Application of the maize pseudorhombic protein ZmDerlin3 gene and its encoded protein in plant heat tolerance breeding

Abstract

This invention discloses maize pseudorhomboid protein. ZmDerlin3 The application of genes and their encoded proteins in heat-resistant plant breeding belongs to the field of genetic engineering technology. This invention clones a maize pseudo-rhomboid protein. ZmDerlin3 The gene, whose nucleotide sequence is shown in SEQ ID NO.1 and whose amino acid sequence is shown in SEQ ID NO.2, provides a maize pseudorhomboid protein. ZmDerlin3 Application of genes and their encoded proteins in plant heat tolerance breeding. Heat shock experiments show that overexpression... ZmDerlin3 The maize seedlings of the wild type showed significantly better growth phenotypes than those of the wild type. ZmDerlin3 The gene positively regulates the heat tolerance of maize. The gene provided by this invention has important application value for breeding new high-yielding and heat-tolerant maize varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a type of corn. ZmDerlin3 Application of genes in heat-resistant plant breeding. Background Technology

[0002] Maize is one of the world's most widely cultivated and important crops, also used for animal feed and industrial production. Maize is a crucial crop supporting my country's agricultural and economic development, but my country's average maize yield is only about 60% of that of the United States. In recent years, global warming has led to frequent extreme heat events, subjecting maize to severe heat stress throughout its entire growth cycle, causing field heat damage such as leaf scorch, empty stalks, barren tips, short skirts, and missing kernels. From 2016 to 2023, high-temperature-induced poor maize grain filling became a common occurrence and showed an increasingly severe trend, seriously threatening my country's maize yield, food security, and national economic development. Analyzing the heat protection mechanisms of maize under high-temperature stress and identifying key heat-resistant genes will provide a theoretical basis and technical support for the genetic improvement of crop heat resistance and bio-breeding, and has significant theoretical and practical implications for ensuring my country's maize production and food security. The rapid degradation of misfolded proteins in the endoplasmic reticulum (ER) under high-temperature stress determines plant survival, and the ER-related protein degradation pathway is a key route for clearing misfolded proteins from the ER. Derlin, a pseudo-rhomboid protein localized to the endoplasmic reticulum (ER), is a key transmembrane channel for recognizing, transporting, and degrading misfolded proteins in the ER, playing a vital role in stress resistance under heat-induced ER stress. As a key functional gene for cellular protein quality control, the heat resistance of Derlin protein is directly related to cell survival, and it has great potential for application in crop heat tolerance breeding. Summary of the Invention

[0003] The purpose of this invention is to provide a corn pseudo-rhomboid protein. ZmDerlin3 New applications of genes in heat-tolerant plant breeding.

[0004] The present invention adopts the following technical solution: a maize pseudorhomboid protein of the present invention ZmDerlin3 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The maize pseudorhomboid protein described in this invention... ZmDerlin3 The encoded protein is a protein as described in (1) or (2) below: (1) a protein consisting of the amino acid sequence of SEQ ID NO.2 in the sequence listing; (2) a protein derived from (1) by substituting and / or adding one to twenty amino acid residues of the amino acid sequence of SEQ ID NO.2 in the sequence listing and having the function of the maize pseudorhomboid protein ZmDerlin3.

[0005] The present invention relates to a plant expression vector containing the aforementioned maize pseudorhomboid protein ZmDerlin3 and / or recombinant Agrobacterium.

[0006] The corn pseudo-rhomboid protein of the present invention ZmDerlin3 The application of the gene, the protein, or the plant expression vector and Agrobacterium in improving plant heat resistance.

[0007] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a corn pseudo-rhomboid protein ZmDerlin3 Genes overexpressed in maize ZmDerlin3 This gene can significantly improve the heat tolerance of seedlings. It can be used for maize inbred line improvement and transgenic breeding to increase maize yield under high-temperature stress. The invention provides… ZmDerlin3 Genes have important applications in improving maize inbred lines and creating highly heat-resistant maize varieties. Attached Figure Description

[0008] Figure 1 For example 2 ZmDerlin3 Gene expression levels under different temperature heat stresses.

