Corn ZmBARK1 sequence and application of encoding protein of corn ZmBARK1 sequence in regulation and control of low temperature resistance of corn in germination period

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a corn ZmBARK1 sequence and an encoding protein thereof in regulating and controlling low temperature resistance in a corn germination period. The relation between the ZmBARK1 gene and the low-temperature resistance of the corn in the germination period is found, and verification shows that after an encoding protein structural domain of the ZmBARK1 gene in the corn is deleted, the low-temperature resistance of the corn in the germination period is remarkably improved, which is of great significance in the field of cultivation of low-temperature-resistant corn varieties. The invention provides a new way for exploring a low-temperature-resistant new material in the corn germination period, lays a genetic material foundation for subsequent research, and provides a good information platform for low-temperature-resistant gene resource reserve in the corn germination period. The method is beneficial to the analysis of a low-temperature-resistant genetic mechanism in the plant germination period and the research of molecular breeding, and lays a theoretical foundation for the breeding of low-temperature-resistant varieties and the improvement of genetic quality of plants such as corn in the germination period.

Description

Technical Field

[0003] The present invention relates to the technical field of genetic engineering, and particularly to the application of the maize ZmBARK1 sequence and its encoded protein in regulating cold tolerance during maize germination. Background Art

[0005] Maize is native to tropical and subtropical regions and is one of the most important food crops in the world. It requires high temperatures to achieve optimal germination and is particularly sensitive to low temperatures. The entire growth period is vulnerable to low-temperature stress. Low-temperature stress during the germination period reduces the germination rate, during the seedling stage reduces the seedling activity, during the flowering stage delays the flowering time, and during the filling stage reduces the seed setting rate. Temperatures between 0 and 15 °C are most harmful during the germination process and have an adverse impact on 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.

[0006] To obtain more food, the sown area of food crops such as maize continues to expand, and the sowing area is gradually expanding to high-latitude and high-altitude regions. However, maize is a thermophilic crop with high temperature requirements, and the low temperatures in high-latitude and high-altitude regions severely restrict the growth and development of maize. China's maize sown area and total output rank second in the world. In 2024, China's maize sown area was approximately 450 million hectares, with a yield of approximately 6.6 tons per hectare. The northern spring maize region, as the main maize production area in the country, has a maize sown area accounting for more than 35% of the national sown area and a yield accounting for more than 40% of the national total output. Taking Heilongjiang as an example, the maize planting area is 93 million mu, accounting for about 1 / 6 of the country, and both the area, yield, and commercialization rate rank first in the country. However, due to the relatively high latitude and insufficient accumulated temperature in the Northeast region, low-temperature cold damage occurs from time to time, and low-temperature stress during the germination period is the most serious adversity disaster in the northern spring maize region. This causes the growth and development of maize to be delayed, the yield to be unstable, and the quality to be low. In severely cold damage years, the maize yield reduction can reach more than 15%. In 2024, the maize planting area in the northern spring maize region (i.e., the Northeast region) decreased by 3.72% compared with 2023. Especially in Heilongjiang region, the yield reduction caused by low temperature during the growing season in Heilongjiang Province can reach 25% to 30%. On the other hand, in order to increase economic benefits and obtain higher yields, farmers continuously increase the planting of medium- and late-maturing maize varieties, resulting in the gradual expansion of the planting boundaries of maize varieties with different maturity periods to areas with lower temperatures, indirectly increasing the risk of maize encountering low temperatures.

[0007] At present, low-temperature chilling injury during the germination period has become one of the main limiting factors for increasing maize yield in the spring maize region of northern China. Therefore, conducting research on the identification of genes related to low-temperature tolerance and their molecular mechanisms during maize germination is of great value for innovating low-temperature tolerance resources and cultivating maize varieties with strong low-temperature tolerance, ensuring the safety of food production in Northeast China and even the whole country. Summary of the Invention

[0009] To solve the problems existing in the prior art, the present invention conducted basic characteristic analysis and preliminary function verification on candidate genes associated with low-temperature stress during maize germination, and found that the expression level of ZmBARK1 is negatively correlated with low-temperature resistance during maize germination. After verification, it was determined that the ZmBARK1 gene is involved in the regulation of low-temperature resistance during maize germination, and this gene is related to the low-temperature resistance during maize germination. Specifically,

[0010] First, the present invention provides the application of ZmBARK1 protein or its coding gene, or a biological material containing ZmBARK1 protein or its coding gene in regulating low-temperature tolerance during plant germination.

