Application of corn biological clock gene Zmtoc1b in improvement of plant drought tolerance

Abstract

The invention is applicable to the technical field of molecular biology and biology, and provides application of a corn biological clock gene Zmtoc1b in improvement of plant drought tolerance, the nucleotide sequence of the corn biological clock gene Zmtoc1b is shown as SEQ ID NO: 1, and the amino acid sequence of a protein Zmtoc1b encoded by the corn biological clock gene Zmtoc1b is shown as SEQ ID NO: 2. The Zmtoc1b gene is cloned from a corn inbred line B73, Arabidopsis thaliana is transformed through an agrobacterium-mediated method, a T3-generation transgenic line with stable inheritance is obtained, drought stress treatment results show that the drought tolerance of the transgenic Arabidopsis thaliana in the seedling stage is obviously higher than that of a wild type, the relative water content and SOD activity of leaves are increased, and the yield of the transgenic Arabidopsis thaliana is increased. Therefore, it is proved that the corn biological clock gene Zmtoc1b can effectively improve the drought tolerance of plants, and a key gene and technical support are provided for improving the stress resistance of the plants.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biotechnology, and in particular relates to the application of the maize biological clock gene Zmtoc1b in improving plant drought resistance. Background Technology

[0002] Maize (Zea mays L.) is an important food, feed, and industrial raw material crop. As a grass crop with high water requirements, maize is extremely sensitive to water stress throughout its growth cycle. Drought is one of the major abiotic stress factors limiting maize production, severely impacting its growth, development, and yield. Therefore, identifying and utilizing drought-resistant gene resources and improving maize drought resistance through molecular breeding is of great significance for ensuring food security.

[0003] toc1 (Timing of CAB Expression 1) is a crucial component of the core oscillator of the biological clock, belonging to the Pseudo-Response Regulator (PRR) family. Its name derives from its regulatory role in the expression of the CAB (Chlorophyll a / b-binding protein) gene. The CAB gene encodes a key protein in the light-harvesting complex, directly influencing photosynthetic efficiency. toc1 precisely controls the diurnal rhythmic expression of photosynthesis-related genes by directly binding to the CAB gene promoter or indirectly regulating related transcription factors, thereby optimizing energy capture efficiency in plants under photoperiods. In Arabidopsis thaliana, toc1 forms a negative feedback regulatory loop with factors such as CCA1 / LHY, precisely regulating the biological clock and influencing biological processes such as photoperiod regulation, flowering time, and hypocotyl elongation. The realization of toc1 function is closely related to its phosphorylation modification. Recent studies have shown that toc1 phosphorylation significantly enhances its binding ability to chromatin, thereby regulating the rhythmic expression of downstream genes within specific time windows.

[0004] In recent years, an increasing number of studies have shown that there is a close relationship between the biological clock and plant adaptation to stress. The biological clock can regulate the rhythmic expression of antioxidant enzyme genes, affecting the plant's ability to scavenge reactive oxygen species at different times. At the same time, biological clock genes also participate in the regulation of the ABA signaling pathway, affecting stomatal opening and closing and water use efficiency. Of particular note is the recent discovery that core biological clock regulators (including toc1, PRRs, etc.) can directly inhibit the expression of autophagy-related genes (ATGs) during the day, while activating these genes at night, so that autophagic flux matches the cellular energy metabolism rhythm. This "biological clock gating" mechanism is an important strategy for plants to adapt to environmental stress. This discovery provides a new theoretical perspective for understanding the regulation of drought resistance by biological clock genes.

[0005] The homolog of toc1 in maize has been named Zmtoc1b. Studies have shown that this gene is rhythmically expressed under different photoperiod conditions, participates in the regulation of maize photoperiod sensitivity, and is significantly associated with the tasseling stage (BTA) and silking stage (BTL). However, the function of the toc1b gene in drought resistance in maize has not been reported in detail. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the maize biological clock gene Zmtoc1b in improving plant drought resistance, thereby addressing the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] Application of the maize biological clock gene Zmtoc1b in improving plant drought resistance. The nucleotide sequence of the maize biological clock gene Zmtoc1b is shown in SEQ ID NO:1, and the amino acid sequence of the protein Zmtoc1b encoded by the maize biological clock gene Zmtoc1b is shown in SEQ ID NO:2.

