Application of a maize ZmRbcL_n1 gene in improving photosynthetic efficiency of plants

Abstract

The application discloses application of a corn ZmRbcL_n1 gene in improvement of photosynthesis efficiency of plants and relates to the technical field of plant genetic engineering. Specifically, the protein coded by the corn ZmRbcL_n1 gene is different from the traditional RbcL protein structure, the protein structure has a molecular chaperone domain for promoting complex assembly, and fluorescence quantification shows that the corn ZmRbcL_n1 gene is highly expressed in leaves, which indicates that the protein may promote photosynthetic carbon fixation of leaves by mechanisms such as promoting RuBisco complex assembly. Further, the application constructs an expression vector for over-expressing the corn ZmRbcL_n1 gene, and the gene is introduced into Arabidopsis thaliana plants by an agrobacterium-mediated method, and it is found that over-expression of the gene can promote biomass accumulation and seed yield of the Arabidopsis thaliana, so that it is indicated that the corn ZmRbcL_n1 gene has great application prospect in improving photosynthesis and increasing carbon fixation efficiency of plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the maize ZmRbcL_n1 gene in improving plant photosynthetic efficiency. Background Technology

[0002] Photosynthesis is the primary driving force behind the accumulation of organic matter on Earth, directly affecting the total biomass of plants and the upper limit of crop yield. Photosynthesis consists of two parts: the light reaction and the dark reaction (Calvin cycle). The Rubisco complex catalyzes the first step of the Calvin cycle, fixing atmospheric CO2 onto a five-carbon substrate. However, the complex has low catalytic efficiency, which limits the efficiency of photosynthesis.

[0003] The RuBisco complex consists of two subunits (the large subunit RbcL and the small subunit RbcS), with RbcL being the catalytically active subunit. RbcL and RbcS require the assistance of accessory proteins such as molecular chaperones to ultimately assemble into the active L8S816 polymature complex. RbcL is evolutionarily highly conserved, and researchers have been searching for more functional RbcL proteins, simultaneously employing targeted screening and site-specific editing to enhance RbcL activity, but with limited success. Identifying and utilizing novel functional RbcL genes could contribute to improving plant photosynthetic efficiency, promoting biological carbon sequestration, and increasing crop yield. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the maize ZmRbcL_n1 gene in improving plant photosynthetic efficiency.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] The primary objective of this invention is to provide the application of the maize ZmRbcL_n1 gene in improving plant photosynthetic efficiency.

[0007] As a further optimization of the present invention, the CDS sequence of the corn ZmRbcL_n1 is shown in SEQ ID NO.1.

[0008] As a further optimization of the present invention, the sequence of the protein encoded by the maize ZmRbcL_n1 gene is shown in SEQ ID NO.4, and the sequence of the protein encoded by the maize ZmRbcL_n1 gene contains a Ubiq_cyt_C_chap molecular chaperone structure, the sequence of which is shown in SEQ ID NO.5.

[0009] As a further optimization of the present invention, the application is specifically to promote at least one of plant biomass accumulation or seed yield.

[0010] As a further optimization of the present invention, the plant is Arabidopsis thaliana or maize.

[0011] As a further optimization of the present invention, the application involves transferring an expression vector overexpressing the maize ZmRbcL_n1 gene into a plant to promote improved photosynthetic efficiency.

[0012] The present invention has the following beneficial effects:

[0013] The maize ZmRbcL_n1 gene provided in this invention encodes a protein with a structure different from traditional RbcL proteins. Its protein structure contains a molecular chaperone domain that promotes complex assembly. Quantitative fluorescence analysis shows that the maize ZmRbcL_n1 gene is highly expressed in leaves, suggesting that its protein may promote leaf photosynthetic carbon fixation through mechanisms such as promoting RuBisco complex assembly. Furthermore, this invention constructed an expression vector overexpressing the maize ZmRbcL_n1 gene and transformed it into Arabidopsis plants using Agrobacterium-mediated transformation. The results showed that overexpression of this gene promoted biomass accumulation and seed yield in Arabidopsis, indicating that the maize ZmRbcL_n1 gene has great application potential in enhancing plant photosynthetic carbon fixation efficiency. Attached Figure Description

[0014] Figure 1 This is a gel electrophoresis image of the amplified ZmRbcL_n1 gene provided by the present invention;

[0015] Figure 2 The ZmRbcL_n1 protein structure analysis provided by this invention;

[0016] Figure 3 Analysis of ZmRbcL_n1 gene expression pattern provided by this invention;

[0017] Figure 4 The carrier spectrum provided for this invention;

[0018] Figure 5 The effects of overexpression of the ZmRbcL_n1 gene provided by this invention on Arabidopsis thaliana phenotype, aboveground biomass accumulation, and total seed weight per plant. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] 1. Materials and Reagents

[0021] Unless otherwise specified, all materials and reagents used in this embodiment are commercially available.