[0009] Figure 2 This shows the subcellular localization of ZmDerlin3 in maize protoplasts in Example 3.

[0010] Figure 3 In the maize overexpression lines in Example 4 ZmDerlin3 The relative expression level.

[0011] Figure 4 For example, wild-type individuals recovered 7 days after heat stress in Example 5. ZmDerlin3 Growth phenotype of overexpression lines.

[0012] Figure 5 For example, wild-type individuals recovered 7 days after heat stress in Example 5. ZmDerlin3 Relative electrical conductivity of leaves in overexpression lines. Detailed Implementation

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0014] The following examples illustrate a gene related to maize heat resistance provided by the present invention. ZmDerlin3 The invention will be described in detail in terms of its application, but this should not be construed as limiting the scope of protection of the invention.

[0015] Unless otherwise specified, the experimental methods described in the following examples are standard methods in plant physiology and molecular biology.

[0016] Unless otherwise specified, all test materials used in the following examples were purchased from conventional biochemical reagent companies.

[0017] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0018] Example 1: Gene Cloning and Vector Construction 1.1 Gene Cloning Seven-day-old maize inbred line H21 seedlings were heat-shocked at 42℃ for 30 min. The second-to-last leaves were then rapidly frozen and ground in liquid nitrogen. Total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, RC411-01). PrimeScript II 1 st cDNA was synthesized using the strand cDNA Synthesis Kit (Takara, 6210A). Cloning primers were designed using Primer 3.0 (Table 1). Using cDNA from the maize inbred line H21 as a template, amplification was performed using PrimeSTARHS DNA Polymerase with GC Buffer (Takara, R044A) high-fidelity enzyme. The PCR amplification reaction system is shown in Table 2. The temperature cycling program was as follows: Step 1: 94℃, 3 min; Step 2: 98℃, 10 s, annealing at 55℃, 10 s, extension at 72℃, 60 s (30 cycles in Step 2); Step 3: 72℃, 5 min, hold at 4℃. After separation and recovery of the PCR product by agarose gel electrophoresis, it was ligated into a T-vector with blunt ends (Transgen, CB111-01), transformed into E. coli, and the gene sequence information was obtained by Sanger sequencing.

[0019] Table 1 Genes ZmDerlin3 CDS cloning primers

[0020] Table 2 PCR amplification reaction system

[0021] 1.2 Construction of ZmDerlin3 fusion with exogenous GFP recombinant vector To carry ZmDerlin3Using the recombinant T-vector of cDNA sequence as a template, and referring to the sequence of the pCAMBIA1300-GFP vector, appropriate restriction enzyme sites were selected. Primers were designed as shown in Table 3 for amplification. PCR amplification was performed according to the reaction system shown in Table 2. The temperature cycling program was as follows: Step 1: 94℃, 3 min; Step 2: 98℃, 10 s, annealing temperature 58℃, 10 s, extension at 72℃, 60 s (30 cycles in Step 2); Step 3: 72℃, 5 min, hold at 4℃. Finally, the PCR product was recovered. The pCAMBIA1300-GFP plasmid was digested with enzymes in a 37℃ water bath for 6-8 h. Homologous recombination ligation was performed using the ClonExpress II One Step Cloning Kit (Novizan, C112). The reaction system is shown in Table 4, and the reaction conditions were: 37℃, 30 min. The ligation product was transformed into E. coli, and colony PCR was performed. After extracting the recombinant plasmid, it was sent to a sequencing company for DNA sequencing and identification, which yielded recombinant vectors with GFP tags fused to the carboxyl or amino terminus of ZmDerlin3.

[0022] Table 3 Primer sequences for the ZmDerlin3 fusion vector with exogenous GFP

[0023] Table 4. Homologous recombination systems constructed using vectors

[0024] 1.3 ZmDerlin3 Construction of overexpression vectors Based on the multiple cloning site of the overexpression vector WMV013 provided by Changzhou Xinmi Biotechnology Co., Ltd., vector sequences containing BamHI and SmaI restriction sites were introduced upstream and downstream of the aforementioned cloned gene, respectively. The specific sequences are listed in Table 5. PCR amplification was performed according to the reaction system shown in Table 2. The WMV013 plasmid was digested in a 37℃ water bath for 6-8 h, and homologous recombination ligation was performed using the ClonExpressII One Step Cloning Kit (Novizan, C112). The reaction system is shown in Table 4, and the reaction conditions were 37℃ for 30 min. The ligation product was transformed into E. coli, and colony PCR was performed. After extracting the recombinant plasmid, it was sent to a sequencing company for DNA sequencing identification. Plasmids with correct sequencing results were used for the next step of maize transformation.