[0009] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0010] This invention cloned the Zmtoc1b gene from the maize inbred line B73 and transformed Arabidopsis thaliana using Agrobacterium-mediated transformation to obtain stably inherited T3 generation transgenic lines. Subcellular localization results showed that the Zmtoc1b protein is located in the cell nucleus. Drought stress treatment results showed that the transgenic Arabidopsis thaliana exhibited significantly higher drought tolerance in the seedling stage than the wild type, with increased relative water content and SOD activity in leaves. This confirms that the maize circadian clock gene Zmtoc1b can effectively improve plant drought tolerance, providing key gene and technical support for improving plant stress resistance. Attached Figure Description

[0011] Figure 1 The gene structure of Zmtoc1b provided in this embodiment of the invention;

[0012] Figure 2 Chromosomal localization of Zmtoc1b provided in embodiments of the present invention;

[0013] Figure 3 The Zmtoc1b promoter cis-acting element provided in the embodiments of the present invention;

[0014] Figure 4 This is a schematic diagram of Zmtoc1b subcellular localization provided in an embodiment of the present invention;

[0015] Figure 5 Phenotypes of various Arabidopsis thaliana lines under drought stress provided in embodiments of the present invention (Bar=1cm).

[0016] Figure 6 This is a statistical diagram illustrating the survival rate of plants under drought stress provided in an embodiment of the present invention.

[0017] Figure 7 The relative water content of various Arabidopsis thaliana lines after drought stress provided in the embodiments of the present invention;

[0018] Figure 8 The SOD content of various Arabidopsis strains after drought stress provided in the embodiments of the present invention;

[0019] In the attached figure, the error bars represent the standard error (SE) of three biological replicates and three technical replicates, and different lowercase letters on the bars indicate significant differences at the p<0.05 level. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0022] Example 1: Cloning of the maize Zmtoc1b gene, including the following steps:

[0023] 1. Total RNA extraction: Total RNA was extracted from the leaves of maize inbred line B73 using the Kangwei Century Ultrapure RNA Extraction Kit. The specific steps were as per the kit instructions. 1 μL of RNA solution was taken and the concentration was measured using a micro spectrophotometer. Another 2 μL of RNA solution was taken for agarose gel electrophoresis to check the integrity of the bands. Once the RNA quality was qualified, it was used for reverse transcription.

[0024] 2. cDNA synthesis: Reverse transcription was performed using the RT mix with DNase (All-in-one) reverse transcription kit. The reaction system was: 4 μL of 5×RT All-in-One Mix, 50 ng - 1 μg of template RNA, 1 μL of DNase, and RNase-free water to a final volume of 20 μL. After gentle mixing, the mixture was briefly centrifuged and placed in a PCR instrument. The reaction program was: 37°C for 2 min, 55°C for 15 min, and 85°C for 5 min. The obtained product was immediately placed on ice or stored at -20°C for later use.

[0025] 3. Gene amplification: Based on the CDS sequence of the Zmtoc1b gene in the maize genome provided by the NCBI and MaizeGDB databases, Gateway-specific cloning primers were designed:

[0026] Zmtoc1b-F:gtacaaaaaagcaggcttcATGGTGGGCGGAGGCGA (as shown in SEQ ID NO:3);

[0027] Zmtoc1b-R:gtacaagaaagctgggttCTACTCTGGAGAAGAAATCATATCT (as shown in SEQ ID NO:4);