[0022] 2. Method

[0023] Unless otherwise specified in this embodiment, all experimental methods can be performed using conventional methods.

[0024] 2.1 Obtaining the maize ZmRbcL_n1 gene

[0025] (1) RNA extraction

[0026] Trizol reagent was used according to the manufacturer's instructions. Approximately 0.1 g of wild-type B73 maize leaves were placed in a 2.0 ml EP tube, steel beads were added, and the mixture was frozen in liquid nitrogen for 5 min. The mixture was then ground into powder using a high-throughput grinder. 1 ml of Trizol reagent was added, and the powder was mixed vigorously. The mixture was centrifuged at 12,000 rpm for 2 min at 4 °C, and the supernatant was transferred to a new EP tube. 200 μl of chloroform was added and mixed vigorously. The mixture was allowed to stand at room temperature until separation, and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new EP tube, and 500 μl of isopropanol was added and mixed thoroughly. The mixture was precipitated at -20 °C for 30 min. The precipitate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol, centrifuged at 12,000 rpm for 5 min at 4 °C, and the ethanol was discarded. The precipitate was dried in a laminar flow hood and then 50 μl of dH2O was added to obtain total RNA from the maize leaves.

[0027] (2) cDNA synthesis

[0028] Using total RNA extracted from maize leaves as a template, the first strand of cDNA was synthesized by reverse transcription using the Vazyme HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper), which served as a template for PCR amplification. For specific operating procedures, please refer to the instruction manual.

[0029] (3) Primer design

[0030] The CDS sequence of the ZmRbcL_n1 gene was obtained by querying the gene number Zm00001d006402. The CDS sequence is shown in SEQ ID NO.1. Amplification primers containing homologous arms were designed using Primer Premier 5.0 software. The PCR primer sequences are shown in Table 1.

[0031] Table 1. PCR primers and their sequence information

[0032]

[0033] (4) PCR amplification

[0034] Using the synthesized cDNA as a template, and employing the designed PCR primers (Table 1), a high-fidelity enzyme 2×Hieff was used. PCR amplification was performed using 2× Hieff Canace Plus PCR Master Mix (With Dye). The PCR reaction mixture consisted of 25 μL of 2× Hieff Canace Plus PCR Master Mix, 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of corn cDNA, and 20 μL of ddH2O. The PCR program was 98℃ for 3 min; 98℃ for 20 s, 60℃ for 20 s, 72℃ for 60 s, for 33 cycles; 72℃ for 5 min. PCR products were then subjected to gel electrophoresis, as shown below. Figure 1 As shown, the target band was recovered by gel cutting and sequenced for verification, and the ZmRbcL_n1 gene was obtained.

[0035] 2.2 Structural analysis of the ZmRbcL_n1 protein

[0036] The ZmRbcL_n1 protein encoded by the ZmRbcL_n1 gene has the amino acid sequence shown in SEQ ID NO.4. The ZmRbcL_n1 protein differs in structure from traditional RbcL proteins. In addition to the RuBisco LargeN and RuBisco Large domains of classic RbcL proteins, its N-terminus also contains a Ubiq_cyt_C_ch ap molecular chaperone structure that promotes complex assembly. Figure 2 The sequence of the Ubiq_cyt_C_chap molecular chaperone structure is shown in SEQ ID NO.5.

[0037] 2.3 Analysis of ZmRbcL_n1 gene expression pattern

[0038] (1) RNA extraction

[0039] Trizol reagent was used according to the manufacturer's instructions. Approximately 0.1 g of roots, stems, and leaves from wild-type B73 maize were placed in 2.0 ml EP tubes, steel beads were added, and the tubes were frozen in liquid nitrogen for 5 min. The mixture was then ground into powder using a high-throughput grinder. 1 ml of Trizol reagent was added, and the mixture was vigorously mixed. The mixture was centrifuged at 12,000 rpm for 2 min at 4 °C, and the supernatant was transferred to a new EP tube. 200 μl of chloroform was added and the mixture was vigorously mixed. The mixture was allowed to stand at room temperature until separation, and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new EP tube, and 500 μl of isopropanol was added and mixed thoroughly. The mixture was precipitated at -20 °C for 30 min. The precipitate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was discarded. 1 ml of 75% ethanol was added to wash the precipitate, and the mixture was centrifuged at 12,000 rpm for 5 min at 4 °C. The ethanol was discarded, and the precipitate was dried in a laminar flow hood and then 50 μl of ddH2O was added to obtain total RNA from different maize tissues.

