Low-light respiratory metabolism module RBCS-NOX and application thereof in improving photosynthetic efficiency of C3 plants

Abstract

The invention discloses a low-light respiratory metabolism module RBCS-NOX and an application of the low-light respiratory metabolism module RBCS-NOX in improving the photosynthetic efficiency of C3 plants. The low-light respiratory metabolism module RBCS-NOX is a fusion protein which is formed by carrying out fusion expression on a Rubisco small subunit RbcS protein and a water-producing type NAD (P) H oxidase (NOX). The module is introduced into C3 plant rice, and the result shows that the photosynthetic rate of an RBCS-NOX transgenic plant is improved, the light respiration rate is reduced, and the ratio of glycine to serine is reduced. The photosynthetic efficiency of the rice is expected to be improved, the correlation mechanism of the photosynthetic efficiency and photorespiration of C3 plants is further clarified, the research results of the gene can provide key technical reserve for high-photosynthetic-efficiency improvement of important crops in China, and the gene has far-reaching significance for deeply clarification of the high-photosynthetic-efficiency mechanism of the crops and crop production.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a low-light respiratory metabolism module RBCS-NOX and its application in improving the photosynthetic efficiency of C3 plants. Background Technology

[0002] Both photosynthesis and photorespiration in plants begin with ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO), a bifunctional enzyme. When performing its carboxylation function, it generates two molecules of 3-phosphoglycerate (3-PGA) which enter the Calvin cycle of photosynthesis. When catalyzing the oxygenation reaction, it generates one molecule of 3-PGA and one molecule of 2-phosphoglycolic acid (2-PG), of which 2-PG is a toxic intermediate metabolite that requires photorespiration for metabolic transformation. However, the RubisCO oxidation reaction consumes 3.25 mol of ATP and 2 mol of NADPH. In C3 crops such as rice and soybean, photorespiration reduces photosynthetic conversion efficiency by 20%-50%, and this reduction is even greater under high temperature and drought conditions. Therefore, photorespiration is an energy- and material-intensive process.

[0003] To address these characteristics of photorespiration, some scientists believe that improving carbon assimilation efficiency, such as altering RuBisCO activity to enhance its carboxylation efficiency, can inhibit photorespiration and increase photosynthetic efficiency. Currently, three main types of Rubisco are known (type I, II, and III). Type I Rubisco is predominant in higher plants, typically an L8S8 protein oligomer composed of eight large subunits (LSU) and eight small subunits (SSU), with a molecular weight of approximately 560 kDa. Sequence alignment and crystal structure analysis have revealed high amino acid sequence and structural similarity among the various subtypes of LSU, and it contains the main catalytic active sites. While SSU is not absolutely necessary for carboxylation, its absence leads to a significant decrease in Rubisco structural stability, substrate binding affinity, and catalytic activity. Some studies also suggest that SSU plays a crucial role in regulating Rubisco activity, content, stability, and assembly.

[0004] Given the aforementioned structural characteristics of Rubisco, scientists have developed several key modification strategies targeting its low carboxylation rate and poor substrate specificity:

[0005] (1) Targeted mutation of the Rubisco encoding gene. Mutations are made in the active site of Rubisco and its surrounding sequences, or point mutations are introduced into the RbcS subunit of the nuclear gene encoding the small subunit, in an attempt to improve the carboxylation capacity of Rubisco. One study used an E. coli screening system to mutate many key sites in Rubisco; however, the carboxylation activity of most mutants did not increase. Only the M6-5 mutant of Synechococcus Rubisco (with two point mutations in the small subunit) showed higher carboxylation efficiency. However, due to the high false positive rate of this screening system and the current lack of understanding of the structure-function relationship of Rubisco, improving the catalytic properties of Rubisco through point mutation remains difficult to achieve in higher plants. Another study predicted and selected a series of RbcL mutation sites associated with C3-to-C4 plants and potentially exhibiting higher carboxylation efficiency, and then introduced these point mutations into tobacco. However, after analysis, these transgenic tobacco Rubisco did not show stronger carboxylation capacity. Because Rubisco has undergone a very long evolutionary process, its amino acid sequence and protein structure have gradually reached a balance in terms of plasticity and stability under current atmospheric conditions. A simple mutation at a single site is unlikely to produce a significantly beneficial change in its catalytic properties. Furthermore, directed evolution can be used to modify Rubisco, which involves simulating the natural environment in the laboratory and accelerating the Rubisco evolution process. However, truly beneficial mutations that meet the target requirements may require a combination of multiple mutation sites. While machine learning can greatly accelerate the mapping from sequence / structure to enzyme function, the vast number of possible combinations leads to challenges such as difficulty in cloning the constructed mutation library and increasing complexity of subsequent screening systems. With advancements in computing power and technology, this method may play an increasingly important role in modifying Rubisco.

