Application of chloroplast localization protein OsRBP1 in improving photosynthetic efficiency and resistance of plants

Abstract

The invention discloses a protein OsRBP1 with an amino acid sequence as shown in SEQ ID NO: 1. The protein OsRBP1 can improve photosynthetic carbon assimilation efficiency of plants, promote growth and development of the plants, increase biomass and improve heat stress resistance of the plants, and has important application value for cultivating new varieties of plants with high photosynthetic efficiency and / or heat resistance.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically, it relates to the application of a chloroplast localization protein OsRBP1 in improving plant photosynthetic efficiency and / or high temperature resistance. Background Technology

[0002] Under the stress of high temperature and high light, the synthesis rate of some important proteins in chloroplasts decreases significantly, and this inhibition seriously affects the photosynthetic carbon assimilation efficiency.

[0003] Before chloroplast protein translation, the mRNA transcribed from chloroplast genes undergoes a complex RNA editing process, with polycistronic pre-mRNA being converted to a monocistronic isoform with the assistance of plassomal ribonucleases. The activity of chloroplast transcripts depends on their structure, protective RNA-binding proteins, and is regulated by some non-specific RNases. Therefore, the expression regulation of chloroplast genes is dominated by post-transcriptional RNA editing events, and these nuclear-encoded chloroplast RNA-binding proteins are primarily responsible for these post-transcriptional RNA editing events. Nuclear-encoded plassomal localization proteins function as RNA chaperones; their function of stabilizing and protecting the correct RNA structure is crucial for translation events in plassomal tissues and helps plants resist the effects of abiotic stresses.

[0004] Chloroplast RNA-binding proteins play an indispensable role in the maturation of chloroplast mRNA. Over the course of evolution, the chloroplast genome developed intron features, requiring intron splicing after transcription. Chloroplast genes are more complex due to the presence of additional eukaryotic gene expression characteristics; therefore, the editing of mRNA precursors by chloroplast RNA-binding proteins facilitates precise regulation of chloroplast gene expression.

[0005] Chloroplast RNA-binding proteins play an important role in chloroplast RNA editing and metabolism, thereby regulating chloroplast gene expression through RNA manipulation: (1) promoting mRNA maturation; (2) stabilizing mRNA structure to promote translation. The regulatory mechanism of chloroplast RNA-binding proteins is closely related to physiological activities such as seed development, germination, and seedling growth in plants, and is also closely linked to photosynthetic efficiency. Most mutants of these proteins exhibit abnormal phenotypes, such as leaf yellowing, embryo lethality, weak plants, and reduced photosynthetic efficiency, while some overexpression mutants can enhance plant resistance to abiotic stress.

[0006] Chloroplast RNA-binding proteins not only edit precursor mRNAs to promote their maturation, but also stabilize the structure of mature mRNAs by editing or binding to them, protecting sensitive restriction enzyme sites from errors during RNA editing and ensuring normal translation function under abiotic stress. Under abiotic stress, the environment within chloroplasts changes, which can easily lead to structural alterations in mRNA and affect normal translation. Therefore, the auxiliary stabilizing function of chloroplast RNA-binding proteins is closely related to plant stress resistance. In translation events, mRNA stability is crucial; the auxiliary stabilizing function of nuclear-encoded chloroplast RNA-binding proteins ensures normal translation events and guarantees plant resistance to abiotic stress, which is beneficial for the normal expression and precise regulation of chloroplast genes.

[0007] While existing research has revealed the roles and regulatory mechanisms of some nuclear-encoded chloroplast RNA-binding proteins, detailed investigations into their regulatory mechanisms remain limited. Furthermore, research on the impact of nuclear-encoded chloroplast RNA-binding proteins on photosynthesis and biomass enhancement is scarce. Therefore, identifying key chloroplast RNA-binding proteins is crucial for understanding chloroplast protein synthesis efficiency and improving crop resilience to high-temperature stress and photosynthetic efficiency. Summary of the Invention

[0008] Based on a summary of published research, we believe that the S1 domain is a key characteristic of this type of chloroplast RNA-binding protein. The S1 domain is complex and exhibits low sequence identity, with multiple residues believed to facilitate RNA binding, and these residues are highly conserved. Therefore, we compared the amino acid sequence of the S1 domain on a rice website and screened for the gene OsRBP1. Experiments revealed that the OsRBP1 gene is crucial for regulating rice growth and development, photosynthetic carbon assimilation efficiency, and the formation of biomass traits. Furthermore, OsRBP1 gene expression in response to heat stress enhances the resistance of transgenic rice to high-temperature stress. Based on this finding, the present invention includes the following technical solution.

