Application of corn ZmRbcLn1 gene in improvement of photosynthesis efficiency of plants

Abstract

The invention discloses application of a corn ZmRbcLn1 gene in improvement of plant photosynthesis efficiency, and relates to the technical field of plant genetic engineering. Specifically, a protein coded by the corn ZmRbcLn1 gene is different from a traditional RbcL protein structure, the structure of the protein has a chaperone structural domain for promoting compound assembly, and fluorescent quantification shows that the corn ZmRbcLn1 gene is highly expressed in leaves, which indicates that the protein can promote photosynthetic carbon sequestration of the leaves by promoting mechanisms such as RuBisco compound assembly. Furthermore, an expression vector of the overexpressed corn ZmRbcLn1 gene is constructed and transferred into an arabidopsis thaliana plant through an agrobacterium-mediated method, it is found that the overexpressed corn ZmRbcLn1 gene can promote arabidopsis thaliana biomass accumulation and seed yield, and therefore it is indicated that the corn ZmRbcLn1 gene has a huge application prospect in the aspects of improving plant photosynthesis and increasing carbon sequestration efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically relates to the application of a maize ZmRbcL_n1 gene in improving the photosynthesis efficiency of plants. Background Art

[0002] Photosynthesis is the source power for the accumulation of organic matter on the earth, and is directly related to the total biomass of plants and the upper limit of crop yields. Photosynthesis consists of two parts: the light reaction and the dark reaction (Calvin cycle). The Rubisco complex catalyzes the first reaction of the "Calvin cycle" to fix atmospheric CO 2 onto a five-carbon substrate. However, the catalytic efficiency of this complex is low, which limits the photosynthesis efficiency.

[0003] The RuBisco complex is composed of two subunits (the large subunit RbcL and the small subunit RbcS). Among them, RbcL is the catalytically active subunit. RbcL and RbcS must be finally assembled into an active L 8 S 8 16 polymerized mature complex with the help of some auxiliary proteins such as molecular chaperones. RbcL is very conservative in evolution. People have been looking for RbcL proteins with stronger functions and at the same time improving the activity of RbcL through directed screening and site-directed editing, but with little success. Identifying new functional RbcL genes and utilizing them will help improve the photosynthesis efficiency of plants, promote biological carbon fixation, and increase crop yields. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a maize ZmRbcL_n1 gene in improving the photosynthesis efficiency of plants.

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

[0006] The first purpose of the present invention is to provide the application of a maize ZmRbcL_n1 gene in improving the photosynthesis efficiency of plants.

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

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

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

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

[0011] As a further optimization solution of the present invention, the application is to transfer an expression vector overexpressing the maize ZmRbcL_n1 gene into a plant to promote the improvement of plant photosynthesis efficiency.

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

[0013] The maize ZmRbcL_n1 gene provided by the present invention encodes a protein with a different structure from the traditional RbcL protein. The structure of its protein has a molecular chaperone domain that promotes complex assembly. And fluorescence quantification 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 the assembly of the RuBisco complex. Further, by constructing an expression vector overexpressing the maize ZmRbcL_n1 gene and transferring it into Arabidopsis thaliana plants through the Agrobacterium-mediated method, it is found that overexpressing this gene can promote the biomass accumulation and seed yield of Arabidopsis thaliana. Thus, it shows that the maize ZmRbcL_n1 gene has great application prospects in improving plant photosynthesis and increasing carbon fixation efficiency. Description of the Drawings

[0014] Figure 1 It is the gel electrophoresis diagram for amplifying the ZmRbcL_n1 gene provided by the present invention;

[0015] Figure 2 It is the structural analysis of the ZmRbcL_n1 protein provided by the present invention;

[0016] Figure 3 It is the analysis of the expression pattern of the ZmRbcL_n1 gene provided by the present invention;

[0017] Figure 4 It is the vector map provided by the present invention;

[0018] Figure 5 It is the effect of overexpressing the ZmRbcL_n1 gene on the phenotype, above-ground biomass accumulation and total seed mass per plant of Arabidopsis thaliana provided by the present invention. Detailed Embodiments

[0019] The following further describes the present application in detail with reference to the drawings. It is necessary to point out here that the following detailed embodiments are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0020] 1. Materials and Reagents

[0021] The materials, reagents, etc. used in this example can be obtained through commercial channels without special instructions.

