Application of maize ribonucleotide reductase large subunit Zmlsc1 gene in plant variety breeding

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a maize ribonucleotide reductase large subunit ZmLSC1 gene in plant variety breeding. The application is particularly applied in obtaining a plant variety with promoted plant growth or a plant variety with slowed leaf senescence. The coding region nucleotide sequence of the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown in SEQ ID NO. 1. The application analyzes a plant with overexpression of the ZmLSC1 gene through plant genetic engineering technology, and first discloses the role of the ZmLSC1 gene in regulating plant growth and development. The gene can be used in molecular breeding to cultivate plants with fast growth and high biomass, and has great application value in plant breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the maize ribonucleotide reductase large subunit ZmLSC1 gene in plant variety breeding. Background Technology

[0002] Traditional breeding methods typically involve crossing naturally mutant lines with varieties exhibiting superior traits, then selecting lines with the desired trait and stable heritability from the offspring to develop new varieties. This hybridization breeding method has yielded numerous excellent varieties. However, traditional breeding methods still have significant drawbacks, such as long breeding cycles, large land areas required, and high human and financial resources, hindering their development. In recent years, the rapid development of genetic engineering and molecular biology has enabled the transfer of target genes into varieties seeking improvement through transgenic technology. This allows for more targeted and efficient alteration of plant traits, providing new avenues for increasing grain yields.

[0003] With the increasing global population and decreasing arable land, there is a growing need to research ways to improve agricultural efficiency. Corn is a major food crop in my country, but its domestic production is low, and the majority of its production relies on imports. High and stable corn yields are closely related to genetically modified varieties, and future research and development of transgenic plants that increase growth and biomass holds promise as a solution to the problem of dwindling food supply.

[0004] Ribonucleotide reductase (RNR) consists of two large subunits (R1) and two small subunits (R2). It catalyzes the reduction of four ribonucleotide diphosphates (NDPs) to their corresponding deoxyribonucleosides (dNDPs). This reaction is the rate-limiting step in the synthesis of DNA precursors and plays an important role in nucleotide metabolism.

[0005] Although studies have shown the effects of altered RNR activity on DNA replication, cell cycle, and DNA loss repair in yeast and human cells, its function and mechanism of action in higher plants are not fully understood. Currently, a few plant RNR-related mutants have been reported. For example, the Arabidopsis RNR1 mutant cls8 causes wrinkled leaves, white pods, and stunted growth. Rice RNRL1 and RNRS1 mutants exhibit white striped leaves and stunted growth. Furthermore, studies have confirmed that some RNR mutants lead to decreased levels of dNTP (deoxyribonucleoside triphosphate) synthesis.

[0006] Therefore, the main characteristics of the RNR mutants identified in flowering plants are stunted growth, yellowing, and abnormal chloroplast development. Existing studies have not included research on the role of RNR in growth regulation, and it remains unclear whether the balance of dNTPs affects plant growth and whether RNR genes can be used to regulate plant growth. Summary of the Invention

[0007] The purpose of this invention is to provide an application of the maize ribonucleotide reductase large subunit ZmLSC1 gene in plant variety breeding, which can obtain plant varieties with promoted plant growth or slowed leaf senescence.

[0008] The present invention also aims to provide a recombinant overexpression vector that can be used in plant breeding to obtain plant varieties in which plant growth is promoted or plant leaf senescence is slowed.

[0009] To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0010] The application of the maize ribonucleotide reductase large subunit ZmLSC1 gene in plant variety breeding, wherein the coding region nucleotide sequence of the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown in SEQ ID NO.1; the application is in obtaining plant varieties in which plant growth is promoted, or in obtaining plant varieties in which leaf senescence is slowed down.

[0011] The gene with the nucleotide sequence shown in SEQ ID NO.1 is the maize (Zea mays L.) Zm00001d045192 gene, with a length of 2448 bp, which we have named ZmLSC1 in this invention. This invention utilizes plant genetic engineering technology and gene cloning to obtain the above sequence, and studies the function of this gene from aspects such as protein subcellular localization and phenotypic analysis of Arabidopsis thaliana ectopic overexpression plants, thereby verifying the function of this gene.

