Plant chlorophyll-de-phytating enzyme cde1 and application thereof

Abstract

This invention relates to the field of agricultural technology, specifically to a plant chlorophyll aphytase CDE1 and its applications. The nucleotide sequence of the plant chlorophyll aphytase CDE1 is shown in SEQ ID NO.1. Overexpression of the plant chlorophyll aphytase CDE1 provided by this invention can improve plant photosynthetic efficiency. Overexpressed CDE1 significantly increases the biomass of Arabidopsis thaliana and can be used to construct high-photometric plants in high-light environments. CDE1, as a chlorophyll aphytase, is highly conserved in higher plants. Research results in Arabidopsis thaliana can be applied to crops such as rice and corn, as well as other fruits and vegetables, and have potential application value for increasing grain yield and enhancing plant stress resistance.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a plant chlorophyll deplantylase CDE1 and its applications. Background Technology

[0002] Chlorophyll (Chl) plays a crucial role in the absorption, transfer, and conversion of light energy during photosynthesis. Higher plants primarily synthesize two types of chlorophyll: Chl a and Chl b. Chl a is mainly distributed in the photosystem (PS) reaction center and the light-harvesting complex (LHC), while Chl b is found only in the LHC. In addition, plants also possess pheophytin a (Phein a), which serves as the first electron acceptor in the photosystem II (PSII) reaction center, binding to the core proteins D1 and D2. Numerous studies have shown that the PSII complex-catalyzed water splitting process to form protons, electrons, and oxygen is the most intense redox reaction discovered in organisms to date, inevitably leading to damage to the reaction center. The D1 protein is most vulnerable to damage, therefore, the continued progress of photosynthesis depends on the PSII repair process.

[0003] The repair of PSII (photoprotective neuropathy) is mainly achieved through the degradation and resynthesis of damaged D1 protein. For a long time, research on the PSII repair process has focused primarily on the degradation, synthesis, and assembly of reaction center proteins. However, little is known about the metabolic involvement of chlorophyll, which binds to the core D1 protein, and how it participates in the degradation and reorganization of the core protein. Previous studies have found that the aphytase reaction catalyzed by Arabidopsis thaliana chlorophyllase 1 (CLH1) releases chlorophyll bound to the D1 protein, promoting the degradation of damaged D1 protein and thus providing photosensitivity in young leaves. Therefore, the degradation and turnover of chlorophyll bound to the core protein plays a crucial role in PSII repair.

[0004] Phytylase-catalyzed chlorophyll deplantation is considered the first step in chlorophyll degradation. Currently, three phytylases have been identified in higher plants: pheophytin hydrolase (PPH), chlorophyll deplantase 1 (CLD1), and chlorophyllase (CLH). PPH mainly plays a role in chlorophyll degradation during leaf senescence; CLD1 participates in chlorophyll degradation under heat stress, but its in vitro activity is much lower than that of CLH; CLH1 participates in chlorophyll degradation during PSII repair in young Arabidopsis leaves, providing photosensitivity protection. Given the limitations of these three enzymes, other chlorophyll deplantases catalyze chlorophyll degradation during PSII repair in mature leaves.

[0005] Therefore, identifying the apoptotic enzymes involved in the PSII repair process is crucial for elucidating the fate of chlorophyll molecules within PSII and is an integral part of understanding the PSII repair mechanism. Improving the PSII repair mechanism and enhancing crop photosynthetic efficiency are of great significance. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a plant chlorophyll apophysylase CDE1 and its applications. Overexpression of CDE1 can improve plant photosynthetic efficiency; overexpressed CDE1 significantly increases the biomass of Arabidopsis thaliana and can be used to construct high-photometric plants in high-light environments. CDE1, as a chlorophyll apophysylase, is highly conserved in higher plants. Research findings in Arabidopsis thaliana can be applied to crops such as rice and maize, as well as other fruits and vegetables, showing potential value in increasing grain yield and enhancing plant stress resistance.

[0007] Therefore, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a plant chlorophyll dephysylase CDE1 in an optional embodiment, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Preferably, the plant chlorophyll deplantidase CDE1 is overexpressed to improve the photosynthetic efficiency of the plant and increase the plant weight of the above-ground parts; the plant includes Arabidopsis thaliana, rice, corn or tomato, and may also include other fruits and vegetables.