[0022] 2. Methods

[0023] For the specific experimental methods not specified in this example, they can all be carried out according to conventional methods.

[0024] 2.1 Obtaining the Maize ZmRbcL_n1 Gene

[0025] (1) RNA Extraction

[0026] It was carried out using Trizol reagent according to the manufacturer's instructions. Take about 0.1 g of wild-type B73 maize leaves and put them into a 2.0 ml EP tube, add steel beads, freeze in liquid nitrogen for 5 min, grind them into powder with a high-throughput grinder, add 1 ml of Trizol reagent, and mix vigorously. Centrifuge at 12,000 rpm at 4℃ for 2 min, and transfer the supernatant to a new EP tube. Add 200 μl of chloroform and mix vigorously, let it stand at room temperature for a while, wait for the mixture to separate, and centrifuge at 12,000 rpm at 4℃ for 10 min. Transfer the supernatant to a new EP tube, add 500 μl of isopropanol and mix well, precipitate at -20℃ for 30 min. Centrifuge at 12,000 rpm at 4℃ for 10 min, discard the supernatant. Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm at 4℃ for 5 min, discard the ethanol, dry it in a laminar flow bench and then add 50 μl of ddH 2 O to obtain the total RNA of maize leaves.

[0027] (2) cDNA Synthesis

[0028] Using the total RNA of maize leaves extracted as a template, reverse transcribe to synthesize the first strand of cDNA using Vazyme reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper), which serves as the template for PCR amplification. The specific operation steps refer to the instruction manual.

[0029] (3) Primer Design

[0030] Query the CDS sequence of the ZmRbcL_n1 gene according to the gene number Zm00001d006402 of the ZmRbcL_n1 gene. The CDS sequence is shown as SEQ ID NO.1. Use Primer Premier 5.0 software to design amplification primers containing homologous arms. 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, with the designed PCR primers (Table 1), high-fidelity enzyme 2×Hieff Plus PCR Master Mix (With Dye) was used for PCR amplification. The PCR reaction system was 25 μL of 2×Hieff Canace Plus PCR Master Mix, 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of maize cDNA, and ddH 2 O 20 μL. The PCR reaction program was 98°C for 3 min; 98°C for 20 s, 60°C for 20 s, 72°C for 60 s, for 33 cycles; 72°C for 5 min. The PCR products were subjected to gel electrophoresis. As Figure 1 shown, the target band was cut out from the gel, recovered, and sequenced for verification to obtain the ZmRbcL_n1 gene.

[0035] 2.2. Structural Analysis of 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 has a different structure from the traditional RbcL protein. In addition to the RuBisco Large N and RuBisco Large domains of the classical RbcL protein, the N-terminus also contains a Ubiq_cyt_C_ch ap molecular chaperone structure that can promote complex assembly ( Figure 2 ), and 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] It was carried out using Trizol reagent according to the manufacturer's instructions. Approximately 0.1 g of the roots, stems, and leaves of wild-type B73 maize were separately placed into 2.0 ml EP tubes, steel beads were added, and they were frozen in liquid nitrogen for 5 min, ground into powder using a high-throughput grinder, 1 ml of Trizol reagent was added, and they were vigorously mixed evenly. Centrifuge at 12,000 rpm at 4 °C for 2 min, and transfer the supernatant to a new EP tube. Add 200 μl of chloroform and mix vigorously, let it stand at room temperature for a while until the mixture is layered, and centrifuge at 12,000 rpm at 4 °C for 10 min. Transfer the supernatant to a new EP tube, add 500 μl of isopropanol and mix well, and precipitate at -20 °C for 30 min. Centrifuge at 12,000 rpm at 4 °C for 10 min, discard the supernatant. Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm at 4 °C for 5 min, discard the ethanol, and after drying in a laminar flow bench, add 50 μl of ddH 2 O to obtain the total RNA of different tissues of maize.

