence and its encoded protein in regulating corn germination stage low temperature tolerance

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a maize ZmBARK1 sequence and application of a protein coded by the ZmBARK1 sequence in regulating low-temperature tolerance of maize in a germination stage. The present application finds a connection between the ZmBARK1 gene and the low-temperature tolerance of maize in the germination stage. After verification, it is found that the low-temperature tolerance of maize in the germination stage is significantly improved after the coding protein domain of the ZmBARK1 gene is deleted in the maize, which has important significance in the field of cultivating low-temperature tolerant maize varieties. The present application provides a new way for exploring new low-temperature tolerant materials of maize in the germination stage, lays a genetic material foundation for subsequent research, and provides a good information platform for low-temperature tolerant gene resources of maize in the germination stage. The present application is helpful for analyzing the genetic mechanism of low-temperature tolerance of plants in the germination stage and the research of molecular breeding, and lays a theoretical foundation for breeding and improving the genetic quality of low-temperature tolerant varieties of maize and other plants in the germination stage.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the maize ZmBARK1 sequence and its encoded protein in regulating low-temperature tolerance during maize germination. Background Technology

[0002] Maize, native to tropical and subtropical regions, is one of the world's most important food crops. It requires high temperatures for optimal germination and is particularly sensitive to low temperatures. Throughout its growth cycle, it is susceptible to low-temperature stress. Low-temperature stress during germination reduces germination rate, during the seedling stage it decreases seedling viability, during flowering it delays flowering time, and during grain filling it reduces seed setting rate. Temperatures between 0 and 15°C are most detrimental during germination, adversely affecting subsequent growth stages, including leaf development, seedling photosynthetic efficiency, physiological and biochemical changes, and root development. Low-temperature stress also leads to significant morphological differences between tolerant and sensitive maize varieties. Globally, this environmental constraint poses a major challenge to agricultural production.

[0003] In order to obtain more food, the planting area of ​​grain crops such as corn has continued to expand, and the planting area is also gradually expanding to high latitude and high altitude regions. However, corn is a warm-loving crop with high temperature requirements, and the low temperature in high latitude and high altitude regions seriously restricts the growth and development of corn.

[0004] Identifying genes related to low-temperature tolerance during maize germination and studying their molecular mechanisms are of great value for the innovation of low-temperature tolerant resources and the breeding of maize varieties with strong low-temperature tolerance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention conducted basic characteristic analysis and preliminary functional verification of candidate genes associated with low-temperature stress during maize germination. It was found that the expression level of ZmBARK1 was negatively correlated with low-temperature resistance during maize germination. After verification, it was confirmed that the ZmBARK1 gene participates in the regulation of low-temperature resistance during maize germination, and this gene is associated with low-temperature resistance during maize germination. Specifically,

[0006] In a first aspect, the present invention provides the application of ZmBARK1 protein or its encoding gene, or biological materials containing ZmBARK1 protein or its encoding gene, in regulating the low-temperature tolerance of plants during germination.

[0007] Secondly, this invention provides the application of ZmBARK1 protein or its encoding gene, or biological materials containing ZmBARK1 protein or its encoding gene, in the cultivation of new plant varieties resistant to low temperatures during the germination period or in the selection of plants resistant to low temperatures during the germination period.

[0008] Thirdly, this invention provides the application of ZmBARK1 protein or its encoding gene, or biological materials containing ZmBARK1 protein or its encoding gene, in the improvement of low-temperature resistant germplasm resources during plant germination.

[0009] In some embodiments, the amino acid sequence of the ZmBARK1 protein is as shown in SEQ ID NO.3, or an amino acid sequence that has more than 90% homology with the shown sequence and has the same protein function. More preferably, the homology is 95%, 96%, 97%, 98%, or 99% or more.

[0010] In the specific implementation process, the amino acid sequence that has more than 90% homology with the sequence shown and has the same protein function is: the amino acid sequence with the same protein function obtained by substituting, deleting or inserting one or more amino acid residues from the amino acid sequence shown in SEQ ID NO.3.