[0010] Secondly, in an optional embodiment, the present invention provides a plant overexpression vector comprising the aforementioned plant chlorophyll deplantidase CDE1.

[0011] Thirdly, in an optional embodiment, the present invention provides a method for constructing the above-mentioned plant overexpression vector, comprising the following steps:

[0012] After cloning the coding region sequence of the above-mentioned plant chlorophyll deplantase CDE1, it was ligated into the binary vector pCambia1300 driven by the 35S promoter to obtain the plant overexpression vector.

[0013] Fourthly, in an optional embodiment, the present invention provides the application of the above-mentioned plant chlorophyll apophysylase CDE1 in improving the photosynthetic efficiency of plants and increasing the weight of the aboveground parts of plants.

[0014] Preferably, the application includes constructing a plant overexpression vector from the above-mentioned plant chlorophyll apophysylase CDE1, transforming it into Agrobacterium, then infecting wild-type Arabidopsis thaliana seeds, disinfecting the collected T0 generation seeds with seed disinfectant for 15 min, washing them three times with sterile water for 3 min each time, then suspending them in sterile water, spreading them evenly on MS medium containing hygromycin and carboxybenzylmycin, transplanting the transformed plants with roots and true leaves into soil culture, extracting RNA for overexpression level identification, harvesting T2 generation seeds, and isolating and identifying transgenic overexpression homozygous lines on resistance plates. The photosynthetic efficiency of the transgenic overexpression homozygous lines is improved, and the weight of the aboveground parts of the plants is increased.

[0015] Fifthly, in an optional embodiment, the present invention provides the application of the above-mentioned plant chlorophyll deplantylase CDE1 in the degradation of chlorophyll.

[0016] Preferably, the degraded chlorophyll comprises degraded chlorophyll a, chlorophyll b, and pheophytin a.

[0017] In a sixth aspect, the present invention provides, in an optional embodiment, the application of the above-mentioned plant chlorophyll deplantylase CDE1 in the PSII repair process.

[0018] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0019]

[0020] Compared with the prior art, the present invention has one of the following beneficial effects:

[0021] 1. The chlorophyll aphytase CDE1 provided by this invention can improve plant photosynthetic efficiency after overexpression. Overexpressed CDE1 significantly increases the biomass of Arabidopsis thaliana and can be used to construct high-photometric plants in high-light environments. CDE1, as a chlorophyll aphytase, is highly conserved in higher plants. The research results in Arabidopsis thaliana can be applied to crops such as rice and corn, as well as other fruits and vegetables, and have potential application value for increasing grain yield and enhancing plant stress resistance. Attached Figure Description

[0022] Figure 1 This refers to the catalysis of Arabidopsis CDE1 on chlorophyll a, chlorophyll b, and pheophytin a substrates in vitro, as shown in Example 1.

[0023] Figure 2 The enzyme kinetics curve of CDE1 in Example 1;

[0024] Figure 3 This serves as a biochemical verification of the site-directed mutagenesis of the CDE1 enzyme active site in Example 1;

[0025] Figure 4 The target sequence location for simultaneous knockout of the CDE1 gene using CRISPR-Cas9 technology in Example 2;

[0026] Figure 5 The mutation details of the CDE1 knockout homozygous mutant constructed using CRISPR-Cas9 technology in Example 2 are shown.

[0027] Figure 6 The phenotype of the cde1-1 mutant obtained by constructing it using CRISPR-Cas9 technology in Example 2 under normal conditions;

[0028] Figure 7 The cell death phenotype of the cde1-1 mutant constructed using CRISPR-Cas9 technology in Example 2 under high light.

[0029] Figure 8 The maximum chlorophyll fluorescence Fv / Fm change of the cde1-1 mutant constructed using CRISPR-Cas9 technology before and after high light intensity in Example 2;

[0030] Figure 9 This refers to the subcellular localization of CDE1 in cells, as shown in Example 2.