[0006] (2) Increasing Rubisco content and activation level. To address the low carboxylation efficiency of Rubisco, plants address the issue by increasing quantity, synthesizing large amounts of Rubisco to maintain normal photosynthesis. Rubisco typically accounts for 20-50% of the total protein in plant leaves. Some studies have attempted to increase the Rubisco content in crops to promote photosynthesis. For example, overexpression of the large and small subunits of Rubisco, as well as the Rubisco assembly chaperone protein RAF1, in C4 maize increased Rubisco content by over 30%, while CO2 assimilation efficiency also increased by 15%. However, overexpression of the small Rubisco subunit or simultaneous overexpression of the small subunit and Rubisco activator enzyme (RCA) in rice did not improve photosynthetic efficiency under normal growth conditions. Furthermore, studies have shown that overexpression of OsRbcL, OsRbcS, and OsRAF1 in rice significantly increased the total Rubisco content, but only under high-nitrogen planting conditions did the photosynthetic rate and yield show a clear advantage. The above research results indicate that even if the Rubisco content in crops can be increased, it may still affect the carbon-nitrogen balance within the plant, making it difficult to improve photosynthetic rate and yield under normal growing conditions. Furthermore, Rubisco requires activation by RCA to function properly, and the degree of activation significantly affects the activity of the carboxylation reaction. Given the increasing frequency of severe weather events such as high temperatures and droughts in the future due to intensified global climate change, introducing RCA, which has better thermal stability, into C3 plants can significantly improve the activation level and catalytic rate of Rubisco, thereby enhancing the overall photosynthetic rate and yield of plants under heat stress.

[0007] (3) Rubisco heterologous recombination modification. Another optimization strategy is to introduce heterologous Rubisco subunits such as RbcL and / or RbcS from C4 plants, which have high carboxylation rates, into C3 target crops. If the Rubisco subunits in C3 plants can be successfully introduced or replaced, theoretically, the carboxylation efficiency of the plants may be increased. Some studies have introduced SSU from C4 crop sorghum into rice. The content and catalytic activity of Rubisco in the transgenic plants were increased compared with wild type, but the photosynthetic rate did not increase. Subsequently, they used CRISPR / Cas9 gene editing technology to further knock out four small subunit genes other than OsRbcS1, namely OsRbcS2-5, in transgenic rice with sorghum SSU. The authors named this plant the CSS plant. The Rubisco leaves of this CSS plant exhibit a hybrid structure composed of rice large subunits and sorghum small subunits. While its carboxylation rate is significantly increased, its affinity and specificity for CO2 decrease significantly. Only under high CO2 concentrations does this rice material show a higher CO2 assimilation rate. However, because Rubisco often requires a series of chaperone proteins to complete normal folding and assembly, and these chaperone proteins are species-specific, in vivo recombination of heterologous Rubisco is hindered. Solving this problem means transferring the entire set of chaperone proteins of the heterologous Rubisco into the target crop via plasmid transformation. However, due to low plasmid transformation efficiency, lack of effective screening markers, and sterility of regenerated plants, plasmid transformation technology has not yet been realized for many crops. Because heterologous Rubisco is difficult to recombinant normally in target crops, some studies have introduced high-carboxylation-rate Rubisco from *Halothiobacillus neapolitanus* into tobacco. Moreover, the large and small subunits of this Rubisco can fold and assemble normally in tobacco chloroplasts without co-conversion of its own chaperone protein, reaching approximately 40% of the wild-type content. However, due to the low affinity of this heterologous Rubisco for CO2, it only exhibits the same growth rate as wild-type tobacco under 1% CO2 conditions. Studies have shown that overexpression of OsRbcS1 in rice, a gene not expressed in leaves and whose encoded protein sequence has low similarity to OsRbcS2–5, has resulted in some Rubisco holoenzymes in the leaves of overexpressing plants composed of the original RbcS and RbcS1 small subunits. This recombinant Rubisco has a higher carboxylation rate, but its affinity and specificity for CO2 are also reduced, resulting in no significant improvement in the photosynthetic efficiency of this transgenic rice under normal atmospheric conditions.From the catalytic properties of the various types of Rubisco mentioned above, it can be seen that there is an inverse relationship between the carboxylation rate and the affinity for CO2. Due to this, the current efforts to modify Rubisco to obtain more ideal catalytic properties, i.e. higher carboxylation efficiency, have not been successful.