[0011] In some embodiments, the coding region nucleotide sequence of the ZmBARK1 protein encoding gene is as shown in SEQ ID NO. 2, or a nucleotide sequence that has more than 90% homology with the shown sequence and encodes a protein with the same function. More preferably, the homology is 95%, 96%, 97%, 98%, or 99% or more.

[0012] In some embodiments, the full-length nucleotide sequence of the ZmBARK1 protein-encoding gene is as shown in SEQ ID NO.1, or a nucleotide sequence that has more than 90% homology with the shown sequence and encodes a protein with the same function. More preferably, the homology is 95%, 96%, 97%, 98%, or 99% or more.

[0013] In the specific implementation process, nucleotide sequences with more than 90% homology to the sequence shown and encoding proteins with the same function include:

[0014] (1) A nucleotide sequence encoding the same protein function obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0015] (2) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 under strict conditions.

[0016] Fourthly, the present invention can provide a method for altering the resistance of plants to low temperatures during germination, including: regulating the expression of the plant ZmBARK1 gene through transgenic methods, gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] In some implementations, reducing the expression of ZmBARK1 enhances the plant's resistance to low temperatures during germination.

[0018] In some embodiments, the transgenic method includes: introducing a recombinant expression vector containing the ZmBARK1 gene into a plant using a plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation to obtain transgenic plant lines. The plasmid is preferably a Ti plasmid.

[0019] In some embodiments, the gene editing method includes: editing the ZmBARK1 gene using DNA homologous recombination technology or CRISPR / Cas technology to obtain gene-edited plant lines. The CRISPR / Cas technology is preferably CRISPR / Cas9 technology.

[0020] In any of the above embodiments, the plant is any of the following: a1) a monocotyledonous plant; a2) a grass; a3) a grass; a4) a maize; a5) maize.

[0021] The biomaterials described in this invention include, but are not limited to, expression cassettes, vectors, or host cells.

[0022] The present invention has the following beneficial effects:

[0023] This invention has discovered the link between the ZmBARK1 gene and the low-temperature tolerance of maize during germination. Verification showed that reducing the expression of the ZmBARK1 gene in maize and deleting its encoded protein domain significantly improved the low-temperature resistance of germinating maize, which is of great significance in the field of breeding low-temperature tolerant maize varieties.

[0024] This invention provides a new approach to exploring new materials resistant to low temperatures during maize germination, lays the genetic material foundation for subsequent research, and provides a good information platform for the reserve of low-temperature resistant gene resources during maize germination.

[0025] This invention contributes to the analysis of the genetic mechanism of low-temperature tolerance during plant germination and to the research on molecular breeding, laying a theoretical foundation for the breeding of low-temperature tolerant varieties and the improvement of genetic quality of plants such as maize during germination.

[0026] This invention advances molecular breeding research on the genetic mechanisms of plant resistance to low temperatures and resistance to abiotic stresses, and provides reliable materials and data support for the study of molecular biological mechanisms to enhance the low-temperature resistance of plants such as maize. Attached Figure Description

[0027] Figure 1 Analysis of the expression pattern of ZmBARK1 gene in maize embryos with different low-temperature tolerance levels under low-temperature treatment during germination, as provided in Example 1 of this invention.

[0028] Figure 2 The secondary structure analysis results of the ZmBARK1 protein provided in Example 2 of the present invention are shown; where h: α-helix structure; e: extended chain region; c: random coil; t: β-sheet.

[0029] Figure 3 This is a comparison of the ZmBARK1 protein provided in Example 2 of the present invention with other IRAK subfamilies; wherein: BAK1, CLV1 and SERK1 are from Arabidopsis thaliana, and IRAK2, IRAK3 and IRAK4 are from humans.

[0030] Figure 4 The subcellular localization results of the ZmBARK1 protein provided in Example 2 of this invention.

[0031] Figure 5 Analysis of the flanking sequences of the ZmBARK1 gene provided in Example 2 of this invention.