[0028] Using maize cDNA as a template, PCR amplification was performed using the high-fidelity enzyme PrimeSTAR® GXL DNA Polymerase. The reaction system consisted of: 10 μL of 5×PrimeSTAR GXL Buffer, 4 μL of cDNA, 4 μL of dNTP Mixture, 2 μL each of forward and reverse primers, 1 μL of PrimeSTAR GXL DNA Polymerase, and 27 μL of sterile distilled water, for a total volume of 50 μL. The reaction program was: 98℃ for 10 s, 57℃ for 15 s, 68℃ for 45 s, for 35 cycles; followed by 68℃ for 5 min. The amplified products were detected by 1% agarose gel electrophoresis, and a specific band appeared at approximately 1500 bp, consistent with the expected size.

[0029] 4. Recovery and Sequencing: The target fragment was recovered by gel extraction using the SanPrep column DNA gel recovery kit, ligated into the entry vector pDONR207 via BP reaction, transformed into DH5α competent E. coli cells, and single colonies were picked for colony PCR detection. Positive colonies were sent to the company for sequencing. The sequencing results showed that the full-length CDS of the Zmtoc1b gene was 1554 bp, encoding 517 amino acids, which was consistent with the expected sequence, indicating that the Zmtoc1b gene was successfully obtained (its nucleotide sequence is shown in SEQ ID NO:1).

[0030] Example 2: Bioinformatics analysis of the maize Zmtoc1b gene:

[0031] Protein information encoded by the Zmtoc1b gene was predicted using online databases such as MaizeGDB and NCBI. The results showed that the full-length cDNA of the Zmtoc1b gene is 2344 bp, with a 5' UTR of 522 bp, a 3' UTR of 268 bp, and a coding region of 1554 bp, encoding 517 amino acids. The physical location of this gene on the chromosome was obtained from the MaizeGDB maize genome database. Figure 1 As shown, the maize Zmtoc1b gene is located on the long arm of chromosome 5. Simultaneously, the distribution information of exons and introns of this gene was obtained using the EnsemblPlants database (e.g., ...). Figure 2As shown in the figure, the Zmtoc1b gene contains six exons and five introns;

[0032] The 2000 bp upstream sequence of the start codon (ATG) of a candidate maize gene was obtained from the Ensembl Plants database. Analysis of its cis-regulatory elements using the PlantCARE online tool revealed that, in addition to common cis-regulatory elements in promoter and enhancer regions such as CAAT-box (existing in AT~TATA-box form) and the core promoter element TATA-box (existing in AT~TATA-box form), the upstream promoter region also involved cis-regulatory elements involved in light response, such as Sp1, G-box, G-Box, and MRE. Cis-regulatory elements involved in plant hormones included the CGTCA-motif and TGACG-motif involved in methyl jasmonate response, and those involved in desaturation. The abscisic acid-responsive cis-acting elements include ABRE, ABRE3a, ABRE4, and AT~ABRE, as well as the gibberellin-responsive element P-box; in terms of abiotic stress response, it contains the drought-induced MYB binding site MBS, the low-temperature-responsive cis-acting element LTR, the abiotic stress-responsive elements DREcore and STRE, and the defense and stress-responsive cis-acting element TC-richrepeats; in addition, it also contains the cis-regulatory elements A-box and BoxII-like sequence, the cis-regulatory element CAT-box associated with meristematic tissue expression, MYB-like elements, MYC elements, and Wbox, WRE3, CCGTCCmotif, and CCGTCC-box, etc. Figure 3 As shown, this gene may be widely involved in biological processes such as hormone signaling responses (ABA, MeJA, GA) and resistance to abiotic stresses (drought, low temperature, oxidative stress).

[0033] Example 3: Subcellular localization of the Zmtoc1b gene, including the following steps:

[0034] 1. Vector construction: The target gene Zmtoc1b was constructed into the expression vector pSATN1-GW using the LR reaction. After the LR reaction was completed, E. coli was transformed. After transformation, the bacterial culture was tested for PCR positivity. The Zmtoc1b-pSATN1-GW plasmid was extracted using a kit and stored at -20℃ for subsequent experiments.