[0040] (2) cDNA synthesis

[0041] Using total RNA extracted from different maize tissues as templates, the first strand of cDNA was synthesized by reverse transcription using the Vazyme HiScriptIII 1st Strand cDNA Synthesis Kit (+gDNA wiper), which served as a template for qPCR amplification. For specific operating procedures, please refer to the instruction manual.

[0042] (3) Primer design for real-time PCR

[0043] Based on the CDS sequence of the ZmRbcL_n1 gene as shown in SEQ ID NO.1, primers for real-time PCR were designed using Primer Premier 5.0 software. The primer sequence information is shown in Table 2.

[0044] Table 2. Primers and their sequence information for quantitative real-time PCR

[0045]

[0046] (4) Real-time PCR

[0047] The AceQ qPCR SYBR Green Master Mix kit was used, with ZmActin as an internal control gene. The relative expression levels of ZmRbcL_n1 in different maize tissues were analyzed using a SLAN 8.2.2 real-time PCR instrument. The experiment included three replicates. Based on the Ct value at the plateau phase of the quantitative amplification curve, 2... -ΔΔCtThe analysis was performed using the following method. The qRT-PCR reaction system consisted of 10 μL of 2×AceQ qPCR SYBR Green Master Mix, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 1 μL of maize cDNA, and 9.2 μL of ddH2O. The PCR reaction program was 95℃ for 5 min; 95℃ for 10 s, 60℃ for 60 s, for 40 cycles.

[0048] The results are as follows Figure 3 As shown, the ZmRbcL_n1 gene is highly expressed in leaves.

[0049] 2.4 Construction of overexpression vectors and their infection in Arabidopsis thaliana

[0050] 2.4.1 Construction of overexpression vectors

[0051] (1) Linearization carrier

[0052] Using pCANBIA1300 as the vector backbone, the promoter of the maize ZmUBI gene (sequence shown in SEQ ID NO. 10) was amplified, and then the CaMV 35S promoter in the vector backbone was replaced to obtain a new overexpression vector 1300-g6-pZmUBI (e.g., Figure 4 (As shown). Using 1300-g6-pZmUBI as a vector, and SpeI and HindIII as restriction enzyme sites, the vector was linearized and large fragments were recovered by gel electrophoresis to obtain the vector digestion product.

[0053] (2) Homologous recombination and transformation

[0054] The ZmRbcL_n1 gene obtained in step 2.1 was homologously recombinated with the vector digestion product obtained in step (1), and the reaction was carried out at 50°C for 30 min, followed by immediate cooling on ice. The recombinant product was transformed into Escherichia coli DH5α competent cells and cultured overnight at 37°C.

[0055] (3) Identification and detection of positive colonies

[0056] Single colonies were selected for PCR verification. Positive bacterial cultures were expanded and plasmids were extracted. The plasmid extraction steps are described in the instruction manual. The samples were then sent to the company for sequencing and were found to be correct.

[0057] 2.4.2 Agrobacterium-mediated transformation

[0058] Remove *E. coli* DH5α competent cells stored at -80℃ and place on ice until thawed. Add 1 μg of the constructed plasmid to the competent cells, gently mix, and incubate on ice for 5 min; then flash freeze in liquid nitrogen for 5 min; incubate in a 37℃ water bath for 5 min, and immediately place on ice for 5 min. Add 500 μl of antibiotic-free LB agar, gently invert and mix several times, and incubate at 28℃, 180 rpm for 3 h. Centrifuge at 3000 rpm for 1 min. Discard the supernatant, retain approximately 200 μl, gently resuspend the cells by pipetting, and plate onto antibiotic-resistant plates containing rifampin, kanamycin, and gentamicin. Incubate at 28℃ until single colonies form.