[0008] Currently, in regulating plant photorespiration metabolism, patent CN 117187291 A discloses a photorespiration metabolic pathway (iGM pathway) driven by the photoinducible promoters pRBCS and pCAB to drive the expression of CrGDH and CmMS, thereby modifying the photorespiration of rice to reduce the photorespiration rate, increase the photosynthetic rate, increase chlorophyll content, and increase biomass. Patent CN 108440672 A discloses a photorespiration metabolic modification pathway (GOC pathway) containing three proteins: OsGLO3, OsCAT2, and OsOXO3, which modifies the photorespiration of C3 plants, thereby reducing photorespiration, increasing photosynthetic efficiency, and ultimately increasing plant biomass. Since the fundamental cause of photorespiration is the competitive binding of CO2 and O2 to Rubisco, theoretically, introducing a module that can reduce the O2 concentration around Rubisco could also reduce photorespiration and increase photosynthesis. However, currently, no low-photorespiration module based on this principle has been provided to influence plant photosynthesis. Summary of the Invention

[0009] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a low-light respiratory metabolism module RBCS-NOX.

[0010] A second objective of this invention is to provide a nucleotide for encoding the aforementioned low-light respiratory metabolic module RBCS-NOX.

[0011] A third objective of this invention is to provide an expression vector comprising a nucleotide sequence encoding the above-mentioned module.

[0012] A fourth objective of the present invention is to provide a recombinant engineered bacterium comprising a nucleotide sequence encoding the above-mentioned module or comprising the above-mentioned expression vector.

[0013] The fifth objective of this invention is to provide an application of a nucleotide sequence encoding the above-mentioned module, or an expression vector containing the above-mentioned module, or a recombinant engineered bacterium containing the above-mentioned module, in improving the photosynthetic rate of plants and reducing the photorespiration rate of photoplants.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] This invention provides a low-light respiratory metabolism module RBCS-NOX, which is a fusion protein and includes, from the N-terminus to the C-terminus, a Rubisco small subunit RbcS, a flexible protein linker Linker, and an aqueous NAD(P)H oxidase NOX.

[0016] Because ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a key enzyme in the carbon assimilation process of photosynthesis, its active site has poor substrate specificity. Therefore, it can catalyze both the carboxylation of ribulose-1,5-bisphosphate (RuBP) and the oxidation of RuBP to 2-phosphoglycolic acid, requiring energy-intensive photorespiration for metabolism. In addition to its poor substrate specificity, Rubisco also has a very low carboxylation rate; its catalytic CO2 carboxylation reaction typically has a turnover number of only 2–10 s. -1 Rubisco oxidation is a key rate-limiting step in the photosynthetic carbon assimilation process. To reduce the rate of oxidation and increase carboxylation efficiency in Rubisco, this invention fuses the water-producing NAD(P)H oxidase NOX protein to the C-terminus of the Rubisco small subunit via a flexible protein linker, forming an RBCS-NOX module. This module allows NOX to be anchored to the periphery of Rubisco via the C-terminus of the RBCS, thereby consuming O2 near the Rubisco active site, making it more prone to carboxylation, and ultimately improving photosynthesis.

[0017] Furthermore, the module also includes a promoter, a terminator, and a protein tag.

[0018] Preferably, the module consists of pRbcS-RbcS-Linker-Flag-NOX-Tnos; where pRbcS is the promoter, Flag is the protein tag, and Tnos is the terminator.