[0035] 2. Maize Protoplast Extraction: Select an appropriate amount of B73 seeds and sow them in nutrient soil. After the maize sprouts emerge, culture the seedlings in darkness until they reach the three-leaf stage. Extract the protoplasts by cutting the middle portion (approximately 6-8 cm) of the second leaf from a healthy maize plant. Use a blade to cut the leaf into strips approximately 0.1-0.5 mm wide. Quickly and gently place these strips into the prepared enzymatic hydrolysate and lyse them in the dark. Place the hydrolysate mixture in a shaker at 28℃ and slowly oscillate at 60-80 rpm for at least 3 hours. When the hydrolysate turns light green, it indicates protoplast release. After shaking culture, take 1... Examine the 0 μL mixture under an optical microscope. Add an equal volume of W5 buffer to the enzyme digest and shake vigorously for 15 seconds to fully release the corn protoplasts. Moisten a sterilized mesh sieve with 2 mL of W5 buffer and filter the enzyme digest containing protoplasts through the mesh sieve into a new Erlenmeyer flask. Transfer the enzyme digest to a 50 mL centrifuge tube, centrifuge at 250 g for 3 min, and carefully remove the supernatant (do not pour directly). Gently add 15 mL of W5 buffer to resuspend the protoplasts. Centrifuge at 450 g for 3 min and remove as much supernatant as possible. Gently resuspend the protoplasts at room temperature with 1 mL of MgCl2 solution.

[0036] The formulation of the maize protoplast extract is shown in Table 1:

[0037] Table 1 Enzyme hydrolysate formulation

[0038] Components volume Final concentration Cellulase R-10 0.15g 1.5% Pectinase R-10 0.04g 0.4% D-Mannitol 0.7288g 0.4M 200mM MES (pH=5.7) 1mL 20mM 200mM KCl 1mL 20mM

[0039] After adding the above components, place the solution in a 55℃ water bath for 10 minutes. During this time, invert the solution to accelerate dissolution. After the enzymatic hydrolysate cools to room temperature, sterilize it using a 0.22µm aqueous filter membrane and filter it into an autoclaved Erlenmeyer flask. Then add the reagents shown in Tables 2-6.

[0040] Table 2

[0041] Components volume Final concentration <![CDATA[1M CaCl2]]> 100μL 10mM BSA 0.01g 0.1% <![CDATA[ddH2O]]> Adjust the volume to 10 mL

[0042] Table 3 W5 solution formulation

[0043] Components volume Final concentration 200mM MES (pH=5.7) 0.2mL 2mM <![CDATA[1mM CaCl2]]> 2.5mL 125mM 2M NaCl 1.54mL 1.54mM 200mM KCl 0.5mL 5mM <![CDATA[ddH2O]]> Adjust the volume to 20 mL

[0044] Table 4 WI solution formulation

[0045] Components volume Final concentration D-Mannitol 0.2733g 0.5M 200mM KCl 0.3mL 20mM 200mM MES (pH=5.7) 0.3mL 20mM <![CDATA[ddH2O]]> Adjust the volume to 3 mL

[0046] Table 5 MMg solution formulation

[0047] Components volume Final concentration D-Mannitol 0.7288g 0.4M <![CDATA[150mM MgCl2]]> 1mL 15mM 200mM MES (pH=5.7) 0.2mL 4mM <![CDATA[ddH2O]]> Adjust the volume to 10 mL

[0048] Table 6 Formulation of 40% PEG Solution

[0049] Components volume Final concentration D-Mannitol 0.3644g 0.2M PEG4000 4g 40% <![CDATA[1M CaCl2]]> 1mL 100mM <![CDATA[ddH2O]]> Adjust the volume to 10 mL

[0050] 3. PEG Transformation of Maize Protoplasts: Add 5-10 ng of the pre-constructed subcellular localization vector to a 2 mL centrifuge tube, then add the prepared protoplasts and mix gently. Add 110 μL of 40% PEG solution to the mixture and quickly stir to mix. Transfect for 10 min in the dark. Add 440 μL of W5 solution to the transfection mixture and mix gently. Centrifuge at 100 g for 3 min, remove the supernatant, gently resuspend in 1 mL of WI solution, and transfer to a 6-well cell culture plate. Incubate at room temperature in the dark for 12-16 h, then observe the fluorescence signal using a fluorescence microscope. The results are as follows: Figure 4 As shown.