[0012] Experiments have shown that overexpression of the ZmLSC1 gene significantly promotes maize plant growth and increases plant size. Arabidopsis thaliana plants ectopically overexpressing ZmLSC1 also exhibited significantly accelerated growth, increased leaf, pod, and plant size, and slower leaf senescence. Therefore, this gene can be used to improve plant growth and biomass, and inhibit leaf senescence.

[0013] Furthermore, the amino acid sequence encoded by the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown in SEQ ID NO.2. The ZmLSC1 gene encodes the maize ribonucleotide reductase large subunit, and its amino acid sequence comprises 815 amino acids. Compared with rice OsRNRL1, OsRNRL2, Arabidopsis thaliana AtRNR1, human HsRNR1, and yeast ScRNR1, it shows 93%, 89%, 86%, 67%, and 56% homology, respectively. ZmLSC1 shows the highest homology with maize RNRL2, with 97% homology in the whole protein sequence. The protein with the sequence described in SEQ ID NO.2 provided by this invention, when overexpressed, can promote plant growth and biomass increase, and slow down leaf senescence.

[0014] The specific method of applying the aforementioned maize ribonucleotide reductase large subunit ZmLSC1 gene is as follows: the maize ribonucleotide reductase large subunit ZmLSC1 gene is introduced into plant cells, tissues or organs, and then the transformed plant cells, tissues or organs are cultivated into plants, and the desired plant varieties are screened out.

[0015] This invention also provides an application of a recombinant expression vector in plant variety breeding, the technical solution of which is as follows:

[0016] The application of a recombinant expression vector in plant variety breeding, wherein the recombinant expression vector contains the maize ribonucleotide reductase large subunit ZmLSC1 gene; the coding region nucleotide sequence of the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown in SEQ ID NO.1; the application is in obtaining plant varieties in which plant growth is promoted, or the application is in obtaining plant varieties in which leaf senescence is slowed.

[0017] The amino acid sequence encoded by the large subunit ZmLSC1 gene of maize ribonucleotide reductase is shown in SEQ ID NO.2.

[0018] The specific application method of the recombinant expression vector is as follows: the recombinant expression vector is introduced into plant cells, tissues or organs, and the transformed plant cells, tissues or organs are then cultured into plants, and the desired plant varieties are screened out.

[0019] The method for preparing the recombinant expression vector includes: designing primers according to the nucleotide sequence shown in SEQ ID NO.1, cloning the maize ribonucleotide reductase large subunit ZmLSC1 gene, and then ligating the cloned maize ribonucleotide reductase large subunit ZmLSC1 gene into the overexpression vector.

[0020] Furthermore, this invention does not specifically limit the type of overexpression vector, and those skilled in the art can choose according to their needs. Preferably, the overexpression vector is one of pCUN-cVENUS, pCAMBIA3300, pCAMBIA1300, and pAM1006. More preferably, the overexpression vector is pCUN-cVENUS, whose backbone is derived from pCAMBIA1300. This invention ligates the nucleotide sequence encoding the amino acids of the ZmLSC1 gene into the plant expression vector pCUN-cVENUS, thereby obtaining a highly expressed recombinant expression vector.

[0021] The recombinant expression vector contains the nucleotide sequence encoding the amino acid of the large subunit ZmLSC1 gene of maize ribonucleotide reductase and an expression regulatory sequence operationally linked to that sequence.

[0022] The expression regulatory sequence is the regulatory sequence that drives the high expression of the large subunit ZmLSC1 gene of maize ribonucleotide reductase.

[0023] Furthermore, in the recombinant expression vector, the expression of the maize ribonucleotide reductase large subunit ZmLSC1 gene is driven by the Ubiquitin promoter. The Ubiquitin promoter used in this invention has high activation efficiency, relatively low methylation level, and stable genetic traits, making it suitable as a promoter for driving ZmLSC1 gene expression.

[0024] By transferring the aforementioned recombinant expression vector into plants, it was found that overexpression of the ZmLSC1 gene significantly promoted maize plant growth and increased size. Arabidopsis thaliana plants ectopically overexpressing ZmLSC1 also exhibited significantly accelerated growth, with larger leaves, pods, and overall plant size. Therefore, this recombinant expression vector can be used to improve plant growth and biomass.