[0011] Second, the present invention provides the application of ZmBARK1 protein or its coding gene, or a biological material containing ZmBARK1 protein or its coding gene in cultivating new plant varieties with low-temperature tolerance during plant germination or selecting plants with low-temperature resistance during plant germination.

[0012] Third, the present invention provides the application of ZmBARK1 protein or its coding gene, or a biological material containing ZmBARK1 protein or its coding gene in improving low-temperature tolerance germplasm resources during plant germination.

[0013] In some embodiments, the amino acid sequence of the ZmBARK1 protein is as shown in SEQ ID NO.3, or an amino acid sequence with a homology of more than 90% to the shown sequence and having the same protein function. More preferably, the homology is 95%, 96%, 97%, 98% or more than 99%.

[0014] In the specific implementation process, the amino acid sequence with a homology of more than 90% to the shown sequence and having the same protein function is: an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 and having the same protein function.

[0015] In some embodiments, the coding region nucleotide sequence of the ZmBARK1 protein coding gene is as shown in SEQ ID NO.2, or a nucleotide sequence with a homology of more than 90% to the shown sequence and encoding the same functional protein. More preferably, the homology is 95%, 96%, 97%, 98% or more than 99%.

[0016] In some embodiments, the full-length nucleotide sequence of the ZmBARK1 protein-coding gene is as shown in SEQ ID NO.1, or a nucleotide sequence having a homology of 90% or more with the shown sequence and encoding the same functional protein. More preferably, the homology is 95%, 96%, 97%, 98% or 99% or more.

[0017] In the specific implementation process, nucleotide sequences having a homology of 90% or more with the shown sequence and encoding the same functional protein include:

[0018] (1) A coding nucleotide sequence having the same protein function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0019] (2) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 under stringent conditions.

[0020] Fourthly, the present invention can provide a method for changing the cold resistance of plants during the germination period, including: regulating the expression of the ZmBARK1 gene in plants by means of transgenic, gene editing, hybridization, backcrossing, self-crossing or asexual reproduction.

[0021] In some embodiments, the cold resistance of plants during the germination period is improved by reducing the expression of ZmBARK1.

[0022] In some embodiments, the transgenic method includes: introducing a recombinant expression vector containing the ZmBARK1 gene into plants by using a plasmid, a plant virus vector, direct DNA transformation, microinjection, a gene gun, electroconductivity or an Agrobacterium-mediated method to obtain transgenic plant lines. The plasmid is preferably a Ti plasmid.

[0023] In some embodiments, the gene editing method includes: editing the ZmBARK1 gene by using DNA homologous recombination technology or CRISPR / Cas technology to obtain gene-edited plant lines. The CRISPR / Cas technology is preferably CRISPR / Cas9 technology.

[0024] In any of the above embodiments, the plant is any one of the following: a1) monocotyledonous plants; a2) plants of the order Poales; a3) plants of the family Poaceae; a4) plants of the genus Zea; a5) maize.

[0025] The biological materials described in the present invention include but are not limited to expression cassettes, vectors, or host cells.

[0026] The present invention has the following beneficial effects:

[0027] The present invention discovers the connection between the ZmBARK1 gene and the cold tolerance during the germination period of maize. After verification, it is found that by reducing the expression of the ZmBARK1 gene in maize and deleting its coding protein domain, the cold resistance of maize during the germination period is significantly improved, which has important significance in the field of cultivating cold-tolerant maize varieties.