[0009] The first aspect of the present invention provides a polypeptide selected from the group consisting of:

[0010] (a) A polypeptide with the amino acid sequence shown in SEQ ID NO:1, which is protein OsRBP1;

[0011] (b) A conserved variant polypeptide derived from (a) having the polypeptide function of (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:1.

[0012] (c) A conserved variant polypeptide derived from (a) having more than 95% homology with the polypeptide sequence defined in (a), preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, more preferably more than 99% homology, and having the function of the polypeptide in (a).

[0013] (d) A derivative polypeptide containing the polypeptide sequence described in (a), (b), or (c).

[0014] The amino acid sequence of protein OsRBP1 is as follows:

[0015] MPPSMAVAAAAAAPRSLLFPVSPPSSQPRRVPALARAGRSQRRHAALARPARRHLDDAMSAGFVRSLNAPPDEDGARSGGRYDPRPGDFAVGVVVSGTEARLDVAVGADRLATLLAKELLPLYRADLPAFAEAAPPRPGSVGAVASPAAGEGDRKPGERGGGRTLVPPGTVVFAEVLGRTLSGRPLLSARRLFRRLAWHRARQVMQLDEPIEVKIYEWNTG GLLTRIEGLRAFLPKFELMDRINTFTDLKNKVGCSIRVCITRLDEETNDLIISEKKAWEMTYLKEGTLLQGIVHKIFPYGAQVRIAGTNRSGLLHISNISRGRVLSVSDI LKIDEEVKVLVVKSNVPDKIALSISDLESTPGLFLSEKARVFSEAEEMAERYREQLPADSKNAKLDADLPGGTIPFDDEATLYANWKWFKFLEDGKLGAVTTEHNGS(SEQ ID NO:1).

[0016] The aforementioned function refers to the ability to bind to chloroplast RNA and promote the increase of chloroplast photocomplex PSII content. Photocomplex PSII is the Photosystem I (PSII) complex reported in patent document CN113462715A.

[0017] A second aspect of the present invention provides a polynucleotide selected from the group consisting of:

[0018] (A) A polynucleotide encoding the polypeptide of claim 1;

[0019] (B) A polynucleotide encoding a polypeptide with the amino acid sequence shown in SEQ ID NO:1;

[0020] (C) The nucleotide sequence is a polynucleotide as shown in SEQ ID NO:2, which is the CDS region sequence of the gene encoding protein OsRBP1;

[0021] (D) A polynucleotide whose nucleotide sequence is ≥95%, preferably ≥96%, preferably ≥97%, preferably ≥98%, more preferably ≥99% homology with the nucleotide sequence shown in SEQ ID NO:2;

[0022] (E) A nucleotide sequence complementary to any of the nucleotide sequences described in (A)-(D).

[0023] The nucleotide sequence SEQ ID NO:2 is:

[0024]

[0025] A third aspect of the invention provides a DNA molecule comprising the polynucleotides described above, such as an expression cassette / expression box for the protein OsRBP1.

[0026] In one embodiment, the DNA molecule comprises an OsRBP1 encoding gene with the nucleotide sequence SEQ ID NO:2 and an upstream pHsfA2 promoter, Ubi promoter, or 35S promoter, which is an expression cassette / expression box for the protein OsRBP1.

[0027] The pHsfA2 promoter is the heat-responsive promoter pHsfA2 promoter reported in the patent document with publication number CN113462715A (derived from the HsfA2 gene of wild-type Arabidopsis thaliana Col-0, whose NCBI accession number is AT2G26150), and its nucleotide sequence is SEQ ID NO:3.