[0025] The application of the maize ribonucleotide reductase large subunit ZmLSC1 gene or recombinant expression vector provided by this invention is any one of the following applications: 1) application in plant variety breeding to increase leaf size; 2) application in plant variety breeding to increase pod length; 3) application in plant variety breeding to increase plant biomass; 4) application in plant variety breeding to delay leaf senescence rate. Preferably, the plant variety is maize.

[0026] This invention utilizes plant genetic engineering technology to analyze the cellular and tissue expression characteristics of the large subunit ZmLSC1 gene of maize ribonucleotide reductase, revealing for the first time the role of the ZmLSC1 gene in regulating plant growth and development. By utilizing the role of this gene and its recombinant expression in regulating plant growth, molecular breeding can be conducted to cultivate plants with accelerated growth and high biomass, possessing significant application value in plant breeding. Attached Figure Description

[0027] Figure 1 Expression level analysis of the Arabidopsis thaliana ZmLSC1 overexpression line in this invention;

[0028] Figure 2 This study analyzes the expression level of the ZmLSC1 overexpression line in maize in this invention.

[0029] Figure 3 Examples of plant height, leaf size, and pod size of the Arabidopsis thaliana ZmLSC1 overexpression line in this invention;

[0030] Figure 4 This is an example of the plant height of the maize overexpression ZmLSC1 line in this invention. Detailed Implementation

[0031] The technical solution and technical effects of the present invention will be further described below with reference to specific embodiments and accompanying drawings. The following description is for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the reagents used in the following embodiments are conventional reagents in the art and can be obtained commercially. Unless otherwise specified, the instruments and equipment used in the following embodiments are conventional instruments and equipment in the art.

[0032] Examples of the application of the maize ribonucleotide reductase large subunit ZmLSC1 gene and its recombinant expression vector

[0033] I. Construction of the large subunit ZmLSC1 gene of maize ribonucleotide reductase and its recombinant expression vector

[0034] The amino acid sequence (as shown in SEQ ID No. 2) and encoding nucleotide sequence (as shown in SEQ ID No. 1) of the ZmLSC1 protein were obtained from the maize genome database (https: / / www.maizegdb.org / ).

[0035] Gene-specific PCR primers were designed based on the nucleotide sequence of the coding region of the ZmLSC1 gene:

[0036] Upstream primer F1: 5'-gcgcgccatttaaatactagtATGTACGTGGTCAAGAGGGACG-3' (as shown in SEQ ID NO. 3);

[0037] Downstream primer R1: 5'-catggtggatcccatactagtGCTTCCACACGCCAGGCA-3' (as shown in SEQ ID NO. 4).

[0038] Total RNA was extracted from the leaves of wild-type maize (B73 ecotype) seedlings at the three-leaf stage and the cDNA sequence reverse transcribed from it was used as a template. The nucleotide sequence of the coding region of the ZmLSC1 gene was amplified and cloned using the above primers and KOD1 high-fidelity enzyme, without the stop codon (in order to enable ZmLSC1 and cVENUS to form a fusion protein).

[0039] For total RNA extraction, 'Promeg' Eastep Super was used to extract total RNA from the leaves of maize seedlings at the three-leaf stage. The extraction steps were performed according to the kit instructions as follows:

[0040] (1) Take about 100mg of fresh corn leaves and quickly put the tissue into a mortar with liquid nitrogen added for grinding. During the grinding process, liquid nitrogen should be added in time to prevent the tissue from melting until the tissue is completely ground into powder.

[0041] (2) Quickly transfer the powdered sample into a sterile 1.5mL centrifuge tube, add 500μL of lysis buffer (provided in the kit) and 500μL of dilution buffer (provided in the kit), mix by repeatedly pipetting, invert the centrifuge tube 3-4 times to mix, and let stand at room temperature for 3-5 minutes.