[0028] The present invention provides a new way to explore new materials for cold tolerance during the germination period of maize, lays a genetic material foundation for subsequent research, and provides a good information platform for the reserve of cold tolerance gene resources during the germination period of maize.

[0029] The present invention contributes to the research on the analysis of the genetic mechanism of cold tolerance during the germination period of plants and molecular breeding, and lays a theoretical foundation for the breeding of cold-tolerant varieties and the improvement of genetic quality during the germination period of maize and other plants.

[0030] The present invention promotes the research on the genetic mechanism of plant resistance to low temperature and molecular breeding of abiotic stress resistance, and provides reliable materials and data support for the research on the molecular biological mechanism of enhancing the low temperature resistance of maize and other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Analysis of the expression pattern of the ZmBARK1 gene in maize embryos with different cold tolerance levels under low temperature treatment during the germination period provided in Example 1 of the present invention.

[0033] Figure 2 Results of the secondary structure analysis of the ZmBARK1 protein provided in Example 2 of the present invention; where h: α-helix structure; e: extended strand region; c: random coil; t: β-sheet.

[0034] Figure 3 Comparison of the ZmBARK1 protein with other IRAK subfamilies provided in Example 2 of the present invention; where: BAK1, CLV1 and SERK1 are from Arabidopsis thaliana, and IRAK2, IRAK3 and IRAK4 are from humans.

[0035] Figure 4 Results of the subcellular localization of the ZmBARK1 protein provided in Example 2 of the present invention.

[0036] Figure 5 Analysis of the flanking sequence of the ZmBARK1 gene provided in Example 2 of the present invention.

[0037] Figure 6Identification of the cold tolerance of the zmbark1 mutant provided in Example 3 of the present invention; wherein: a. Sequence analysis of the ZmBARK1 mutation site and the encoded nuclear genome fragment. b. Characteristics of the cold tolerance phenotype of zmbark1 seeds during germination. c. Phenotypic evaluation of WT and zmbark1 during 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.

[0038] Figure 7 Identification of the cold tolerance of the zmbark1 mutant provided in Example 4 of the present invention; wherein: a. Characteristics of the cold tolerance phenotype of zmbark1 at the seedling stage. b. Phenotypic evaluation of WT and zmbark1 at the seedling stage 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 manners

[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0041] In the following embodiments, the instrument equipment involved is conventional instrument equipment unless otherwise specified; the reagents involved are commercially available conventional reagents unless otherwise specified; the test methods involved are conventional methods unless otherwise specified.

[0042] Example 1 Analysis of the expression pattern of the ZmBARK1 gene

[0043] 1. Analysis of the expression pattern of the ZmBARK1 gene

[0044] In the present invention, healthy seeds of the highly cold-tolerant maize inbred line B73 and the low-temperature highly sensitive maize inbred line Mo17 were used as test materials, disinfected with 75% alcohol for 10 minutes, and then rinsed with sterile water for the germination test. The seeds were soaked at 25°C for 6 hours, placed on moist germination paper, and covered with another wet paper. For qRT-PCR analysis, maize embryos were collected at 1, 2, and 4 h after treatment at low temperature (10°C) and normal temperature (25°C). Each treatment was repeated 3 times biologically, and the 2 −△△CT method was used to evaluate the relative transcription level. The results are as Figure 1As shown in the figure, after 1 and 2 hours of treatment, the expression levels of ZmBARK1 in B73 and Mo17 both decreased, and there were extremely significant differences (P < 0.01). After 4 hours of treatment, the expression level of Mo17 increased, while the expression level of B73 decreased significantly (P < 0.01). The results indicate that under low-temperature treatment, the expression of ZmBARK1 in the highly low-temperature-tolerant maize inbred line B73 is lower than that in the highly low-temperature-sensitive maize inbred line Mo17.

[0045] Example 2 Analysis of the basic characteristics of the ZmBARK1 gene

[0046] 1. Secondary structure analysis of the ZmBARK1 protein

[0047] According to the amino acid sequence encoded by ZmBARK1 of the present invention, the secondary protein structure was predicted using the SOPMA online website. The results are as Figure 2 shown. The ZmBARK1 protein is mainly composed of random coils (58.36%), followed by α-helices (30.50%), and then by extended strand regions (11.14%), and there is no other secondary structure, indicating that the ZmBARK1 protein may have a stable spatial conformation based on the α-helix structure.