[0028] A fourth aspect of the present invention provides a recombinant plasmid comprising the DNA molecule described above, wherein the recombinant plasmid is an overexpression vector formed by cloning the DNA molecule described above onto a plasmid vector suitable for expression in Agrobacterium, such as pHB-YFP, pHB-FLAG, pBin19, pUN1301, fluorescent reporter vectors pGreenII0800-LUC, pCAMBIA3300, pCAMBIA1301, pCAMBIA2301, pBI121, pTF102, and other vectors used for plant transgenic purposes or modified vectors.

[0029] A fifth aspect of the present invention provides a microbial engineered bacterium, which is a transformant containing the recombinant plasmid described above, for mediating the transfer of the recombinant plasmid described above into plants such as rice or Arabidopsis thaliana. Preferably, the microorganism is Agrobacterium, such as Agrobacterium tumefaciens, Agrobacterium EHA105, or Agrobacterium GV3101. For example, the above-mentioned recombinant plasmid is transferred into an Agrobacterium strain using a freeze-thaw method to form a microbial engineered bacterium.

[0030] Preferably, the microbial host of the above-mentioned engineered microorganisms is Agrobacterium, such as Agrobacterium tumefaciens, Agrobacterium EHA105, or Agrobacterium GV3101.

[0031] The sixth aspect of the invention provides the use of the polypeptides, polynucleotides, DNA molecules, recombinant plasmids, or microbial engineered bacteria described above in improving the photosynthetic efficiency of plants such as rice, in increasing the biomass of plants such as rice, in improving the heat tolerance of plants such as rice, i.e., resistance to heat stress, or in breeding new varieties of plants such as rice with high photosynthetic efficiency and / or high-temperature tolerance.

[0032] In one application embodiment, the plant may be a monocotyledonous plant, preferably a grass crop. Crops include, but are not limited to, rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, and cruciferous plants (e.g., Arabidopsis thaliana), with rice being the preferred crop.

[0033] Specifically, the above application involves overexpressing the aforementioned polypeptide, such as OsRBP1, in plants such as rice through the following methods, thereby improving the photosynthetic efficiency of plants such as rice, increasing the biomass of plants such as vegetables, enhancing the heat tolerance of plants such as rice (i.e., resistance to heat stress), and cultivating new varieties of plants such as rice with high photosynthetic efficiency and / or high-temperature tolerance:

[0034] (i) The gene encoding a polynucleotide such as protein OsRBP1 as described above is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely OsRBP1 overexpression vector. Plants are then transformed by Agrobacterium-mediated transformation to obtain transgenic plants that overexpress polypeptides such as OsRBP1 as described above.

[0035] (ii) By using gene editing technology, the gene encoding a polynucleotide such as protein OsRBP1 as described above is cloned into a plant chromosome to obtain a transgenic plant that overexpresses the gene encoding a polypeptide such as OsRBP1 as described above.

[0036] (iii) Place the existing OsRBP1 coding gene in the plant genome under the regulation of a functionally enhanced promoter, such as the pHsfA2 promoter, the 35S promoter, or the Ubi promoter.

[0037] Optionally, the gene editing technology described in step (ii) above may be selected from the group consisting of: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0038] This invention reveals the important function of the OsRBP1 protein in chloroplast development in rice, demonstrating that this protein is crucial for rice growth, development, and photosynthesis. We constructed transgenic rice with pHsfA2::OsRBP1 cDNA, which increased the photosynthetic efficiency and biomass of rice, improved its resistance to high-temperature stress, promoted food production, and provided a new carbon fixation solution for addressing the greenhouse effect. Attached Figure Description

[0039] Figure 1The growth phenotypes of ZH11 rice OsRBP1 gene knockout mutants and wild-type ZH11 are shown. a: ZH11 wild-type and OsRBP1 knockout mutants osrbp1-1 and osrbp1-2 after 24 days of field growth; b: ZH11 wild-type and OsRBP1 knockout mutants osrbp1-1 and osrbp1-2 after 75 days of field growth; c: Statistical bar graph of carbon dioxide assimilation rate of ZH11 wild-type and OsRBP1 knockout mutants osrbp1-1 and osrbp1-2 measured at the grain-filling stage of rice. (n=6, leaf position: flag leaf). Figure 1 The comparison images show that the OsRBP1 knockout mutant grows weaker than the wild type.