[0042] (3) Centrifuge at 12,000 rpm for 5 minutes at room temperature, then carefully aspirate the supernatant into a sterile 1.5 mL centrifuge tube. Add 0.5 times the volume of anhydrous ethanol to the supernatant and quickly pipette. Transfer the mixture to the centrifuge column provided in the kit and centrifuge at 12,000 rpm for 1 minute. Discard the filtrate and return the centrifuge column to the collection tube. Add 600 μL of RNA washing buffer (provided in the kit; ethanol needs to be added before use) to the centrifuge column, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate.

[0043] (4) Digestion with DNase I (DNase I should be freshly prepared). Take a sterilized 1.5 mL centrifuge tube and add the following reagents.

[0044] The following is the amount of DNase I incubation solution needed to extract one tube of RNA.

[0045]

[0046] (5) Add 50 μL of freshly prepared DNase I incubation solution to the center of the centrifuge column and incubate at room temperature for 15 minutes. Add 600 μL of RNA wash buffer to the centrifuge column, centrifuge at 12000 rpm for 45 seconds, and discard the filtrate. Add 600 μL of RNA wash buffer to the centrifuge column, centrifuge at 12000 rpm for 45 seconds, and discard the filtrate. Finally, reposition the centrifuge column onto the collection tube and centrifuge at 12000 rpm for 2 minutes. Transfer the centrifuge column to the elution tube (provided in the kit), add 50 μL of nuclease-free water to the center of the centrifuge column membrane, incubate at room temperature for 2 minutes, centrifuge at 12000-14000 xg for 1 minute, and store the RNA at -70℃. Repeating this step can increase the RNA recovery concentration.

[0047] The reverse transcription process was performed according to the instructions for Novozymes HiScript II Reverse Transcriptase (Glycerol-free). The specific steps are as follows: (1) RNA template denaturation: Add 5 μg of the RNA extracted in the previous step and Oligo(dT) to the PCR tube. 23 (1) Add 1 μL of VN (50 μM), and finally add RNase-free ddH2O to 13 μL. Then place it in a metal bath at 65 °C for 5 min, quickly place it in an ice-water bath to cool, and let it stand on ice for 2 min. (2) Prepare the first-strand cDNA synthesis solution: Add 4 μL of 5×HiScript II Buffer, 1 μL of dNTP Mix (10 mM each), 1 μL of HiScript II Reverse Transcriptase, and 1 μL of RNase inhibitor (40 U / μL) to the PCR tube from the previous step, and mix well by pipetting. (3) Perform the first-strand cDNA synthesis reaction under the following conditions: 25 °C for 5 min; 50 °C for 45 min; 85 °C for 2 min; store at -20 °C.

[0048] PCR amplification of reverse transcription products: 2 μL of cDNA template obtained in the previous step, 0.6 μL of upstream primer F1, 0.6 μL of downstream primer R1, 10 μL of KOD One PCR Master Mix, and 6.8 μL of ultrapure water. The PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 5 s, 68℃ for 10 s, 35 cycles; 68℃ for 10 min, 4℃ for 30 min. The PCR products were recovered by agarose gel electrophoresis using a Kangwei Century Agarose Gel DNA Recovery Kit.

[0049] The recovered product obtained from the above steps was ligated into the pCUN-cVENUS vector using a one-step cloning reaction, and the ubiquitin promoter was ligated to the front of its sequence. Vectors with positive insert fragments were screened and sequenced. The plasmid with correct sequencing was named pUBI-ZmLSC1-cVENUS and used to construct a recombinant expression vector for the ZmLSC1 gene. This vector was used as the final vector for the next step of the experiment.

[0050] The pCUN-cVENUS vector used in this invention has a backbone derived from pCAMBIA1300, and is a plant binary expression vector. The transgenic plant resistance selection marker in the pCUN-cVENUS vector has been replaced with glufosinate-ammonium (Basta) instead of hygromycin (Hyg), facilitating transgenic plant selection. After linking to the ubiquitin promoter, the target gene can be stably and highly expressed in transgenic materials.