[0019] Furthermore, the RbcS protein is rice OsRbcS2; the amino acid sequence of rice OsRbcS2 is shown in SEQ ID No. 1.

[0020] Furthermore, the OsRbcS2 nucleotide sequence is shown in SEQ ID No. 2.

[0021] Furthermore, the aqueous NAD(P)H oxidase NOX is the NAD(P)H oxidase of *Lactobacillus Sanfranciscensis*, named LsNOX2, and the amino acid sequence of LsNOX2 is shown in SEQ ID No. 3. LsNOX2 is an oxidoreductase with flavin adenine dinucleotide (FAD) as a coenzyme (electron donor). This enzyme can utilize both NADH and NADPH as substrates. mThe values ​​were very similar, at 6.1 μM and 6.7 μM respectively. The most unique feature of this enzyme is that it reduces O2 to H2O while oxidizing NAD(P)H, unlike most other NOX enzymes which generate reactive oxygen species such as hydrogen peroxide or superoxide anions. The specific reaction process is as follows: one molecule of NAD(P)H first reacts with O2 to produce H2O2, but the H2O2 is not released. Instead, it continues to react with the thiol group of the active site Cys42 to generate a sulfonic acid group and one molecule of H2O. Subsequently, a second molecule of NAD(P)H reduces the sulfonic acid group to a thiol group, releasing a second molecule of H2O. Therefore, the overall reaction is the reduction of O2 by two molecules of NAD(P)H to two molecules of H2O. Due to the excellent catalytic ability of LsNOX2 and the fact that the byproduct is H2O rather than the cell-harmful H2O2, this enzyme is often used as an effective NAD(P)H byproduct. + Regenerative enzymes have significant potential applications in biosynthetic pathways. This invention fuses LsNOX2 to the C-terminus of OsRbcS2 via a flexible protein linker (GGGGS)3 to form an RBCS-NOX module. This module allows LsNOX2 to anchor to the periphery of Rubisco via the C-terminus of OsRbcS2, thereby consuming O2 near the Rubisco active site, making it more prone to carboxylation, ultimately reducing photorespiration and increasing photosynthesis.

[0022] Furthermore, the nucleotide sequence encoding the LsNOX2 protein has been codon-optimized using rice codon preference, and the nucleotide sequence is shown in SEQ ID No. 4.

[0023] Preferably, the coding sequence of LsNOX2 is amplified using conventional methods, and the required primers are as follows:

[0024] RBCS2-NOX-F:5'-GAGTTCTGGTGGCAACGGAGGCGGAGGCTCCGGC-3'

[0025] RFN-POX-R:5'-CAATTCACACTTGTAGGATCCTTACTTGTGCGCCTTATCCGCTTGGGCTTGCG-3'

[0026] Furthermore, the promoter is the rice RbcS self-promoter, and the nucleotide sequence is shown in SEQ ID No. 6. It exhibits some photoinducibility and is mainly expressed in green tissues such as stems and leaves.

[0027] Furthermore, the terminator is the tNOS terminator of the Agrobacterium taurine synthase gene, which is commonly used in transgenic crops.

[0028] Furthermore, the protein tag may be a gene that expresses an enzyme or a luminescent compound that can produce color changes in a plant, a marker for antibiotic resistance, or a marker for chemical resistance, etc.

[0029] Furthermore, the antibiotic resistance markers include gentamicin markers, kanamycin markers, hygromycin markers, etc.

[0030] Furthermore, the chemical reagent resistance marker genes include herbicide resistance genes, etc.

[0031] Preferably, the protein tag is a 1×Flag tag, and the nucleotide sequence of the 1×Flag is shown in SEQ ID No. 8.

[0032] Preferably, the nucleotide sequence of the flexible protein linker is shown in SEQ ID No. 7.

[0033] The present invention provides a nucleotide for encoding the aforementioned low-light respiratory metabolic module RBCS-NOX, the nucleotide sequence of which is shown in SEQ ID No. 5.

[0034] The present invention provides an expression vector comprising a nucleotide sequence encoding the aforementioned low-light respiratory metabolic module RBCS-NOX.

[0035] Furthermore, the expression vector is pOx or other derived plant expression vectors.