[0040] Figure 2 The subcellular localization of the OsRBP1 protein was shown. After transforming the 35S::OsRBP1-GFP plasmid into tobacco, observation of the epidermis revealed strong, punctate fluorescence signals that largely overlapped with chloroplast fluorescence. Subsequent observation of protoplasts from transiently transformed tobacco leaves yielded results consistent with those observed in the epidermis and stomata, showing strong, punctate fluorescence signals that largely overlapped with chloroplast fluorescence. The fluorescence data indicate that the OsRBP1 protein is localized in the chloroplasts.

[0041] Figure 3 The structural differences in thylakoid membranes of chloroplasts between wild-type and osrbp1-1 seedlings were revealed. Ultrathin sections were prepared from leaves of 26-day-old wild-type and osrbp1-1 seedlings. Electron microscopy revealed that the wild-type thylakoid stacks were uniform, with good continuity between different stacks and no breaks, and the starch granules were completely consumed or transferred at night. In contrast, the chloroplasts of the mutant osrbp1-1 showed many defects, with blurred chloroplast membrane boundaries compared to the wild-type. Notably, the thylakoid stacks were disordered, with discontinuous connections between different stacks, and some stacks were dense, which is detrimental to normal photosynthesis. Furthermore, after nighttime consumption, the starch granules in the chloroplasts were not completely consumed or transferred. Figure 3 This indicates that knockout of the OsRBP1 gene causes a defect in the formation of chloroplast thylakoid stacks.

[0042] Figure 4Images of thylakoid membrane protein complexes extracted from leaves of wild-type and mutant osrbp1-1 seedlings by polyacrylamide gel electrophoresis (PAGE) are shown. a) Fresh leaves of 0.8 g each from 26-day-old wild-type and osrbp1-1 seedlings were harvested, ground, and thylakoid membrane proteins were extracted. After chlorophyll quantification of both samples, thylakoid membrane protein complexes were separated by blue natural polyacrylamide gel electrophoresis (PAGE). The mutant osrbp1-1 showed lower levels of five thylakoid membrane protein complexes (PSII super complex, PSI+PSII core dimer, PSII monomer, and CP43-less-PSII) than the wild-type, and all four complexes were associated with PSII. This result suggests that the OsRBP1 gene selectively regulates photosynthetic biosynthesis. b) Detailed Western spectroscopy analysis was performed on different core proteins in the photosynthetic complex. After performing serial dilution electrophoresis on the two samples, it was found that the content of the internal control ATPβ in both samples was basically the same. However, compared with the wild type, the content of the three core subunits of the PSII complex, D1, D2, and CP47, decreased significantly. This indicates that the OsRBP1 mutation has an adverse effect on the accumulation of PSII core protein content. Figure 4 Photographic comparisons show that the OsRBP1 mutation leads to a reduction in the content of the PSII photocomplex and its protein subunits.

[0043] Figure 5 The statistical results of photoreactivity of wild-type rice and mutants osrbp1-1 and osrbp1-2 are shown. The photoreactivity of the flag leaf of both mutants and wild-type rice was measured using a PAM2500 instrument. In a and b: we found that under a fixed light intensity, the Fv / Fm ratio and the actual electron yield (Φ) of the photorecombinant system II of the mutants... PSII All were significantly lower than the wild type. (n=5, PAR=618μmol photons m) -2 s -1 **P value < 0.01, ***P value < 0.001, Student's ttest); c, d: We measured the numerical trends of PSII quantum yield and electron transport rate under progressively increasing light intensity. Analysis of the figures shows that the actual electron yield and electron transport efficiency of photorecombination system II are significantly lower than those of the wild type. (n = 5). Figure 5 This indicates that knockout of OsRBP1 leads to a decrease in the photoreaction efficiency of rice.