[0051] II. Obtaining transgenic Arabidopsis and maize overexpressing the ZmLSC1 gene

[0052] (1) Agrobacterium-mediated transformation

[0053] The pUBI-ZmLSC1-cVENUS vector was transformed into Agrobacterium GV3101 competent cells using the heat shock method. The transformed cells were then plated onto solid LB medium containing 50 mg / L gentamicin, 50 mg / L rifampin, and 50 mg / L kanamycin. After incubation at 28°C with shaking for 2 days, positive clones were screened using primers F1 and R1. The obtained positive clones were inoculated into liquid LB medium containing 50 mg / L gentamicin, 50 mg / L rifampin, and 50 mg / L kanamycin. After incubation at 28°C with shaking for 24 hours, 0.5 mL of the bacterial culture was collected and the culture was preserved for use in Arabidopsis transformation.

[0054] (2) Arabidopsis thaliana transformation

[0055] Add 20 μL of the bacterial culture obtained in the above steps to 10 mL of liquid LB medium containing 50 mg / L gentamicin, 50 mg / L rifampin, and 50 mg / L kanamycin, and incubate at 28°C with shaking for 36 h. Transfer the entire bacterial culture to 50 mL of liquid LB medium containing 50 mg / L gentamicin, 50 mg / L rifampin, and 50 mg / L kanamycin, and incubate at 28°C with shaking for 12 h. Centrifuge the resulting 50 mL bacterial culture at 4000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in 50 mL of infection buffer. The infection buffer formulation (1 L) is as follows: 50 g sucrose, 2.2 g MS powder, 200 μL Silwet-L77, and water to a final volume of 1 L.

[0056] Select healthy Arabidopsis plants that have bolted for one to two weeks, remove the siliques and open flowers, leaving the apical meristem and flower buds, and soak the treated Arabidopsis inflorescences in 50 mL of resuspended bacterial solution for 1 minute. Then, treat the soaked plants in the dark at 22°C for 24 hours, while wrapping them with plastic wrap to maintain humidity, and then place them under light to grow.

[0057] (3) Screening of transformed plants

[0058] (3.1) Obtaining ZmLSC1 transgenic Arabidopsis plants

[0059] After the transformed Arabidopsis plants continued to grow for about a month, mature seeds were collected and aliquoted into 1.5 mL centrifuge tubes. 1 mL of 2% sodium hypochlorite (containing 0.5% Triton X-100) was added, and the tubes were shaken for sterilization for 15 min. The seeds were then washed five times with sterile ultrapure water. The sterilized seeds were evenly placed on 1 / 2 MS medium containing 50 mg / L herbicide and 100 mg / L herbicide, and treated in the dark at 4°C for 48 h. Afterward, they were cultured under light at 22°C for 6-7 days. Positive seedlings with normal growth were selected and transplanted into soil. When the seedlings reached about 21 days of growth, DNA was extracted from true leaves of about 0.5 cm². PCR was performed using primers F1 and R1 to screen for positive plants.

[0060] (3.2) Identification of ZmLSC1 transgenic Arabidopsis thaliana

[0061] Genetic segregation ratio method for identifying insertion copy number: According to genetic principles, self-crossing after a single copy insertion will produce a 3:1 segregation ratio in the offspring. Combined with statistical methods, the number of resistant and non-resistant seedlings on antibiotic culture medium was counted. The segregation ratio method was used to identify transgenic plants with a single copy insertion (single-copy ZmLSC1 transgenic Arabidopsis thaliana), which was then used for homozygous selection.

[0062] Screening of homozygous lines of transgenic Arabidopsis thaliana ZmLSC1 OE-9 and ZmLSC1 OE-23: After the above identification and analysis, two single-copy ZmLSC1 transgenic Arabidopsis thaliana lines were randomly selected and designated as ZmLSC1 OE-9 and ZmLSC1 OE-23 (T1 generation), respectively. They were sown on MS medium containing 40 mg / L Basta herbicide. After two consecutive generations of screening, the parental plants in which all self-pollinated progeny grew normally (i.e., all progeny were herbicide resistant) were designated as homozygous lines. Finally, T3 generation transgenic Arabidopsis thaliana ZmLSC1 OE-9 and ZmLSC1 OE-23 homozygous single-copy plants were obtained and used as experimental materials for subsequent experimental analysis.