[0025] Table 5 Primer sequences for constructing the recombinant vector pCUB10800-Ubiquitin1-ZmDerlin3-Flag

[0026] Example 2 Real-time quantitative PCR detection ZmDerlin3 Expression patterns under thermal stress Select plump, uniformly sized, and healthy maize inbred line H21 seeds, disinfect them by soaking in 0.1% HgCl2 for 10 min, rinse three times with deionized water, and sow them in a solid culture medium containing vermiculite and nutrient soil. When maize seedlings reach the two-leaf stage, they are subjected to heat shock treatment at different temperatures of 37℃, 42℃, and 45℃. Samples are continuously taken and ground into powder in liquid nitrogen. Total RNA is extracted using the Trizol extraction kit, cDNA first-strand synthesis is performed using the Prime Script RT reagent Kit (Takara, RR047B), and real-time quantitative PCR is performed using the TB Green Premix Ex Taq II kit (Takara, RR820A). All experimental procedures are performed in accordance with the kit instructions.

[0027] Detection ZmDerlin3 The primers for the gene are: (SEQ ID NO.3): 5'-AAGCAAGGCATATGGGAC-3' (SEQ ID NO.4): 5'-TCATCAGAAGGCGAATAC-3' Internal reference gene Zmβ-actin The primers are (SEQ ID NO.5): 5'-TGTCCATCACTTGTGAAGCCTCCT-3' (SEQ ID NO.6): 5'-ACGACCTTAGCCAATATCGCACCA-3' See example results Figure 1 In the leaves of corn seedlings ZmDerlin3 Gene expression levels increased significantly after heat shock treatment at different temperatures; with prolonged heat shock treatment, ZmDerlin3 The expression levels showed a trend of first increasing and then decreasing; among them, after treatment at 37℃ and 42℃ for 60 min, ZmDerlin3 The expression level reached its peak after treatment at 45℃ for 240 min. Therefore, ZmDerlin3 The expression level of the gene was significantly upregulated inducible by heat stress, indicating that the gene is involved in the plant's response to heat stress.

[0028] Example 3 Subcellular localization analysis of protein ZmDerlin3 Maize seedlings were cultured in the dark for 10 days. Upper leaves were cut into 0.5 mm segments and immersed in an enzymatic hydrolysate (containing 1.5% Cellulase R10 and 0.4% Macerozyme R10). After vacuuming, the mixture was shaken in the dark for 3 hours (30℃, 100 rpm). The hydrolysate was filtered through 40 μm nylon gauze, and the filtrate was centrifuged at 4℃ (400 rpm, 5 min), discarding the supernatant. The precipitate was washed twice with pre-cooled resuspension and incubated on ice for 30 min. After washing, the protoplasts were centrifuged to remove the supernatant. Transformation solution was added at a plasmid:protoplast ratio of 1:5, followed by an equal volume of 40% PEG4000 solution. The mixture was gently mixed and incubated in a 22.5℃ water bath for 15-20 min. The protoplasts were collected by centrifugation and observed under a laser confocal microscope (excitation wavelength of ZmDerlin3-GFP: 488 nm; excitation wavelength of endoplasmic reticulum marker protein mCherry-HDEL: 587 nm).

[0029] See example results Figure 2 GFP represents green fluorescence, ER represents red fluorescence of endoplasmic reticulum marker protein, DIC represents bright field, and merge represents image overlay. The results show that the green fluorescence of the empty GFP control group in the first row is distributed in the nucleus and cytoplasm. The second row NGFP-ZmDerlin3 and the third row ZmDerlin3-CGFP represent ZmDerlin3 with GFP fused to the N-terminus and C-terminus, respectively. It can be seen that its green fluorescence and the red fluorescence of the endoplasmic reticulum marker protein mCherry-HDEL show obvious overlap, indicating that ZmDerlin3 is specifically located in the endoplasmic reticulum.