[0044] Figure 6This study shows the construction of the pHsfA2::OsRBP1 vector, a comparison of agronomic traits between transgenic rice (OE) overexpressing OsRBP1 and wild-type rice DJ, and the transcriptional level of OsRBP1 in the field. A: Schematic diagram of pHsfA2::OsRBP1 vector construction; B: Three lines (OE1, OE12, OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice grown in the field for 24 and 75 days; C: Real-time quantitative PCR of OsRBP1 gene in the three lines (OE1, OE12, OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice (n=3, *P value<0.05, Student's t test). Error bars indicate standard errors. Figure 6 This indicates that the transgenic rice with pHsfA2::OsRBP1 exhibits stronger growth than the wild type, and the expression level of its OsRBP1 gene is higher than that of the wild type.

[0045] Figure 7 This paper compares the photosynthetic efficiency and biomass of transgenic rice (OE) overexpressing OsRBP1 with that of wild-type rice. Specifically, a: CO2 assimilation rate (n=6, leaf position: flag leaf) of three transgenic rice lines (OE1, OE12, OE21) measured during the grain-filling stage of DJ wild-type and pHsfA2::OsRBP1; b: Aboveground biomass (b, n=3) of wild-type rice and transgenic lines (OE1, OE12, and OE21) grown in the Shanghai Songjiang field (N30°56′36″, E121°07′26″) of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, in 2024 (*Pvalue<0.05, Student's t test). Error bars indicate standard errors. Figure 7 This indicates that the photosynthetic efficiency and biomass of the pHsfA2::OsRBP1 transgenic rice are significantly higher than those of the wild type.

[0046] Figure 8 This study compares the photoreaction efficiency of OsRBP1-overexpressing transgenic rice (OE) with that of wild-type rice under heat stress. Flag leaves of wild-type and pHsfA2::OsRBP1 transgenic rice were heat-treated (55℃ for 2 h), and after a 0.5 h recovery period at room temperature, the photoreaction efficiency of the flag leaves of both transgenic and wild-type rice was measured using a PAM2500 instrument. In a and b, we found that under a fixed light intensity, the Fv / Fm ratio and the actual electron yield (Φ) of the photorecombinant system II of the mutant were significantly different. PSII Both were significantly higher than the wild type. (n=5, PAR=618μmol photons m) -2 s -1*P value<0.05**P value<0.01,***P value<0.001,Student's t test,Error bars indicate standard errors); c, d: We measured the numerical trends of PSII quantum yield and electron transport rate under progressively increasing light intensity. Analysis of the figure shows that the actual electron yield and electron transport efficiency of photorecombination system II are higher than those of the wild type (n=5). Figure 8 This indicates that the pHsfA2::OsRBP1 transgenic rice has a stronger resistance to high temperatures than the wild type. Detailed Implementation

[0047] Endosymbiotic events have endowed plants with the photosynthetic capabilities of cyanobacteria, but this also places higher demands on the precision of chloroplast gene regulation. However, regulation solely by cyanobacteria is far from sufficient, leading to the development of internuclear and plastid coordination during the long evolutionary process. Current research indicates that nuclear-encoded chloroplast RNA-binding proteins can precisely regulate chloroplast RNA metabolism, thereby achieving the function of regulating chloroplast genes. Existing research shows that chloroplast RNA-binding proteins are closely related to chloroplast gene expression, primarily responsible for the stability of mRNA maturation and translation events. Their regulatory mechanisms include: intron splicing, proper folding of mRNA precursor terminal structures, protection of sensitive restriction enzyme sites, binding to terminals to maintain mRNA structural stability, and initiation of translation events, etc. Therefore, the regulation of chloroplast RNA-binding proteins is crucial for normal photosynthesis, and identifying key chloroplast RNA-binding proteins related to improving photosynthetic efficiency in crops under high temperatures is an extremely important fundamental scientific challenge.

[0048] Nuclear-encoded chloroplast RNA-binding proteins are crucial in the translation of chloroplast-encoded proteins and the formation of photosynthesis-related protein complexes. We believe the S1 domain is an important feature of these proteins. Based on the amino acid sequence of the S1 domain, we compared it with the rice website and screened and cloned the OsRBP1 gene, which can bind chloroplast RNA, from rice ZH11. We hope to enhance the photosynthetic efficiency, increase yield, and improve heat stress resistance of rice by studying the function and regulatory mechanism of the OsRBP1 gene in rice.