[0063] (3.3) Obtaining and identifying ZmLSC1 transgenic maize plants

[0064] The creation of transgenic maize plants was completed by Wuhan Aidi Crystal Co., Ltd., using B104 as background material. The identification method involved PCR amplification of the fragment of the herbicide Basta resistance gene Bar contained in the vector. The method steps are as follows:

[0065] (3.3.1) Obtaining transgenic maize seedlings:

[0066] (a) Disinfection: Select corn ears of suitable embryo age, remove the corn husks, and disinfect them. (b) Embryo Removal: Remove the embryos from the disinfected ears using an embryo removal knife and place them in a sterile tube containing the infection solution for later use. (c) Agrobacterium Culture and Infection Solution Preparation: Streak Agrobacterium solution onto a resistance plate and incubate overnight to complete activation. Pick colonies from the activated plate, suspend the colonies in the infection solution, mix well, and adjust the OD value of the solution for later use in infection. (d) Co-culture: Discard the infection solution in the sterile centrifuge tube from step (b), add Agrobacterium suspension with an appropriate OD value, tighten the centrifuge tube cap, gently shake to suspend the embryos, let stand for several minutes, discard the Agrobacterium suspension, transfer the embryos to a co-culture plate, and wait for the Agrobacterium solution on the embryo surface to dry to complete the low-temperature co-culture. (e) Recovery: After co-culture, transfer the embryos to recovery medium for appropriate recovery culture. (f) Screening: Transfer the embryos from the recovery medium to the screening medium to obtain resistant callus. (g) 7. Regeneration and seedling emergence: Use tweezers to transfer the resistant callus to the regeneration medium to induce seedling emergence. (h) Rooting: After successful induction of T0 seedlings, transfer the seedlings to a rooting tank for rooting induction. When the seedlings have grown to 3-4 young leaves, samples can be taken for PCR testing.

[0067] (3.3.2) PCR detection of Bar gene expression:

[0068] Plant DNA was extracted using the CTAB method: (a) Grinding: Take a sample, grind it with CTAB, and then add an appropriate amount of CTAB solution; (b) Water dissolution: 30 min, shake once every 10 min; (c) After cooling the sample to room temperature, add chloroform-isoamyl alcohol, the same volume as CTAB, and shake for 20 min; (d) Centrifuge for 10 min, and transfer the supernatant to a new EP tube; (e) Add 0.7 times the amount of isopropanol (pre-cooled in a -20℃ freezer), shake gently, observe the filamentous material, and place in the freezer to stand for 30 min; (f) Centrifuge for 5 min, and discard the supernatant; (g) Wash with 70% alcohol, pipette to suspend it, and add ddH2O and RNase to each tube; (h) Detection: The agar content in the gel is 1%. The presence of the Bar gene in T0 seedlings was amplified and detected by conventional PCR using Bar gene-specific primers. Two positive seedlings with bands detected by PCR were named LSC1OX-1 and LSC1OX-2, respectively.

[0069] III. Detection of ZmLSC1 gene expression levels in transgenic Arabidopsis and maize plants

[0070] Leaves were harvested from positive transgenic Arabidopsis thaliana plants, and cDNA was obtained from the transgenic plants using the same method as described above. Gene-specific real-time quantitative PCR primers qPCR-F and qPCR-R were designed based on the cDNA sequence of the ZmLSC1 gene. The expression level of the ZmLSC1 gene in transgenic Arabidopsis thaliana was detected by real-time quantitative PCR. The Arabidopsis thaliana eIF4A gene was used as the internal control gene (primers qPCR-F2 and qPCR-R2 were used). When detecting the expression level of the ZmLSC1 gene in transgenic maize, the maize Actin1 gene was used as the internal control gene (qPCR-F3 and qPCR-R3). The primer sequences are as follows:

[0071] qPCR-F: 5'-GGTCTTACCTGTTGAAG-3' (as shown in SEQ ID NO.5);

[0072] qPCR-R: 5'-TGGTTCTTTCTCAGATCA-3' (as shown in SEQ ID NO. 6);

[0073] qPCR-F2: 5'-TCCAGGCATTGTCCACAGAA-3' (as shown in SEQ ID NO.7);

[0074] qPCR-R2: 5'-ACCTGCTCCTCCTTAGACAT-3' (as shown in SEQ ID NO. 8);