[0031] Figure 10The results of semi-quantitative RT-PCR detection in transgenic CDE1 overexpressing plants in Example 3;

[0032] Figure 11 The aboveground biomass of the transgenic CDE1 overexpressing plant in Example 3 is greater than that of the wild type. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] In the following examples, the Arabidopsis thaliana solid medium was 1 / 2 MS, with the following formulation: 1% sucrose, 2.15 g / L Murashige and Skoog basal salt mixtures (Sigma), 0.7% Phytol agar (Duchefa Biochemie), pH = 5.7. The MS plates with seeds were placed on a light rack and cultured at 22℃ ± 2, 70-80 μmol / L. -2 s -1 Cultivation was carried out under light intensity and light cycle (light / dark) conditions of 16 h / 8 h. After 7-10 days, the plants were transplanted into the soil with soil conditions of black soil:vermiculite:perlite = 5:4:1. The greenhouse cultivation conditions were the same as described above.

[0037] Escherichia coli TOP10 strains were purchased from Thermo Fisher Scientific (Invitrogen); Agrobacterium strain GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0038] Construction of overexpression vectors for plant chlorophyll apophysylase CDE1 and screening of plants overexpressing the CDE1 gene

[0039] After cloning the CDE1 coding region sequence, it was ligated into the binary vector pCambia1300 driven by the 35S promoter to obtain the CDE1 overexpression vector.

[0040] The CDE1 overexpression vector was transformed into Agrobacterium, and then wild-type Arabidopsis seeds were inoculated. The collected T0 generation Arabidopsis seeds were disinfected with seed disinfectant for 15 min, washed three times with sterile water for 3 min each time, and then suspended in sterile water. They were evenly spread on MS medium containing hygromycin and carboxybenzin. Transformed plants with roots and true leaves were transplanted into soil for culture. RNA was extracted for overexpression level identification. After harvesting T2 generation seeds, transgenic overexpression homozygous lines were isolated and identified on resistance plates to obtain overexpressing gene plants.

[0041] Construction of a knockout vector for the plant chlorophyll apoptase CDE1 and screening of plants with the CDE1 gene knocked out.

[0042] Using Arabidopsis thaliana as the wild-type Columbia ecotype Col-0 background, gene knockout was performed using CRISPR-Cas9 technology. Suitable target sequences were searched on the target sequence design website CRISPR-GE (http: / / skl.scau.edu.cn / ). The target sequence needed to meet the following characteristics: (1) located on an exon and close to the 5'UTR region; (2) low off-target probability; (3) GC content between 40% and 70%. The selected CDE1 gene knockout target site in this embodiment is described in [reference needed]. Figure 4 The selected target sequence is then ligated into the AtU6:sgRNA-pYAO:Cas9 vector plasmid to construct the gene knockout vector.

[0043] Transform the correctly sequenced monoclonal microplasmid into Agrobacterium and infect plants to harvest T0 generation seeds. T0 generation seeds are screened on hygromycin resistance plates, and resistant seedlings are transplanted into soil for growth. Sampling and identification can be performed after approximately two weeks. If the transgenic plant has a phenotype, select 2-3 phenotyped plants and extract DNA; if not, select 15 positive seedlings from the same target sequence for DNA extraction in the first batch. Use M13F+sgRNA-R to identify transgenic insertion. Design primers that cross the target sequence to amplify the DNA fragment for PCR amplification. The fragment length is typically 400-800 bp, ensuring that one sequencing reaction can detect the target sequence region. Send 5 positive seedlings for the first batch for each target sequence. If no ideal edited plant is obtained, send another 5 seedlings, with a maximum of 15 seedlings sent. If only heterozygous plants are obtained from the T1 generation, propagate for the first generation. The resulting T2 generation plants are first identified by M13F+sgRNA-R (or AtyaoR universal primers) PCR. Plants without transgenic insertion are Cas9-free plants. Amplify the target sequence region of Cas9-free plants and sequence to identify homozygotes. Send 5 plants for the first test. If no ideal homozygous plants are obtained, send another 5 plants for the test. The maximum number of plants sent for the test shall not exceed 15.