[0049] To this end, we constructed a mutant (osrbp1) that knocked out the OsRBP1 gene; and constructed a transgenic rice (OE) that overexpressed the OsRBP1 protein by constructing a vector pHsfA2::OsRBP1 overexpressing the OsRBP1 gene. In field experiments, we compared the agronomic traits, photosynthetic efficiency, and heat stress response of these mutants with wild-type rice.

[0050] Our research data confirms that the OsRBP1 gene plays a crucial role in the growth and development of rice. Loss of function of the OsRBP1 gene leads to a significant reduction in photosynthetic efficiency, stunted growth, and delayed development in mutants. In the seedling stage, OsRBP1 knockout mutants generally exhibit stunting, yellowing leaves, and defects in thylakoid membrane stacking. Furthermore, mature mutant plants are shorter and thinner than the wild type, with fewer tillers. Transient expression experiments in tobacco confirmed that the OsRBP1 protein is located in chloroplasts. Blue natural polyacrylamide gel electrophoresis and Western blot experiments revealed that OsRBP1 knockout significantly reduced the content of the PSII photocomplex and its protein subunits D1, D2, and CP47. This genetic evidence suggests that OsRBP1 is a key factor regulating photosynthetic efficiency and growth and development in rice; therefore, this invention reveals an OsRBP1-dependent pathway for regulating high photosynthetic efficiency in plants.

[0051] Research in rice also indicates that the OsRBP1 gene plays a crucial role in the development of the photosynthetic complex PSII and chloroplast development in rice. It is also essential for regulating rice growth and development, photosynthetic carbon assimilation efficiency, and the formation of biomass traits, increasing photosynthetic efficiency and biomass, and enhancing rice's heat resistance. The study found that OsRBP1 gene expression in response to heat stress improves the efficiency of the PSII complex in rice photosynthesis under high temperatures, enhancing rice's resistance to heat stress, thereby increasing the number of tillers, photosynthetic efficiency, and biomass in transgenic rice. Yield measurements of OsRBP1-overexpressing transgenic lines were validated in a field experiment in Songjiang, Shanghai, over one growing season (2024), with biomass increases ranging from 6.9% to 14.8%.

[0052] In summary, our research includes the following:

[0053] 1. By comparing the S1 domain, a key gene, OsRBP1, was identified to regulate plant photosynthetic efficiency, biomass, and / or heat resistance.

[0054] 2. A novel mechanism of the OsRBP1 protein was revealed. The OsRBP1 gene plays a key role in the development of the rice chloroplast photocomplex PSII and has an important promoting effect on the regulation of rice growth and development, photosynthetic carbon assimilation efficiency and the formation of biomass traits.

[0055] 3. The promoter pHsfA2 of the heat shock transcription factor gene HsfA2 (NCBI accession number AT2G26150), which strongly responds to high-temperature stress and expresses at high levels, was combined with the OsRBP1 gene. This newly constructed gene expression cassette, pHsfA2::OsRBP1 cDNA, was successfully integrated into the genome of a transgenic rice plant using a plant binary expression vector, and expressed in response to heat stress. Field yield measurements showed that this operation was beneficial in increasing the number of tillers, photosynthetic efficiency, and biomass in transgenic rice. This concept may provide a new carbon fixation solution for addressing the global greenhouse effect.

[0056] 4. Through heat treatment of detached leaves and related data measurement of PSII complex, it was found that OsRBP1 gene expression in response to heat stress is beneficial to improving the resistance of transgenic rice to high temperature stress.

[0057] The amino acid sequence of protein OsRBP1 is SEQ ID NO:1. Those skilled in the art can expect that conserved variants of OsRBP1 have the same function, which is reasonable.