[0036] The present invention provides a recombinant engineered bacterium containing a nucleotide sequence encoding the aforementioned low-light respiratory metabolic module RBCS-NOX or containing the aforementioned expression vector.

[0037] This invention also provides an application of a nucleotide sequence encoding the aforementioned low-photorespiration metabolic module RBCS-NOX, or an expression vector comprising the aforementioned, or a recombinant engineered bacterium comprising the aforementioned, in improving the photosynthetic rate of plants and reducing the photorespiration rate of light-loving plants. In practical applications, this metabolic module can be transferred into different C3 plants to cultivate ideal varieties with enhanced photosynthetic capacity.

[0038] Therefore, the present invention also provides the application of a nucleotide sequence encoding the aforementioned low photorespiratory metabolic module RBCS-NOX, or an expression vector comprising the aforementioned expression vector, or a recombinant engineered bacterium comprising the aforementioned recombinant engineered bacteria, in the preparation of transgenic plants with increased photosynthetic rate and decreased photorespiratory rate.

[0039] Furthermore, the preparation method can be achieved by transforming the plant into plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation.

[0040] Furthermore, the above applications can increase plant biomass.

[0041] Furthermore, the aforementioned plants are C3 plants.

[0042] Preferably, the C3 plant is rice.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention provides an RBCS-NOX low-photorespiration metabolic module. The RBCS-NOX module is a fusion protein formed by fusing the Rubisco small subunit RbcS protein with water-producing NAD(P)H oxidase (NOX). When this module is introduced into C3 plants, the plants exhibit increased photosynthetic rate and decreased photorespiration rate. In practical applications, this metabolic module can be transferred into different C3 plants to cultivate ideal varieties with enhanced photosynthetic capacity. This invention has significant application value and importance in elucidating the mechanism of high light efficiency in rice, reducing carbon loss during photorespiration, and improving the photosynthetic capacity of rice. Attached Figure Description

[0045] Figure 1 This is the core component of the pOx vector. Among them, P... 35S For the 35S enhanced promoter of cauliflower mosaic virus; T 35S 35S terminator for cauliflower mosaic virus; Pubi is the promoter of the maize ubiquitin gene; Tnos is the nos terminator; MCS is the multiple cloning site; HPT is the hygromycin resistance gene; LB is the left boundary; RB is the right boundary.

[0046] Figure 2 This is the core component of the RBCS-NOX-pOX vector. Among them, P... 35S For the 35S enhanced promoter of cauliflower mosaic virus; T 35S The terminator is 35S terminator for cauliflower mosaic virus; PrbcS is the promoter of the RbcS2 gene; Tnos is the nos terminator; HPT is the hygromycin resistance gene; Flag is the 1×Flag tag; LB is the left boundary; RB is the right boundary.

[0047] Figure 3 Electrophoresis results for detecting amplified target bands.

[0048] Figure 4 The predicted three-dimensional structure of the RBCS-NOX fusion protein.

[0049] Figure 5 Comparison of the three-dimensional structures of RbcS in the fusion protein with those of its homologous RbcS model.

[0050] Figure 6The results show the transcription and protein levels of RBCS-NOX plants.

[0051] Figure 7 The results of CN-PAGE staining of crude enzyme solution from RBCS-NOX transgenic plants are shown. Among them, WT is wild type; RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5) are RBCS-NOX-pOx transgenic lines.

[0052] Figure 8 The results are Western Blots of crude enzyme solution from RBCS-NOX transgenic plants after CN-PAGE. Among them, WT is wild type; RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5) are RBCS-NOX-pOx transgenic lines.

[0053] Figure 9 The photosynthetic parameters and photorespiration rate of leaves in RBCS-NOX-pOx transgenic plants were measured during the heading and flowering stages. Figure 9 In this context, a represents net photosynthetic rate; b represents stomatal conductance; c represents intercellular carbon dioxide concentration; and d represents photorespiration rate. WT represents wild type; RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5) are RBCS-NOX-pOx transgenic lines. Different letters indicate significant differences (n=30, P<0.05, Duncan test).