[0048] 2. Homologous alignment analysis of the ZmBARK1 protein

[0049] In the present invention, the protein sequence of ZmBARK1 and the protein sequences of other IRAK subfamilies were obtained through the NCBI and UniProt databases; among them: BAK1, CLV1, and SERK1 are from Arabidopsis thaliana, and IRAK2, IRAK3, and IRAK4 are from humans, and sequence alignment was performed using MEGA 11 and GeneDoc software. The results are as Figure 3 shown. Comparative protein sequence analysis shows that the ZmBARK1 protein has the highest similarity with BAK1 (SERK3, somatic embryogenesis receptor-like kinase 3, AT4G33430) in Arabidopsis thaliana in its conserved domain.

[0050] 3. Subcellular localization of the ZmBARK1 protein

[0051] The pCUB-eGFP-3×FLAG empty vector of the present invention was digested with BamH I restriction endonuclease, and the pCUB-eGFP-3×FLAG vector fragment was recovered by gel extraction. According to the coding region sequence of the ZmBARK1 gene (SEQ ID NO.2), homologous recombination primers as shown in SEQ ID NO.5 and SEQ ID NO.6 were designed, and the coding region of the ZmBARK1 gene (SEQ ID NO.2) was inserted into the pCUB-eGFP-3×FLAG vector, followed by transformation. Single colonies were picked and sequenced to construct the pCUB-ZmBARK1-eGFP-3×FLAG recombinant vector; the subcellular localization was determined by transfecting maize protoplasts, and the fluorescence signal was observed under a laser confocal microscope. The green fluorescence signal of the control sample transfected with the empty vector was present in 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 protein encoded by the ZmBARK1 gene was localized on the nucleus and plasma membrane ( Figure 4 ).

[0052] 4. Analysis of promoter elements of the ZmBARK1 gene

[0053] In the present invention, PlantCARE was used to analyze the 2.0 kb upstream region of the start codon of ZmBARK1 as SEQ ID NO.4 to identify the cis-acting elements in the flanking sequence of the gene. The results are as Figure 5 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 CAAT box, binding sites for MYB and G-box. These findings indicate that the promoter of ZmBARK1 may be a stress-inducible promoter.

[0054] Example 3 Identification of the cold tolerance of the zmbark1 mutant during germination

[0055] 1. Identification of the cold tolerance of the zmbark1 mutant during germination

[0056] To verify whether ZmBARK1 plays a real function in response to low temperature stress, an independent ZmBARK1 gene EMS mutant with the background of B73, named EMS4-1a088b, was purchased from the maize EMS-induced mutant library (http: / / maizeems.qlnu.edu.cn / ). This mutant was sold by the mutant library MEMD and was publicly available. According to the MEMD website, the mutation site of the mutant EMS4-1a088b is located in the second exon of ZmBARK1, causing the base G at the 174bp of the CDS sequence to mutate into A, resulting in premature termination of translation of the encoded protein and loss of function of this gene. This mutant was named zmbark1. First, the authenticity of the mutation site of this EMS mutant was identified. The total DNA of the leaves of zmbark1 plants at the two-leaf stage was extracted by the CTAB method in the present invention. Detection primers were designed according to the mutation site as shown in SEQ ID NO.7 and SEQ ID NO.8, and PCR amplification and sequencing were carried out to determine the successfully mutated zmbark1 mutant. Seeds were disinfected with 75% alcohol for 10 minutes and then rinsed with sterile water for germination test. The seeds were soaked at 25°C for 6 hours, placed on moist germination paper, and covered with another wet paper (50 seeds / replicate). For low temperature treatment, the seeds were incubated at 10°C for 31 days and then recovered at 15°C for 7 days. The control treatment was incubated at 25°C for 6 days. Traits at the germination stage were measured, and all measurements used the mean ± standard deviation of 3 independent experiments. Six indexes such as 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) were calculated. The results are as Figure 6 shown. Sequencing confirmed the presence of a single-base mutation (G mutated into A) in the second exon of ZmBARK1, resulting in the replacement of tryptophan at the 58th position by a stop codon. This mutation led to the deletion of 320 amino acids of the ZmBARK1 protein, including the key conserved domain, indicating that ZmBARK1 could not function completely. The phenotypic evaluation under low temperature stress at the germination stage showed that compared with the wild type (WT) B73, the germination and growth of the zmbark1 mutant were significantly enhanced, mainly manifested in that the RGR, RSL, RRL, RRSA, and RSVI of zmbark1 were significantly higher than those of WT (P < 0.01). These results indicate that ZmBARK1 is a negative regulator of low temperature tolerance at the germination stage, and the deletion of the domain in the mutant enhances the germination and growth of maize under low temperature stress.