[0030] Example 4 ZmDerlin3 Obtaining maize overexpression lines 4.1 Maize genetic transformation The built ZmDerlin3 The overexpression vector was sent to Changzhou Xinmi Biotechnology Co., Ltd., where it was transformed into maize inbred line KN5585 to obtain transgenic T0 generation seeds.

[0031] 4.2 Identification and screening of transgenic positive lines Transgenic T0 generation seeds were self-pollinated for two generations, and positive lines were identified by PCR. DNA was extracted from the leaves of transgenic maize seedlings using the CTAB method, and specific primers for the ZmDerlin3-Flag fusion gene were designed. The primer sequences are shown in Table 6. Positive lines were identified by PCR. The PCR system was as shown in Table 2, and the temperature cycling program was as follows: Step 1: 94℃, 3 min; Step 2: 98℃, 10 s, annealing at 58℃, 10 s, extension at 72℃, 60 s (30 cycles in Step 2); Step 3: 72℃, 5 min, hold at 4℃. The presence or absence of the positive gene was detected by agarose gel electrophoresis, and sequencing was performed for comparison. Following the real-time quantitative PCR method in Example 2, positive lines in different transgenic maize lines were identified by PCR. ZmDerlin3 The expression level was detected, and the results are shown in [the table / document]. Figure 3 Among the three overexpression homozygous lines ZmDerlin3 The expression level was significantly increased, which can be used for subsequent heat resistance identification experiments.

[0032] Table 6. Specific primer sequences for the ZmDerlin3-Flag fusion gene

[0033] Example 5: Identification of the heat resistance of ZmDerlin3 KN5585 inbred line wild type, the above ZmDerlin3 Three portions of overexpressed maize seeds were germinated and then sown in a nutrient soil-vermiculite solid culture medium.

[0034] Take wild type and the above ZmDerlin3 Seven-day-old maize seedlings of the overexpression line were subjected to heat shock at 55℃ for 13 hours in a plant light incubator until the leaves were completely scorched and the stem base was bent. The seedlings were then removed and placed in a 23℃ greenhouse for recovery culture for 7 days, during which the maize phenotype was observed. The relative conductivity of the second-to-last leaf was measured. Relative conductivity reflects the degree of damage to the plant cell membrane under stress; the greater the damage, the higher the relative conductivity. The specific method was as follows: the second-to-last maize leaf was cut into three sections and soaked in 20 mL of ddH2O. The sections were allowed to stand at room temperature for 30 min, and the initial conductivity Ec1 was measured using a conductivity meter. The sections were then boiled in a boiling water bath for 10 min, cooled, and ddH2O was added to a final volume of 20 mL. After mixing, the final conductivity Ec2 was measured. The relative conductivity REC (%) was calculated using the formula: REC (%) = (Ec1 / Ec2) × 100%. At least three biological replicates were performed for each treatment.

[0035] See example results Figure 4 After heat treatment and subsequent recovery cultivation, wild-type maize seedlings showed slow growth, and their leaves exhibited severe scorching, yellowing, and curling. ZmDerlin3 The growth status of overexpressing maize inbred lines was significantly better than that of wild-type lines. Figure 4 The relative conductivity of the two inverted lobes was measured separately. ZmDerlin3 The relative electrical conductivity of leaves in maize inbred lines overexpressing this gene was significantly lower than that of wild-type maize, indicating that... ZmDerlin3 Overexpression of maize inbred lines resulted in less damage to leaf cell membranes under heat stress. Figure 5 ).

Claims

1. A maize pseudo-rhomboid protein-coding gene ZmDerlin3 Applications in improving the heat stress tolerance of maize include: Construct containing ZmDerlin3 Plant overexpression vectors for genes, to obtain Zmderlin3 The maize material overexpressing the gene, wherein the heat stress conditions are: heat shock at 55℃ for 13 hours followed by recovery culture for 7 days, and the... ZmDerlin3 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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