[0054] Figure 10 This study aimed to determine the free amino acid content in RBCS-NOX-pOx transgenic plants. Among them, Figure 10 In the table, a represents glycine content; b represents serine content; and c represents the glycine / serine ratio. WT represents wild type; RBCS-NOX18-1-1 (RLN18-1-1) represents the RBCS-NOX-pOx transgenic line. n = 3, **P < 0.01, t-test. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0057] The primer synthesis and sequencing work used in this invention were completed by Guangzhou Ruibo Biotechnology Co., Ltd.

[0058] Example 1: Obtaining the low-light metabolism module RBCS-NOX and its expression vector

[0059] (1) Obtaining the RBCS-NOX-pOx expression vector

[0060] Primers were designed based on the cDNA sequences of pRbcS, OsRbcS2, and LsNOX2 provided by NCBI (http: / / www.ncbi.nlm.nih.gov / ). The primer sequences are as follows:

[0061] PRO-RBCS2-F:5'-CATGATTACGAATTCGAGCTCATCGGGTCGAGGTGAA C-3'

[0062] RBCS2-SIG-R:5'-CATCACGGAGGGGGCCATCTCTGCAGCTCACCAAGCT CTC-3'

[0063] RBCS2-ORF-F:5'-CTTGGTGAGCTGCAGATGGCCCTCCGTGATGG CGTCGTC-3'

[0064] RBCS2-ORF-R:5'-GGAGCCTCCGCCTCCGTTGCCACCAGACTCCTCGCA GCCCG-3'

[0065] RBCS2-NOX-F:5'-GAGTTCTGGTGGCAACGGAGGCGGAGGCTCCGGC-3'

[0066] RFN-POX-R:5'-CAATTCACACTTGTAGGATCCTTACTTGTGCGCCTTATCCGCTTGGGCTTGCG-3'

[0067] Using rice genomic DNA as a template, the OsRbcS promoter (pRbcS) was amplified using conventional methods under the guidance of primers PRO-RBCS2-F and RBCS2-SIG-R.

[0068] Using rice cDNA as a template, the signal peptide and coding sequence of OsRbcS2 were amplified using conventional methods under the guidance of primers RBCS2-ORF-F and RBCS2-ORF-R.

[0069] After codon optimization of the LsNOX2 coding sequence of Lactobacillus sanguanisi according to the codon preference of rice, its coding gene sequence was obtained by full sequence synthesis technology. Under the guidance of primers RBCS2-NOX-F and RFN-POX-R, the coding sequence of LsNOX2 was amplified using conventional methods.

[0070] After the reaction, the PCR amplification products were subjected to 1% agarose gel electrophoresis, and the DNA fragments of pRbcS (approximately 1600 bp), OsRbcS2 (approximately 500 bp), and LsNOX2 (approximately 1400 bp) were recovered and purified.

[0071] The RBCS-NOX coding sequence was obtained by sequentially tandemly connecting OsRbcS2, the flexible protein Linker ((GGGGS)3), the 1×Flag tag, and LsNOX2 using overlap-PCR technology. The RBCS-NOX sequence was then cloned into the rice expression vector pYLox.5 (pOx, donated by Professor Liu Yaoguang of South China Agricultural University). The original constitutive promoter pUbi was replaced with the photoinducible promoter pRbcS by double enzyme digestion and recombination ligation. The constructed vector was referred to as RBCS-NOX-pOX and sent to Guangzhou Ruibo Biotechnology Co., Ltd. for sequencing.

[0072] like Figure 1 As shown, this is the core portion of the pOx carrier. Figure 2 The image shows the core portion of the RBCS-NOX-pOX vector. Electrophoresis was performed to detect the PCR amplification results, as shown below. Figure 3 As shown, this indicates that the DNA fragments pRbcS, OsRbcS2, and LsNOX2 were successfully amplified.

[0073] Example 2: Structural prediction of the RBCS-NOX fusion protein

[0074] The RBCS-NOX fusion protein sequence was input into I-TASSER (https: / / seq2fun.dcmb.med.umich.edu / / I-TASSER / ), and a 3D structure prediction map of the fusion protein was obtained. The linker and LsNOX2 were then removed from the result, and the image was rotated to obtain I-TASSER OsRbcS. Simultaneously, the OsRbcS protein sequence was input into SWISS-MODEL (https: / / swissmodel.expasy.org / ), and homology alignment modeling was performed to obtain SWISS-MODEL OsRbcS.