[0058] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. A "conservative variant polypeptide" may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having substituent groups in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the sequence of this polypeptide (e.g., a leader sequence or secretory sequence, or a sequence used to purify the polypeptide, or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Examples include (1) a polypeptide formed by substitution, deletion, or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the amino acid sequence represented by the enzyme, and having the function of the wild-type enzyme; (2) a polypeptide having more than 50% (preferably more than 60%; more preferably more than 70%; more preferably more than 80%; more preferably more than 85%; more preferably more than 90%; more preferably more than 95%; more preferably more than 98%; more preferably more than 99%) similarity to the polypeptide of the sequence represented by the enzyme, and having the function of the wild-type enzyme; or (3) a polypeptide formed by adding a tag sequence to the N or C terminus of the polypeptide of the sequence represented by the enzyme, or by adding a signal peptide sequence to its N terminus. These fragments, derivatives, and analogs are well known to those skilled in the art in accordance with the teachings herein. The term "variation" or "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably positive mutations, i.e., mutations that enhance function. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains and therefore generally includes the substitution of amino acids in polypeptides with amino acids from the same or similar amino acid definition class. However, as used herein, a conserved mutation does not include hydrophilic-hydrophilic, hydrophobic-hydrophobic, hydroxyl-containing-hydroxyl-containing, or small residue-small residue substitutions if a conserved mutation can be alternatively expressed as aliphatic-to-aliphatic, nonpolar-to-nonpolar, polar-to-polar, acidic-to-acidic, basic-to-basic, aromatic-to-aromatic, or restriction residue-to-restriction residue substitutions. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.Exemplary conservative substitutions can be performed, for example, according to the table below.

[0059] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0060] To achieve the expression of OsRBP1 in plants such as rice, its encoding gene is used as a foreign gene to construct a gene expression cassette or expression construct, which is a DNA molecule. The expression cassette / expression construct is then operatively linked to a plasmid vector through subcloning to obtain a recombinant plasmid. The recombinant plasmid is then transformed into a host cell to obtain a transformant, i.e., a genetically engineered bacterium or recombinant bacterium, or it can be transformed into a plant through Agrobacterium-mediated transformation to obtain a transgenic plant.

[0061] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0062] As used herein, the terms “(disease resistance) improved,” “enhanced,” or “strengthened” can mean an increase of at least 10% relative to a reference level (such as wild-type rice ZH11 or DJ), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times relative to a reference level.

[0063] In this document, for the sake of simplicity, the name of a protein, such as OsRBP1, and its encoding gene (DNA), OsRBP1, are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing OsRBP1 as a regulatory factor function or category that binds to chloroplast RNA and regulates the cultivation and content of the chloroplast photocomplex PSII, it refers to a protein; when used as a gene description, it refers to the gene encoding that protein.

[0064] As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system containing all the necessary elements required to express the target protein OSRBP1. Typically, it includes the following elements: a promoter, a gene sequence encoding a polypeptide, and a terminator; additionally, it may optionally include a signal peptide encoding sequence such as mCherry (red fluorescent protein), GFP (green fluorescent protein), or YFP (yellow fluorescent protein); these elements are operatively linked.

[0065] As used herein, the term "expression construct" or "expression building block" refers to a recombinant DNA molecule containing a desired nucleic acid coding sequence (e.g., SEQ ID NO:2) and may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector (plasmid vector).

[0066] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, then the nucleic acid is exogenous to that host cell. A particular sequence is "exogenous" to the cell or organism in which it is inserted.

[0067] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO:2, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0068] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the polypeptide OsRBP1. The nucleic acid constructs can be manipulated according to the different expression vectors or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0069] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequence SEQ ID NO:2 of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Expression of the OsRBP1 gene of the present invention is typically achieved by operably linking the nucleic acid sequence SEQ ID NO:2 of the present invention to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0070] Gene knock-in vectors can be used to integrate the polynucleotide sequence SEQ ID NO:2 described herein into a region of interest in the host genome. Typically, gene knock-in vectors contain the polynucleotide sequence described herein, as well as 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequence described herein, and 3' homologous arms. When using gene knock-in vectors, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequence into the site of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing guide RNAs targeting the target gene, resulting in increased homologous recombination efficiency in the modified region, thus homologously recombinating the target fragment SEQ ID NO:2 contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0071] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin resistance, or chloramphenicol for *E. coli*, *Agrobacterium*, etc.

[0072] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0073] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0074] When OsRBP1 is used to improve plant agronomic traits or create new heat-resistant plant varieties, it is preferred to transfer it into plant plants through the traditional Agrobacterium-mediated transformation method. For this purpose, it is necessary to construct recombinant Agrobacterium engineered strains that express OsRBP1.