[0057] Example 4 Identification of the low temperature tolerance of the zmbark1 mutant at the seedling stage

[0058] 1. Identification of the low temperature tolerance of the zmbark1 mutant at the seedling stage

[0059] The 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 to take photos and collect phenotypic data. The following were calculated: RSL: relative shoot length, RSFW: relative shoot fresh weight, RSDW: relative shoot dry weight, RRL: relative root length, RRV: relative root volume, RRFW: relative root fresh weight, and RRDW: relative root dry weight. The results are as Figure 7 shown. Under the growth conditions of 25°C for 4 days, there was no significant difference in the growth of seedlings between WT and zmbark1. Under the growth conditions of 6°C for 7 days, compared with WT, the seedlings of zmbark1 were more robust and the leaf damage induced by low temperature was reduced. 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 low-temperature tolerance at the seedling stage. The deletion of this mutant domain enhances the growth of maize seedlings under low-temperature stress, further indicating its key role in the response to low-temperature stress during plant germination and the seedling stage.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements 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. 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.

2. Use of ZmBARK1 protein or its encoding gene, or biological materials containing ZmBARK1 protein or its encoding gene, in cultivating new varieties of plants that are resistant to low temperatures during plant germination or in breeding plants that are resistant to low temperatures during plant germination.

3. Application of ZmBARK1 protein or its encoding gene, or biological materials containing ZmBARK1 protein or its encoding gene in improving germplasm resources that are resistant to low temperatures during plant germination.

4. The use according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the ZmBARK1 protein is shown in SEQ ID NO. 3, or an amino acid sequence with a homology of more than 90% to the shown sequence and having the same protein function.

5. The use according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the coding region of the ZmBARK1 protein encoding gene is shown in SEQ ID NO. 2, or a nucleotide sequence having a homology of more than 90% with the shown sequence and encoding a protein with the same function.

6. The use according to any one of claims 1 to 3, characterized in that: The full-length nucleotide sequence of the ZmBARK1 protein encoding gene is shown in SEQ ID NO.1, or a nucleotide sequence having a homology of more than 90% with the shown sequence and encoding a protein with the same function.

7. A method for changing the low temperature resistance of plants during the germination period, characterized in that: include: The expression of plant ZmBARK1 gene can be regulated through transgenic technology, gene editing, hybridization, backcrossing, selfing or asexual reproduction.

8. The method according to claim 7, characterized in that The transgenic method includes: using plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to introduce the recombinant expression vector containing the ZmBARK1 gene into plants to obtain transgenic plant strains.

9. The method according to claim 7, characterized in that: The gene editing method includes: using DNA homologous recombination technology or CRISPR / Cas technology to edit the ZmBARK1 gene to obtain a gene-edited plant strain.

10. The use according to any one of claims 1 to 6, or the method according to any one of claims 7 to 9, characterized in that: The plant is any one of the following: a1) monocotyledonous plants; a2) plants of the order Poaceae; a3) plants of the family Poaceae; a4) plants of the genus Zea; and a5) corn.

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