[0075] The results are as follows Figure 4 and Figure 5As shown, both have two α-helices and one β-fold. Apart from the random curling, their three-dimensional structures are also quite similar. It is preliminarily believed that the fusion of LsNOX2 into OsRbcS through the Linker has no effect on the conformation of OsRbcS.

[0076] Example 3: Obtaining and phenotypic analysis of transgenic plants containing the RBCS-NOX-pOx vector.

[0077] (1) Obtaining transgenic plants

[0078] The RBCS-NOX-pOx vector, which modifies the photorespiratory metabolic pathway of RBCS-NOX, was transformed into callus tissue of mature embryos of the japonica rice variety Zhonghua 11 using Agrobacterium-mediated transformation, as described in the following literature (Hiei et al, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J. 1994, 6: 271-282). After pre-differentiation and differentiation, transformed plants were obtained, and the hygroscopic resistance gene HPT was identified by PCR.

[0079] The primer sequences for PCR amplification of HPT are as follows:

[0080] HPT-F: 5'-CTGAACTCACCGCGACGTCTGTC-3'

[0081] HPT-R: 5'-TAGCGCGTCTGCTGCTCCATACA-3'

[0082] The PCR amplification conditions were: 95℃ for 2 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 15 sec, 35 cycles; 72℃ for 5 min.

[0083] Transgenic plants that tested positive by PCR were collected, and genomic DNA was extracted from the leaves of T1 generation transgenic plants for Southern Blot analysis to determine the copy number of the inserted gene. Single-copy transgenic plants were selected, and seeds were collected from each plant. After germination, more than 100 seeds were selected and screened using hygromycin. If no seeds died, it indicated that the seeds were homozygous. The homozygous transgenic seeds and wild-type rice Zhonghua 11 seeds were selected for germination.

[0084] Rice was cultured to the 4-leaf stage using Kimura B nutrient solution (pH adjusted to 4.8). Total RNA and total protein were then extracted from rice leaves, and qRT-PCR and Western Blot were used to detect the expression of the target gene at the RNA and protein levels.

[0085] The specific formula for Kimura B nutrient solution is as follows: (NH4)2SO4 (0.365mM), KH2PO4 (0.182mM), KNO3 (0.183mM), K2SO4 (0.086mM), Ca(NO3)2 (0.366mM), MgSO4 (0.548mM), EDTA-FeIII (0.020mM), MnCl2·4H2O (0.091×10-3mM), ZnSO4·7H2O (0.77×10-3mM). -3 mM), CuSO4·5H2O (0.32×10) -3 mM), H3BO3 (0.0462mM), (NH4)6Mo7O 24 .4H2O(0.145×10 -3 mM).

[0086] After callus induction and differentiation, a total of 24 transformed plants were obtained. The hygroscopic enzyme resistance gene HPT was identified by PCR, and all of them were positive.

[0087] like Figure 6 As shown, RNA and protein levels in T2 generation plants (RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5)) were detected, revealing that the target gene was expressed at both RNA and protein levels.

[0088] (2) Detection of chimeric Rubisco-NOX holoenzyme

[0089] To further analyze whether the RBCS-NOX module can form a chimeric Rubisco-NOX holoenzyme, total protein was extracted from the leaves of transgenic plants and subjected to clear-native polyacrylamide gel electrophoresis (Clear-Native PAGE). Preliminary observation was performed by staining with Coomassie Brilliant Blue R-250. Simultaneously, Western blotting was conducted using Flag and OsRbcS monoclonal antibodies to further verify the existence of the RBCS-NOX complex.

[0090] like Figure 7As shown, two bands with significantly different migration rates appeared in the transgenic lines RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5), while no bands with larger molecular weights appeared in WT. By comparing with the marker, it can be preliminarily identified that the band in WT is the background Rubisco in rice (approximately 560 kD), while the larger molecular weight bands in the transgenic lines are chimeric Rubisco-NOX holoenzymes.