[0040] (2) cDNA synthesis

[0041] Using the total RNA of different tissues of maize obtained by extraction as a template, reverse transcription was performed using the Vazyme reverse transcription kit HiScriptIII 1st Strand cDNA Synthesis Kit (+gDNA wiper) to synthesize the first strand of cDNA, which was used as the template for qPCR amplification. The specific operation steps refer to the instruction manual.

[0042] (3) Design of fluorescence quantitative PCR primers

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

[0044] Table 2. Fluorescence quantitative PCR primers and their sequence information

[0045]

[0046] (4) Fluorescence quantitative PCR

[0047] Using the AceQ qPCR SYBR Green Master Mix kit, with ZmActin as the internal reference gene, the relative expression levels of ZmRbcL_n1 in different tissues of maize were analyzed using a SLAN8.2.2 fluorescence quantitative PCR instrument. The experiment included 3 replicates. According to the Ct value when the quantitative amplification curve reached the plateau, 2 -ΔΔCtAnalysis was performed by the method. The qRT-PCR reaction system was 10 μL of 2×AceQ qPCR SYBR Green Master Mix, 0.4 μL of the upstream primer, 0.4 μL of the downstream primer, 1 μL of maize cDNA, and 9.2 μL of ddH 2 O. The PCR reaction program was 95°C for 5 min; 95°C for 10 s, 60°C for 60 s, for 40 cycles.

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

[0049] 2.4. Construction of the overexpression vector and infection of Arabidopsis thaliana

[0050] 2.4.1. Construction of the overexpression vector

[0051] (1) Linearized vector

[0052] Using pCANBIA1300 as the vector backbone, after amplifying the promoter of the maize ZmUBI gene (the sequence is shown in SEQ ID NO. 10), the CaMV 35S promoter in the vector backbone was replaced to obtain the new overexpression vector 1300-g6-pZmUBI (as Figure 4 shown). Using 1300-g6-pZmUBI as the vector, with SpeI and HindIII as the restriction enzyme sites, linearization was performed and the large fragment was 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 above was subjected to homologous recombination with the vector digestion product obtained in step (1), reacted at 50°C for 30 min, and then immediately placed on ice for cooling. The recombinant product was transformed into competent Escherichia coli DH5α cells and cultured overnight at 37°C.

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

[0056] Single clone colonies were picked for PCR verification. After the positive bacterial liquid was expanded and cultured, plasmid extraction was performed. The plasmid extraction steps are shown in the instruction manual, and the company was sent for correct sequencing.

[0057] 2.4.2. Agrobacterium transformation

[0058] Take out the Escherichia coli DH5α competent cells stored at -80°C and thaw them on ice. Add 1 μg of the constructed plasmid to the competent cells, gently mix by flicking, and incubate on ice for 5 min; quickly freeze in liquid nitrogen for 5 min; incubate in a 37°C water bath for 5 min, immediately place on ice, and let stand for 5 min. Add 500 μl of antibiotic-free LB, gently invert and mix several times, and shake at 28°C and 180 rpm for 3 h. Centrifuge at 3000 rpm for 1 min. Discard the supernatant, leave about 200 μl, gently pipette to resuspend the cells, spread them on a resistance plate containing rifampicin, kanamycin, and gentamicin, and culture at 28°C until single colonies form.

[0059] 2.4.3. Infect Arabidopsis thaliana

[0060] Pick Agrobacterium and inoculate it into 20 ml of LB liquid medium containing rifampicin, kanamycin, and gentamicin resistance, and culture overnight at 200 rpm. Re-inoculate 20 ml of the small culture broth into 200 ml of LB liquid medium, shake the bacteria at 28°C and 200 rpm until the OD 600 is 1.0 - 1.6. Centrifuge at 3000 rpm for 10 min at room temperature. Discard the supernatant. Add about 50 ml of infection washing solution (5% sucrose solution) to resuspend the bacterial solution, 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) to resuspend the bacterial solution, and let stand at room temperature for 1 h. Immerse all the inflorescences of Arabidopsis thaliana in the infection solution and infect for 15 - 30 s. Incubate in the dark for 24 h, grow under weak light for 24 h, and then culture under normal light.