[0075] qPCR-F3: 5'-TCACCCTGTGCTGCTGACCG-3' (as shown in SEQ ID NO. 9);

[0076] qPCR-R3: 5'-GAACCGTGTGGCTCACACCA-3' (as shown in SEQ ID NO.10);

[0077] The reaction conditions using the above primers are as follows:

[0078] Real-time quantitative PCR reaction system: 5 μL of 2x qPCR SYBR Green Master Mix (YEASEN); 0.5 μL of forward primer (20 μmol); 0.5 μL of reverse primer (20 μmol); 1 μL of cDNA template; ddH2O to 10 μL. Three replicates were set up. After gentle shaking, the mixture was used in the experiment with a Roche LightCycler 96 real-time PCR instrument. The reaction program adopted was the real-time quantitative PCR program: 95℃ for 10 s; 94℃ for 5 s, 35 cycles; 60℃ for 20 s, fluorescence value read; 95℃ for 10 s; 65℃ to 95℃, 0.5℃ increments, fluorescence value read per cycle.

[0079] After the PCR reaction, at 2 -ΔΔCt The expression of the ZmLSC1 gene in different lines was analyzed and compared as a relative difference to measure the level of gene transcription. Ct value was the number of cycles when the fluorescence signal of the PCR reaction reached the set threshold, and ΔCt value was the difference between the Ct value of the specific primer and the Ct value of the Actin1 primer.

[0080] Real-time quantitative PCR was used to detect the relative expression of the ZmLSC1 gene in wild-type (Col) and transgenic Arabidopsis thaliana plants, as follows: Figure 1 As shown in the figure. The relative expression of the ZmLSC1 gene in ZmLSC1-overexpressing maize lines was also detected, and the results are shown in the figure. Figure 2 As shown.

[0081] Depend on Figure 1 It can be seen that, relative to the wild type, the ZmLSC1 gene is expressed in both the No. 2 transgenic line ZmLSC1 OE-9 and the No. 3 transgenic line ZmLSC1 OE-23.

[0082] Depend on Figure 2 It can be seen that, compared with the wild-type B104 material (WT), the expression level of the ZmLSC1 gene increased in both overexpression lines, with LSC1OX-1 showing an increase of about six times and LSC1OX-2 showing an increase of about four times.

[0083] IV. Phenotypic Analysis of Arabidopsis and Maize with Overexpression of ZmLSC1 Gene

[0084] Wild-type and ZmLSC1 overexpression transgenic positive plants were sterilized with 2% sodium hypochlorite and then grown on 1 / 2 MS medium at 22°C under normal light for 7 days. It was found that the root length was significantly increased compared to the wild-type plants, and the true leaves emerged earlier and were larger. When the cotyledons had fully opened and the roots were approximately 0.5 cm long, they were transplanted into nutrient soil and grown at 22°C under normal light for approximately 35 days. The overall phenotype of the plants was then observed to differ from that of the wild-type. The results are as follows: Figure 3 As shown.

[0085] Depend on Figure 3 It was found that the plant height of Arabidopsis thaliana plants overexpressing ZmLSC1 was significantly increased compared to the wild type. Measurements of the plant heights of the two overexpressing lines and the wild-type Arabidopsis thaliana after 45 days of growth revealed that the average plant height of the wild-type was approximately 25 cm, while lines 2 (ZmLSC1OE-9) and 3 (ZmLSC1OE-23) were 35 cm and 32 cm, respectively. Figure 3 (Right). When the seeds were harvested after 60 days of growth, it was found that the pods of the two Arabidopsis ZmLSC1 overexpression lines were significantly longer than those of the wild type. The average length of the wild type was 1.12 cm, while that of line 2 (ZmLSC1 OE-9) and line 3 (ZmLSC1 OE-23) was 1.35 cm and 1.27 cm, respectively. Figure 3 (Lower left). It was also found that the leaves of the overexpression line senescent about 7 days later than those of the wild type.

[0086] Furthermore, phenotypic analysis was performed on the two ZmLSC1 overexpressing maize lines, and the results are as follows: Figure 4 As shown.