[0044] Example 1

[0045] Basic biochemical enzymatic characteristics of the CDE1 gene, a plant chlorophyll deplantylase

[0046] To investigate the function of the Arabidopsis thaliana chlorophyll aphytase CDE1, the basic biochemical enzymatic characteristics of CDE1 were analyzed. Chlorophyll a (Chlorophyll a, Chl a) and chlorophyll b (Chlorophyll b, Chlb) were purchased from Sigma-Aldrich, and pheophytin a (Phein a) was purchased from Fujifilm. The concentrations of Chl a, b, and Pheiin a were calculated based on the standard curve. The chlorophyll standards were prepared to a concentration of 625 μM and reacted with purified CDE1 protein at 28°C for 20 min. The reaction was terminated with four volumes of acetone, and the characteristics of the reaction products were examined using HPLC. For the examination of enzyme kinetic parameters, the standards were serially diluted to 625 μM, 125 μM, 75 μM, 25 μM, 5 μM, and 1 μM, and reacted with purified CDE1 protein under the same conditions. The extract obtained after terminating the reaction was filtered through a 0.22 μm membrane (Millipore, USA) and can be directly used for high-performance liquid chromatography (HPLC) and spectroscopic analysis of samples.

[0047] HPLC was performed using an Agilent 1100 HPLC system (Agilent, Germany), equipped with an Agilent G1311A pump, an autosampler, and a G1314A UV-vis variable wavelength detector. Chromatographic conditions were: C18 column (4.6 mm × 250 mm, Nucleodur, Germany), detection temperature 35℃, and flow rate 0.5 mL / min. -1 The injection volume was 20 μL, and the UV detection wavelength was 435 nm / 635 nm. The mobile phase and elution program used a gradient elution with two solvents: A, methanol:water (80:20, v / v); B, ethyl acetate. For the first 20 min, solvent A was continuously added to solvent B until the ratio reached 50:50. Elution continued at this ratio for another 20 min. The types of compounds in the sample were roughly determined by comparing the peak times with the retention times of the standards. See [Results are attached]. Figures 1-3 .

[0048] like Figure 1 As shown, CDE1 can degrade chlorophyll a, chlorophyll b, and pheophytin a in vitro.

[0049] like Figure 2As shown, at the same substrate concentration (625 μM), CDE1 exhibits high catalytic activity towards chlorophyll a and pheophytin a, and the enzyme kinetic curves show that its activity towards pheophytin a reaches saturation very quickly, indicating that pheophytin a is the optimal substrate for CDE1.

[0050] like Figure 3 As shown, CDE1 possesses conserved serine S219, aspartic acid D399, and histidine H427 sites. An expression vector was constructed using site-directed mutagenesis, and enzyme activity was detected in vitro. The results showed that neither the DN nor the MBP tag catalyzed chlorophyll. Mutating the S219 or D399 sites completely eliminated the hydrolytic activity of the recombinant protein; mutagenesis of the H427 site resulted in the recombinant protein retaining only about 10% of its activity. This confirms that S219, D399, and H427 are the core catalytic sites of CDE1.

[0051] Example 2

[0052] Arabidopsis CDE1 knockout mutant phenotype

[0053] Design a target sequence (TCTCTCTCTGGTGGCTTCC) that targets CDE1, the location of which on the gene is as follows: Figure 4 As shown, the gene knockout vector AtU6:sgRNA-pYAO:Cas9 was constructed, and Agrobacterium-mediated transformation of wild-type Arabidopsis plants was performed, yielding T0 generation seeds. The T0 generation seeds were screened on a hygromycin-resistant medium. After transplanting and growth, leaf DNA was extracted from the plants, and PCR was used to identify genomic changes in the target sequence region, screening for homozygous mutants of the CDE1 gene. The mutation sites in the cde1-1 homozygous mutant are shown below. Figure 5 As shown.

[0054] like Figure 6 As shown, the CDE1 knockout mutant cde1-1, generated by CRISPR-Cas9 gene editing, showed no significant difference in phenotype compared to the wild type after 14 days of growth under normal conditions. When the CDE1 gene was reintroduced into the cde1-1 mutant, the phenotype of the reintroduced plant was restored, indicating that the phenotype of cde1-1 is caused by the CDE1 gene mutation.