[0051] Example 4: Construction of plant expression vector and obtaining transgenic Arabidopsis thaliana, including the following steps:

[0052] 1. Construction of the introductory vector: The target fragment was extracted using a SanPrep column-based DNA gel extraction kit and ligated into the introductory vector pDONR207 via a backpropagation (BP) reaction. The fragment was then transformed into competent DH5α *E. coli* cells. The correctly sequenced introductory vector pDONR207-Zmtoc1b culture was amplified and the plasmid was extracted. The extracted pDONR207-Zmtoc1b plasmid was then ligated into the expression vector pEarleyGate101 via a backpropagation (LR) reaction. The LR reaction mixture consisted of pDONR207-Zmtoc1b. 3 μL of mtoc1b plasmid, 1 μL of pEarleyGate101 empty vector plasmid, and 1 μL of LRClonase™II Enzymemix were added, for a total volume of 5 μL. After ligation at 25°C for 1 h, the cells were transformed into DH5α competent cells, plated on LB agar plates containing the corresponding antibiotics, and cultured at 37°C for 12-16 h. Single colonies were picked for colony PCR detection using the same primers as in Example 1. Positive clones were obtained, and plasmids were extracted and sequenced for verification. The recombinant plasmid pEarleyGate101-Zmtoc1b was successfully obtained.

[0053] 2. Agrobacterium transformation: The recombinant plasmid pEarleyGate101-Zmtoc1b was transformed into Agrobacterium GV3101 competent cells. 5 μL of the plasmid was added to 50 μL of GV3101 competent cells, mixed, and then incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, incubated in water at 37℃ for 5 min, and incubated on ice for 5 min. 700 μL of antibiotic-free YEP liquid medium was added, and the cells were cultured at 28℃ and 180 rpm for 2-3 h with shaking. After centrifugation at 6000 rpm for 2 min, the cells were resuspended in about 100 μL of medium and spread on YEP solid medium containing the corresponding antibiotic. The cells were incubated upside down at 28℃ for 24-36 h. Positive clones were identified by colony PCR to obtain Agrobacterium strains containing pEarleyGate101-Zmtoc1b.

[0054] 3. Arabidopsis genetic transformation: Arabidopsis (Columbia ecotype) was transformed using the flower-dipping method. Positive Agrobacterium tumefaciens bacterial suspension was inoculated into 50 mL of LB liquid medium containing the appropriate antibiotics and cultured until OD600 = 1.2-1.6. The bacteria were collected by centrifugation at 4℃ and 6000 rpm for 10 min. The bacterial cells were resuspended in a resuspension solution (5% sucrose + 0.02% Silwet L-77) until OD600 ≈ 0.8. Unflowered flower buds of Arabidopsis (after removing any pods and flower buds) were immersed in the Agrobacterium tumefaciens bacterial suspension for 90 seconds. After infection, the cells were cultured in the dark for 24 hours. The cells were re-immersed after one week. The infected Arabidopsis were cultured normally until maturity, and the T0 generation seeds were harvested.

[0055] 4. Some of the harvested T0 generation seeds were screened by Basta and identified by PCR to obtain T1 generation plants transgenic with the Zmtoc1b gene. After two generations, T3 generation Arabidopsis thaliana plants transgenic with the Zmtoc1b gene were obtained for subsequent phenotypic experiments.