[0061] 2.4.4. Screening and identification of positive seedlings

[0062] Take an appropriate amount of seeds in the ultra-clean bench, use the seed disinfectant solution to wash away impurities and shriveled seeds, and rotate and disinfect for 20 min on a rotary mixer. Repeat once after changing the disinfectant solution, and then rinse 5 times with ddH 2 O. Transfer them to a 1 / 2MS medium containing Hgy resistance after pipetting evenly with a 1 ml pipette, spread them evenly on the plate, let it dry, then seal the plate with a sealing film, wrap it with tin foil, and stratify at 4°C in the dark for 48 h. Treat with light in the greenhouse for 2 h, and then culture in the greenhouse in the dark. After culturing in the dark for about 2 - 4 d, transfer the seedlings with elongated hypocotyls to small pots and cover them with a transparent lid. Grow under weak light for two days and then grow under normal light. After two weeks, cut a small amount of Arabidopsis thaliana leaves to extract genomic DNA, perform PCR identification with primers, and observe and record the phenotypes.

[0063] 2.4.5. Fluorescent quantitative PCR analysis

[0064] The specific operation steps are the same as those in 2.3 above. Using the AceQ qPCR SYBR Green Master Mix kit, the relative expression levels of ZmRbcL_n1 in Arabidopsis thaliana leaves were analyzed using the SLAN 8.2.2 fluorescence quantitative PCR instrument. The experiment included 3 replicates. According to the Ct values at the plateau stage of the quantitative amplification curve, the 2 -ΔΔCt method was used for analysis.

[0065] According to the results of fluorescence quantitative PCR analysis, two Arabidopsis thaliana plants with high expression of the ZmRbcL_n1 gene were found, and they were numbered OE-2# and OE-8# respectively.

[0066] 2.5 Effects of Overexpressing the ZmRbcL_n1 Gene on Biomass Accumulation and Seed Yield of Arabidopsis thaliana

[0067] The OE-2# and OE-8# plants were self-crossed and then cultured under normal light. After the seeds of the T1 generation matured, the above-ground parts and pods of each individual plant were collected, air-dried. After the above-ground parts were completely dry, their total biomass was weighed and counted. After the seed pods were completely dry, the seed pods were removed, and the total weight of the seeds of each individual plant was weighed and counted. In addition, Arabidopsis thaliana plants with normal expression of the ZmRbcL_n1 gene were used as the control group, and the same treatment as described above was carried out on the control group.

[0068] The results are as Figure 5 shown. Compared with the control group, the Arabidopsis thaliana plants OE-2# and OE-8# overexpressing the ZmRbcL_n1 gene grew more vigorously and accumulated more biomass (dry weight), and the total weight of the seeds per individual plant was also significantly increased. This indicates that the ZmRbcL_n1 gene has great application prospects in enhancing plant photosynthesis and increasing carbon fixation efficiency.

[0069] The above-described embodiments only represent several implementation modes of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Application of maize ZmRbcL_n1 gene in improving plant photosynthesis efficiency.

2. The use according to claim 1, characterized in that: The CDS sequence of corn ZmRbcL_n1 is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The sequence of the protein encoded by the maize ZmRbcL_n1 gene is shown in SEQ ID NO.

4. The sequence of the protein encoded by the maize ZmRbcL_n1 gene contains a Ubiq_cyt_C_chap molecular chaperone structure. The sequence of the Ubiq_cyt_C_chap molecular chaperone structure is shown in SEQ ID NO.

5.

4. The use according to claim 1, characterized in that: The application is specifically to promote at least one of plant biomass accumulation or seed yield.

5. The use according to claim 1, characterized in that: The plant is Arabidopsis thaliana or corn.

6. The use according to claim 1, characterized in that: The application is to transfer the expression vector for overexpressing the corn ZmRbcL_n1 gene into the plant to promote the improvement of the photosynthesis efficiency of the plant.

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