[0032] Figure 6 This invention provides an example of identifying the low-temperature tolerance of the zmbark1 mutant during germination, as provided in Example 3. The identification includes: a. Sequence analysis of the ZmBARK1 mutation site and its encoded nuclear genome fragment; b. Characteristics of the low-temperature tolerance phenotype during zmbark1 seed germination; c. Phenotypic evaluation of WT and zmbark1 germination under low-temperature stress. RGR: Relative germination rate, RSL: Relative shoot length, RRL: Relative root length, RRSA: Relative root surface area, RRV: Relative root volume, RSVI: Relative simple vigor index.

[0033] Figure 7 This invention provides an example of identifying the low-temperature tolerance of the zmbark1 mutant during the seedling stage, as provided in Example 4. Specifically: a. Characteristics of the low-temperature tolerance phenotype of zmbark1 seedlings; b. Phenotypic evaluation of WT and zmbark1 seedlings under low-temperature stress. RSL: Relative seedling length, RSFW: Relative seedling fresh weight, RSDW: Relative seedling dry weight, RRL: Relative root length, RRV: Relative root volume, RRFW: Relative root fresh weight, RRDW: Relative root dry weight. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods unless otherwise specified.

[0036] Example 1: Analysis of ZmBARK1 gene expression patterns

[0037] 1. Analysis of ZmBARK1 gene expression patterns

[0038] This invention uses healthy seeds of the high-temperature-tolerant maize inbred line B73 and the low-temperature-sensitive maize inbred line Mo17 as test materials. Germination tests were conducted by disinfecting the seeds with 75% alcohol for 10 minutes and then rinsing them with sterile water. The seeds were soaked at 25°C for 6 hours, placed on moist germination paper, and covered with another moist paper. For qRT-PCR analysis, maize embryos were collected at 1, 2, and 4 hours after low-temperature (10°C) and room-temperature (25°C) treatments. Each treatment was performed in triplicate, using 2... −△△CT The relative transcriptional level was assessed using a method. Results are as follows: Figure 1 As shown, the expression levels of ZmBARK1 in both B73 and Mo17 decreased significantly after 1 and 2 hours of treatment (P < 0.01), while the expression level of Mo17 increased and the expression level of B73 decreased significantly after 4 hours of treatment (P < 0.01). These results indicate that under low-temperature treatment, the expression of ZmBARK1 in the high-temperature-tolerant maize inbred line B73 was lower than that in the low-temperature-sensitive maize inbred line Mo17.

[0039] Example 2: Analysis of the basic characteristics of the ZmBARK1 gene

[0040] 1. Secondary structure analysis of ZmBARK1 protein

[0041] This invention uses the SOPMA online website to predict secondary protein structures based on the amino acid sequence encoded by ZmBARK1. The results are as follows: Figure 2 As shown, the ZmBARK1 protein is mainly composed of random coils (58.36%), followed by α-helices (30.50%), and then extended strand regions (11.14%), with no other secondary structures. This suggests that the ZmBARK1 protein may be a stable spatial conformation based on the α-helix structure.

[0042] 2. Homology comparison analysis of ZmBARK1 protein

[0043] This invention obtained the protein sequence of ZmBARK1 and other IRAK subfamily proteins from the NCBI and UniProt databases; among them, BAK1, CLV1, and SERK1 are from Arabidopsis thaliana, and IRAK2, IRAK3, and IRAK4 are from humans. Sequence alignment was performed using MEGA 11 and GeneDoc software. The results are as follows: Figure 3 As shown, comparative protein sequence analysis revealed that the ZmBARK1 protein has the highest similarity in its conserved domain to BAK1 (SERK3, somatic embryogenesis receptor-like kinase 3, AT4G33430) in Arabidopsis thaliana.