[0056] Example 5: Drought tolerance analysis of transgenic Arabidopsis thaliana:

[0057] Drought stress treatment during the seedling stage: Wild-type and transgenic Arabidopsis thaliana plants cultured to 3 weeks old were subjected to drought treatment, with watering stopped for 7 days, followed by rehydration for 3 days. Survival rates were recorded. The results showed that after drought treatment, wild-type plants wilted severely, and most died after rehydration; transgenic lines showed less wilting, and their survival rate after rehydration was significantly higher than that of wild-type plants. Figures 5-6 This indicates that overexpression of the Zmtoc1b gene can effectively enhance the tolerance of Arabidopsis thaliana to drought stress.

[0058] Example 6: Determination of physiological and biochemical indicators in transgenic Arabidopsis thaliana under drought stress:

[0059] 1. Relative moisture content determination: Leaves of wild-type and transgenic Arabidopsis thaliana were harvested after 7 days of drought treatment and immediately weighed as fresh weight (FW). After soaking in ultrapure water at room temperature for 24 hours, the leaves were weighed as saturated fresh weight (SFW). The leaves were then dried at 37℃ for 2 days until constant weight and weighed as dry weight (DW). Relative moisture content (%) was calculated as [(FW-DW) / (SFW-DW)]×100%. Results showed that under normal conditions, there was no significant difference in relative moisture content among the different lines; after drought treatment, the relative moisture content of the transgenic lines was significantly higher than that of the wild type. Figure 7 );

[0060] 2. Antioxidant enzyme activity assay: The SOD activity of leaves after 7 days of drought treatment was determined using a superoxide dismutase (SOD) activity assay kit (colorimetric method). Specific procedures were followed according to the kit instructions. Results showed that under normal conditions, there was no significant difference in SOD activity among the different lines; after drought treatment, the SOD activity of the transgenic lines was significantly higher than that of the wild type. Figure 8 ).

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the maize biological clock gene Zmtoc1b in improving plant drought tolerance, characterized in that, The nucleotide sequence of the maize clock gene Zmtoc1b is shown in SEQ ID NO:1, and the amino acid sequence of the protein Zmtoc1b encoded by the maize clock gene Zmtoc1b is shown in SEQ ID NO:

2.

2. The application of the maize circadian rhythm gene Zmtoc1b according to claim 1 in improving plant drought resistance, characterized in that, Includes the following steps: Unopened flower buds of Arabidopsis thaliana were immersed in Agrobacterium tumefaciens containing the Zmtoc1b gene using the flower-dipping method. The infected Arabidopsis thaliana were cultured normally until maturity, and T0 generation seeds were harvested. The T0 generation seeds were screened by Basta and identified by PCR to obtain T1 generation plants transgenic with the Zmtoc1b gene. After two generations, T3 generation plants transgenic with the Zmtoc1b gene were obtained.

3. The application of the maize circadian rhythm gene Zmtoc1b according to claim 2 in improving plant drought resistance, characterized in that, The method for constructing Agrobacterium containing the Zmtoc1b gene includes the following steps: Initial vector construction: The target gene Zmtoc1b was ligated into the vector pDONR207 to obtain the pDONR207-Zmtoc1b plasmid, and then ligated into the expression vector pEarleyGate101 via LR reaction to obtain the recombinant plasmid pEarleyGate101-Zmtoc1b. Agrobacterium transformation: The recombinant plasmid pEarleyGate101-Zmtoc1b was transformed into Agrobacterium GV3101 competent cells to obtain Agrobacterium strains containing pEarleyGate101-Zmtoc1b.

4. The application of the maize circadian rhythm gene Zmtoc1b according to claim 3 in improving plant drought resistance, characterized in that, In the step of constructing the introductory vector, the system of the LR reaction is as follows: 3 μL of pDONR207-Zmtoc1b plasmid, 1 μL of pEarleyGate101 empty vector plasmid, 1 μL of LR Clonase™ II Enzyme mix, and a total volume of 5 μL.

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