[0055] like Figure 7 As shown, wild-type plants, cde1-1 mutant plants, and Arabidopsis thaliana plants with the CDE1 gene reintroduced were grown normally on 1 / 2 MS medium for 10 days, then transplanted into soil and cultured for another 14 days before being transferred to 1000 μmol / L medium. -2 s -1When treated under high light, the cde1-1 mutant exhibited a clear cell death phenotype, while the CDE1 gene reintroduction in plants restored the mutant phenotype.

[0056] like Figure 8 As shown, wild-type plants, cde1-1 mutant plants, and CDE1 overexpressing plants were transferred to 1000 μmol / L PVC. -2 s -1 When treated under high light, the photosynthetic efficiency of the cde1-1 mutant decreased significantly, while the maximum photosynthetic efficiency of PSII (Fv / Fm) in the CDE1 overexpressing plants was higher than that of the WT plants, indicating that the CDE1 overexpressing plants have higher photosynthetic efficiency under high light.

[0057] like Figure 9 As shown, the promoter and genomic coding sequence of the CDE1 gene were constructed into the pCambia1300 vector, transformed into the cde1-1 mutant, and transgenic Arabidopsis plants were obtained. Observation under a laser confocal fluorescence microscope revealed that CDE1 is located on the chlorophyll inner membrane and thylakoid membrane under normal growth conditions.

[0058] Example 3

[0059] The phenotype of Arabidopsis CDE1 gene overexpressing transgenic plants was studied, and the function of CDE1 was further investigated by constructing overexpressing transgenic lines.

[0060] like Figure 10 As shown, the relative expression level of CDE1 gene mRNA in CDE1 overexpression lines was detected, and it was found that the relative expression level of CDE1 mRNA was significantly higher than that of wild type.

[0061] like Figure 11 As shown, the CDE1 overexpression lines obtained through transgenic technology have a larger aboveground biomass than the wild-type lines.

[0062] The above results indicate that CDE1 overexpression can improve the photosynthetic efficiency of plants and increase the plant weight of the aboveground parts.

[0063] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A plant chlorophyll aphytylase CDE1, characterized in that, The nucleotide sequence of the plant chlorophyll deplantylase CDE1 is shown in SEQ ID NO.

1.

2. The plant chlorophyll aphytase CDE1 according to claim 1, characterized in that, The plant chlorophyll apophysylase CDE1, through overexpression, improves the photosynthetic efficiency of plants and increases the plant weight of the above-ground parts. The plants mentioned include Arabidopsis thaliana, rice, corn, or tomato.

3. A plant overexpression vector, characterized in that, Includes the plant chlorophyll deplantylase CDE1 as described in claim 1 or 2.

4. A method for constructing the plant overexpression vector according to claim 3, characterized in that, Includes the following steps: After cloning the coding region sequence of the plant chlorophyll deplantase CDE1 as described in claim 1, it was ligated into the binary vector pCambia1300 driven by the 35S promoter to obtain the plant overexpression vector.

5. The application of the plant chlorophyll deplantidase CDE1 as described in claim 1 or 2 in improving the photosynthetic efficiency of plants and increasing the weight of the aboveground parts of plants.

6. The application according to claim 5, characterized in that, The application includes constructing a plant overexpression vector of the plant chlorophyll apophysylase CDE1 as described in claim 1 or 2, transforming it into Agrobacterium, then infecting wild-type Arabidopsis thaliana seeds, disinfecting the collected T0 generation seeds with seed disinfectant for 15 min, washing them three times with sterile water for 3 min each time, then suspending them in sterile water, spreading them evenly on MS medium containing hygromycin and carboxybenzylmycin, transplanting the transformed plants with roots and true leaves into soil culture, extracting RNA for overexpression level identification, harvesting T2 generation seeds, and isolating and identifying transgenic overexpression homozygous lines on resistance plates. The photosynthetic efficiency of the transgenic overexpression homozygous lines is improved, and the weight of the aboveground parts of the plants is increased.

7. The application of the plant chlorophyll deplantylase CDE1 as described in claim 1 or 2 in the degradation of chlorophyll.

8. The application according to claim 7, characterized in that, The degraded chlorophyll includes degraded chlorophyll a, chlorophyll b, and pheophytin a.

9. The application of the plant chlorophyll deplantylase CDE1 as described in claim 1 or 2 in the PSII repair process.

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