[0044] 3. Subcellular localization of ZmBARK1 protein

[0045] This invention involves digesting the empty pCUB-eGFP-3×FLAG vector with BamHI restriction endonuclease and recovering the pCUB-eGFP-3×FLAG vector fragment using a gel. Based on the ZmBARK1 gene coding region sequence (SEQ ID NO.2), homologous recombination primers as shown in SEQ ID NO.5 and SEQ ID NO.6 were designed to insert the ZmBARK1 gene coding region (SEQ ID NO.2) into the pCUB-eGFP-3×FLAG vector. Transformation was performed, single colonies were picked, and sequencing was conducted to construct the pCUB-ZmBARK1-eGFP-3×FLAG recombinant vector. Subcellular localization was determined by transfecting maize protoplasts. Fluorescence signals were observed using laser confocal microscopy. The green fluorescence signal of the control group transformed with the empty vector was present within the maize protoplasts, while the green fluorescence signal of the ZmBARK1 fusion protein appeared in the nucleus and plasma membrane regions of the protoplasts, indicating that the ZmBARK1 gene-encoded protein is located in the nucleus and plasma membrane. Figure 4 ).

[0046] 4. Promoter element analysis of the ZmBARK1 gene

[0047] This invention identifies cis-acting elements in gene flanking sequences by using PlantCARE analysis of a 2.0 kb upstream region of the ZmBARK1 start codon, as shown in SEQ ID NO. 4. The results are as follows: Figure 5 As shown, promoter analysis using PlantCARE identified stress-responsive elements in the ZmBARK1 promoter, such as ABRE, SA, MEJA, and auxin-responsive elements, as well as regulatory elements such as the binding sites of the CAAT box, MYB, and G-box. These findings suggest that the ZmBARK1 promoter may be a stress-inducible promoter.

[0048] Example 3: Identification of the low-temperature tolerance of the zmbark1 mutant during germination.

[0049] 1. Identification of low-temperature tolerance of zmbark1 mutant during germination period

[0050] To verify whether ZmBARK1 functions effectively under low-temperature stress, an independent ZmBARK1 gene EMS mutant, EMS4-1a088b, with a B73 background, was purchased from the maize EMS-induced mutant library (http: / / maizeems.qlnu.edu.cn / ). This mutant is sold by the MEMD mutant library and is publicly available. According to the MEMD website, the mutation site of mutant EMS4-1a088b is located on the second exon of ZmBARK1, causing a G-to-A mutation at 174 bp of the CDS sequence, resulting in premature termination of translation of the encoded protein and loss of gene function. This mutant was named zmbark1. First, the authenticity and effectiveness of the EMS mutant were determined. Total DNA was extracted from two-leaf stages of zmbark1 plants using the CTAB method. Primers were designed based on the mutation site, as shown in SEQ ID NO.7 and SEQ ID NO.8, for PCR amplification and sequencing to identify the successfully mutated zmbark1 mutant. Seeds were disinfected with 75% alcohol for 10 minutes, then rinsed with sterile water for germination tests. Seeds were soaked at 25°C for 6 hours, placed on moistened germination paper, and covered with another moistened paper (50 seeds / replication). For the low-temperature treatment, seeds were incubated at 10°C for 31 days, then restored at 15°C for 7 days. The control treatment was incubated at 25°C for 6 days. Germination traits were measured, and all measurements were expressed as the mean ± standard deviation of three independent experiments. Six indices were calculated: relative germination rate (RGR), relative shoot length (RSL), relative root length (RRL), relative root surface area (RRSA), relative root volume (RRV), and relative simple vigor index (RSVI). Results are as follows: Figure 6 As shown, sequencing confirmed a single-base mutation (G to A) in exon 2 of ZmBARK1, resulting in the replacement of the stop codon with tryptophan at position 58. This mutation leads to a 320-amino acid deletion in the ZmBARK1 protein, including a key conserved domain, indicating that ZmBARK1 cannot function fully. Phenotypic assessment under germination-stage low-temperature stress showed that compared with wild-type (WT) B73, the zmbark1 mutant exhibited significantly enhanced germination and growth, mainly manifested in significantly higher RGR, RSL, RRL, RRSA, and RSVI values ​​in zmbark1 compared to WT (P < 0.01). These results indicate that ZmBARK1 is a negative regulator of germination-stage low-temperature tolerance, and the domain deletion in the mutant enhanced germination and growth of maize under low-temperature stress.