[0087] Depend on Figure 4 It was found that the overexpressing maize plants were significantly taller than the wild type. After weighing the third leaf, it was found that the fresh weight of the LSC1OX-1 leaf was about twice that of the wild type leaf, while the fresh weight of the LSC1OX-2 leaf was about 1.5 times that of the wild type. At the same time, the leaves of the overexpressing lines were significantly longer and wider than those of the wild type.

[0088] In summary, this invention, through plant genetic engineering technology, analyzes the cellular and tissue expression characteristics of the large subunit ZmLSC1 gene of maize ribonucleotide reductase, revealing for the first time the role of the ZmLSC1 gene in regulating plant growth and development. Utilizing the role of this gene and its recombinant expression in regulating plant growth, molecular breeding can be conducted to cultivate plants with accelerated growth and high biomass, possessing significant application value in plant breeding.

Claims

1. Use of the maize ribonucleotide reductase large subunit ZmLSC1 gene in plant variety breeding, characterized in that, The coding region nucleotide sequence of the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown as SEQ ID NO. 1; the application is the application in obtaining a plant variety with promoted plant growth, wherein the plant variety with promoted plant growth is maize or Arabidopsis thaliana, and the promotion of plant growth is realized by overexpressing the ZmLSC1 gene in the plant; or the application is the application in obtaining a plant variety with slowed leaf senescence, wherein the plant variety with slowed leaf senescence is Arabidopsis thaliana, and the slowing of leaf senescence is realized by overexpressing the ZmLSC1 gene in the plant.

2. Use of the maize ribonucleotide reductase large subunit ZmLSC1 gene according to claim 1 for breeding of plant varieties, characterized in that, The application is specifically in the following manner: introducing the maize ribonucleotide reductase large subunit ZmLSC1 gene into a plant cell, tissue or organ, cultivating the transformed plant cell, tissue or organ into a plant, and screening to obtain the desired plant variety.

3. Use of a recombinant expression vector in breeding of plant varieties, characterized in that, The recombinant expression vector comprises a maize ribonucleotide reductase large subunit ZmLSC1 gene; the coding region nucleotide sequence of the maize ribonucleotide reductase large subunit ZmLSC1 gene is shown as SEQ ID NO. 1; the application is the application in obtaining a plant variety with promoted plant growth, wherein the plant variety with promoted plant growth is maize or Arabidopsis thaliana, and the promotion of plant growth is realized by overexpressing the ZmLSC1 gene in the plant; or the application is the application in obtaining a plant variety with slowed leaf senescence, wherein the plant variety with slowed leaf senescence is Arabidopsis thaliana, and the slowing of leaf senescence is realized by overexpressing the ZmLSC1 gene in the plant.

4. Use of a recombinant expression vector according to claim 3 in the breeding of plant varieties, characterized in that The application is specifically in the following manner: introducing the recombinant expression vector into a plant cell, tissue or organ, cultivating the transformed plant cell, tissue or organ into a plant, and screening to obtain the desired plant variety.

5. Use of a recombinant expression vector according to any one of claims 3 to 4 in the breeding of plant varieties, characterized in that, The preparation method of the recombinant expression vector comprises the following steps: designing primers according to the nucleotide sequence shown as SEQ ID NO. 1, cloning the maize ribonucleotide reductase large subunit ZmLSC1 gene, and then connecting the cloned maize ribonucleotide reductase large subunit ZmLSC1 gene to an overexpression vector.

6. Use of a recombinant expression vector according to any one of claims 3 to 4 in the breeding of plant varieties, characterized in that The recombinant expression vector comprises the maize ribonucleotide reductase large subunit ZmLSC1 gene sequence and an expression regulatory sequence operatively linked to the gene sequence.

7. Use of a recombinant expression vector according to claim 6 in the breeding of plant varieties, characterized in that The expression regulatory sequence is a regulatory sequence for driving high expression of the maize ribonucleotide reductase large subunit ZmLSC1 gene.

8. Use of a recombinant expression vector according to claim 7 in the breeding of plant varieties, characterized in that In the recombinant expression vector, the Ubiquitin promoter is used to drive the expression of the maize ribonucleotide reductase large subunit ZmLSC1 gene.

Images