[0059] 2.4.3 Infection of Arabidopsis thaliana

[0060] Pick a small amount of Agrobacterium and inoculate it into 20 ml of LB liquid medium containing rifampicin, kanamycin, and gentamicin resistance, and incubate overnight at 200 rpm. Then, re-inoculate 20 ml of the small culture medium into 200 ml of LB liquid medium and shake at 28°C and 200 rpm until OD reaches the target value. 600 The value was 1.0-1.6. Centrifuge at 3000 rpm for 10 min at room temperature. Discard the supernatant. Add approximately 50 ml of infection washing buffer (5% sucrose solution), resuspend the bacterial culture, and centrifuge at 3000 rpm for 10 min at room temperature. Add 100 ml of infection solution (5% sucrose solution containing 0.1 mM AS), resuspend the bacterial culture, and incubate at room temperature for 1 h. Immerse the entire Arabidopsis inflorescence in the infection solution for 15-30 s. Incubate in the dark for 24 h, then grow under low light for 24 h, followed by normal light culture.

[0061] 2.4.4 Screening and Identification of Positive Seedlings

[0062] Take an appropriate amount of seeds in a clean bench, wash away impurities and shriveled seeds with seed disinfectant, and sterilize by rotation in a rotary mixer for 20 minutes. Repeat once after changing the disinfectant, and then rinse 5 times with ddH2O. Mix thoroughly with a 1ml pipette and transfer to 1 / 2 MS medium containing Hgy resistance. Spread evenly on the plate, let it dry, seal the plate with sealing film, wrap it with aluminum foil, and stratify at 4℃ in the dark for 48 hours. Then treat with light in a greenhouse for 2 hours, and then place it in a greenhouse for in the dark. After about 2-4 days of incubation in the dark, transplant the seedlings with elongated hypocotyls into small pots and cover them with transparent lids. Let them grow under low light for two days, then under normal light. Two weeks later, cut a small amount of Arabidopsis leaves to extract genomic DNA, perform PCR identification using primers, and observe and record the phenotype.

[0063] 2.4.5. Quantitative Real-Time PCR Analysis

[0064] The specific operating steps are the same as described in section 2.3 above. The AceQ qPCR SYBR Green Master Mix kit was used, and the relative expression level of ZmRbcL_n1 in Arabidopsis leaves was analyzed using a SLAN 8.2.2 real-time PCR instrument. The experiment included three replicates. Based on the Ct value at the plateau phase of the quantitative amplification curve, 2... -ΔΔCt The method is used for analysis.

[0065] Based on the results of quantitative real-time PCR analysis, two Arabidopsis thaliana plants with high expression of the ZmRbcL_n1 gene were identified and numbered OE-2# and OE-8#, respectively.

[0066] 2.5 Effects of ZmRbcL_n1 gene overexpression on biomass accumulation and seed yield in Arabidopsis thaliana

[0067] After self-pollination, OE-2# and OE-8# plants were cultured under normal light. Once the seeds of the T1 generation matured, the aboveground parts and pods of individual plants were collected, dried, and weighed after complete drying to determine the total biomass. After complete drying, the seed pods were removed, and the total weight of seeds per plant was determined. Additionally, Arabidopsis plants normally expressing the ZmRbcL_n1 gene served as a control group, receiving the same treatment as described above.

[0068] The results are as follows Figure 5 As shown, Arabidopsis plants OE-2# and OE-8# overexpressing the ZmRbcL_n1 gene exhibited more vigorous growth and accumulated more biomass (dry weight) compared to the control group, with a significant increase in the total seed weight per plant. This indicates that the ZmRbcL_n1 gene has great potential for enhancing plant photosynthesis and increasing carbon fixation efficiency.

[0069] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of the maize ZmRbcL_n1 gene in improving plant photosynthetic efficiency, characterized in that... The plant in question is Arabidopsis thaliana or maize. The sequence of the protein encoded by the maize ZmRbcL_n1 gene is shown in SEQ ID NO.4, and the sequence of the protein encoded by the maize ZmRbcL_n1 gene contains the Ubiq_cyt_C_chap molecular chaperone structure.

2. The application according to claim 1, characterized in that, The CDS sequence of the maize ZmRbcL_n1 gene is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The sequence of the Ubiq_cyt_C_chap molecular chaperone structure is shown in SEQ ID NO.

5.

4. The application according to claim 1, characterized in that, The application specifically refers to promoting at least one of plant biomass accumulation or seed yield.

5. The application according to claim 1, characterized in that, The application involves transferring an expression vector overexpressing the maize ZmRbcL_n1 gene into plants to improve photosynthetic efficiency.

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