[0051] Example 4: Identification of the low-temperature tolerance of the zmbark1 mutant during the seedling stage

[0052] 1. Identification of the low-temperature tolerance of the zmbark1 mutant during the seedling stage

[0053] Zmbark1 mutant seedlings were grown in sterile sand at 25°C. When they had two fully expanded true leaves, they were treated at 6°C for 7 days (16 h light / 8 h dark) and at 25°C for 4 days (16 h light / 8 h dark), respectively. Three independent biological replicates were selected, photographs were taken, and phenotypic data were collected. RSL (relative seedling length), RSFW (relative seedling fresh weight), RSDW (relative seedling dry weight), RRL (relative root length), RRV (relative root volume), RRFW (relative root fresh weight), and RRDW (relative root dry weight) were calculated. Results are as follows: Figure 7 As shown, under 25℃ and 4-day growth conditions, there was no significant difference in seedling growth between WT and zmbark1. Under 6℃ and 7-day growth conditions, zmbark1 seedlings were more robust than WT seedlings, with reduced low-temperature-induced leaf damage. Except for RRFW, other traits of zmbark1, such as RSL, RSFW, RSDW, RRL, RRV, and RRDW, were significantly higher than those of WT (P < 0.01). These findings indicate that ZmBARK1 is a negative regulator of seedling low-temperature tolerance, and the deletion of this mutant domain enhanced the growth of maize seedlings under low-temperature stress, further demonstrating its crucial role in low-temperature stress response during plant germination and seedling stages.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of ZmBARK1 protein or its coding gene, or biological material containing ZmBARK1 protein or its coding gene in regulating low temperature tolerance of plants in the germination stage, characterized in that, The application relates to a method for improving the resistance of a plant to low temperature in the germination stage by reducing the expression of a ZmBARK1 gene; the amino acid sequence of the ZmBARK1 protein is shown as SEQ ID NO.

3. The coding region nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 2; the full-length nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 1; and the plant is corn.

2. Use of ZmBARK1 protein or its coding gene, or biological material containing ZmBARK1 protein or its coding gene in breeding new varieties of plants resistant to low temperature at germination stage or in selecting plants resistant to low temperature at germination stage, characterized in that, The application relates to a method for improving the resistance of a plant to low temperature in the germination stage by reducing the expression of a ZmBARK1 gene; the amino acid sequence of the ZmBARK1 protein is shown as SEQ ID NO.

3. The coding region nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 2; the full-length nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 1; and the plant is corn.

3. Application of ZmBARK1 protein or its coding gene or biological material containing ZmBARK1 protein or its coding gene in improvement of low-temperature tolerance germplasm resources in plant germination stage, characterized in that, The application relates to a method for improving the resistance of a plant to low temperature in the germination stage by reducing the expression of a ZmBARK1 gene; the amino acid sequence of the ZmBARK1 protein is shown as SEQ ID NO.

3. The coding region nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 2; the full-length nucleotide sequence of the ZmBARK1 protein coding gene is shown as SEQ ID NO. 1; and the plant is corn.

4. A method of altering the performance of a plant against low temperature during the germination stage of the plant, characterized by, The application relates to a method for improving the resistance of a plant to low temperature in the germination stage by reducing the expression of a ZmBARK1 gene; the coding region nucleotide sequence of the ZmBARK1 gene is shown as SEQ ID NO. 2; the full-length nucleotide sequence of the ZmBARK1 gene is shown as SEQ ID NO. 1; and the plant is corn.

5. The method of claim 4, wherein, The ZmBARK1 gene is edited by using a DNA homologous recombination technology or a CRISPR / Cas technology to obtain a gene editing plant strain.

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