[0091] like Figure 8 As shown, when using OsRbcS antibody detection, two bands with significantly different migration rates appeared in the transgenic lines RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5), while only one band with a smaller molecular weight was observed in WT. This further confirmed the results of cosmetic staining, namely that WT only contained background Rubisco, while both transgenic lines showed background Rubisco and Rubisco-NOX chimeras. When using Flag antibody detection, the band size of the transgenic lines was found to be similar to the band size of the complex detected by RbcS antibody, further confirming that the larger molecular weight band was a Flag-tagged Rubisco-NOX chimera. Therefore, it is believed that the introduced RBCS-NOX module can participate in the assembly of the Rubisco holoenzyme and form a chimeric Rubisco-LsNOX2 holoenzyme.

[0092] (3) Measurement of photosynthetic parameters and photorespiration rate of transgenic plants

[0093] The introduction of the RBCS-NOX module consumes O2 close to that of Rubisco, making it more prone to carboxylation reactions. Theoretically, this can reduce photorespiration and increase the photosynthetic rate. Therefore, the net photosynthetic rate and photorespiration rate of the flag leaf of the RBCS-NOX transgenic plant during the heading and flowering stage were measured using a Li-Cor portable photosynthesis meter LI-6800. Data recording began after the instrument readings stabilized.

[0094] The results are as follows Figure 9As shown, compared to the wild type, the net photosynthetic rate of the transgenic line RBCS-NOX18-1-1 (RLN18-1-1) was significantly increased by 17.37%, while the transgenic line RBCS-NOX22-1-5 (RLN22-1-5) showed an increasing trend. The stomatal conductance of the transgenic line RBCS-NOX18-1-1 (RLN18-1-1) was significantly increased by 14.67%; in addition, the intercellular CO2 concentration of the transgenic line RBCS-NOX22-1-5 (RLN22-1-5) was decreased. Subsequently, the photorespiration rate of the transgenic plants was measured using a hypoxia method, and statistical analysis revealed that the photorespiration rates of the transgenic lines RBCS-NOX18-1-1 (RLN18-1-1) and RBCS-NOX22-1-5 (RLN22-1-5) were significantly reduced by 16.12% and 11.53%, respectively, compared to the wild type.

[0095] The glycine and serine contents of RBCS-NOX transgenic plants were determined using an automated amino acid analyzer.

[0096] The results are as follows Figure 10 As shown, the glycine / serine ratio of the RBCS-NOX transgenic line RBCS-NOX18-1-1 (RLN18-1-1) decreased significantly by -19.06% compared to the wild type.

Claims

1. A low light respiratory metabolism module RBCS-NOX, characterized in that, The module is a fusion protein, comprising Rubisco small subunit RbcS, flexible protein linker Linker and water-producing NAD(P)H oxidase NOX from N terminus to C terminus.

2. Low light respiratory metabolism module RBCS-NOX according to claim 1, characterized in that, The module further comprises a promoter, a terminator and a protein tag.

3. Low light respiratory metabolism module RBCS-NOX according to claim 2, characterized in that, The module consists of pRbcS-RbcS-Linker-Flag-NOX-Tnos.

4. Low photorespiratory metabolism module RBCS-NOX according to any one of claims 1 to 3, characterized in that, The Rubisco small subunit RbcS is rice OsRbcS2; the amino acid sequence of the rice OsRbcS2 is shown in SEQ ID No.

1.

5. The low photorespiration module RBCS-NOX according to any one of claims 1 to 3, characterized in that, The water-producing NAD(P)H oxidase NOX is NAD(P)H oxidase of Lactobacillus Sanfranciscensis, named LsNOX2; the amino acid sequence of the LsNOX2 is shown in SEQ ID No.

3.

6. A nucleotide for encoding the low-light respiration metabolism module RBCS-NOX according to any one of claims 1-5, wherein the nucleotide sequence of the RBCS-NOX is shown in SEQ ID No.

5.

7. An expression vector comprising the nucleotide according to claim 6.

8. A recombinant engineering bacterium comprising the nucleotide according to claim 6 or the expression vector according to claim 7.

9. Use of the nucleotide according to claim 6 or the expression vector according to claim 7 or the recombinant engineering bacterium according to claim 8 in increasing photosynthetic rate of plants or reducing light respiration rate of plants.

10. Use of the nucleotide according to claim 6 or the expression vector according to claim 7 or the recombinant engineering bacterium according to claim 8 in preparing transgenic plants with increased photosynthetic rate or reduced light respiration rate.

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