[0075] In this article, the terms "recombinant bacteria (strain)" and "(genetically) engineered bacteria (strain)" have the same meaning, both referring to strains of wild-type Agrobacterium, such as Agrobacterium tumefaciens, that have been genetically modified to contain the OsRBP1 overexpression vector.

[0076] Based on the function of protein OsRBP1 in improving plant photosynthetic efficiency and enhancing plant resistance to heat stress, it can be used to improve plant agronomic traits, promote plant biomass increase, and create high photosynthetic efficiency and / or heat-resistant plant germplasm resources.

[0077] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0078] 1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the coding sequence of the polypeptide OsRBP1;

[0079] 2) Contact plant cells, tissues, or organs with Agrobacterium in step 1) to transfer the coding sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0080] 3) Select plant cells or tissues into which the coding sequence is transferred; 4) Regenerate plants from the plant cells or tissues in step 3).

[0081] The technical effects of the present invention are mainly reflected in the following aspects:

[0082] (1) A new gene, OsRBP1, which plays a key role in the growth and development of rice, was screened based on the amino acid sequence of the S1 domain.

[0083] (2) A new mechanism of OsRBP1 protein was revealed. The OsRBP1 gene is crucial for the regulation of rice growth and development, photosynthetic carbon assimilation efficiency and the formation of biomass traits.

[0084] (3) Expression of the OsRBP1 gene in response to heat stress is beneficial to improving the photosynthetic efficiency and biomass of transgenic rice;

[0085] (4) Expression of the OsRBP1 gene in response to heat stress is beneficial to improving the resistance of transgenic rice to high temperature stress.

[0086] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0087] Example

[0088] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0089] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0090] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0091] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0092] The primer synthesis and gene sequencing in the examples were commissioned to Sangon Biotech (Shanghai) Co., Ltd. and BGI Genomics.

[0093] The molecular biology methods and transgenic plant construction methods, including the construction of OsRBP1 overexpression recombinant plasmids, the construction of CRISPR-Cas9 system plasmids for OsRBP1 gene knockout, and gene editing technologies, are performed using techniques commonly used in this field. For example, methods for assessing plant photosynthetic efficiency and detecting heat stress responses can be referenced in our research group's patent document CN113462715A, some of which is incorporated herein by reference.

[0094] Some of the PCR primers used in the examples are listed in Table 1.

[0095] Table 1. Some PCR primers used in the examples

[0096] Primer name Sequence (5'-3') PBR1-F CTCACCACTGGCGAAGATGCC PBR1-R TCAGCTGCCATTGTGTTCTGTTGTTAC q-PBR1-F ACGAGCCAATTGAGGTCAAAA q-PBR1-R GTATGCTGCAACCAACTTTGTT pHsfA2-F ACGCGTCGACCTTTTGCCAATTCCCTTGTCCTC pHsfA2-R CGGGATCCTTTCGTTGTTTATCTCAAATC OsU31-sgF TGGCGTGGCGTGCTCGGCCGCACGCTGAG OsU31-sgR AAACCTCAGCGTGCGGCCGAGCACGCCAC MYC-qRT-R CAAGTCCTCTTCAGAAATGAGCT

[0097] In Table 1, "-F" in the name represents positive; "-R" represents negative.

[0098] Example 1: Screening and cloning of the OsRBP1 gene

[0099] Under abiotic stresses of high temperature and high light, the synthesis rate of some important proteins in chloroplasts decreases significantly, severely impacting photosynthetic carbon assimilation efficiency. Therefore, improving the translation efficiency of proteins within chloroplasts is a crucial scientific question for enhancing photosynthetic efficiency, yield, and resistance in plants under stress. Based on this, we selected RNA-binding proteins with S1 domains encoded by nuclear genes and located within chloroplasts as our research focus. According to numerous studies in our laboratory and by previous researchers, the S1 domain typically facilitates RNA-protein binding, promoting RNA structural stability. We selected two RNA-binding proteins with S1 domains for our study, including OsRBP1. Using CRISPR-Cas9 technology, we constructed mutants of the genes encoding these two S1-domain RNA-binding proteins. Currently, only the OsRBP1 mutant exhibits a significantly reduced photosynthetic carbon assimilation rate and stunted growth. Therefore, we conducted a systematic study on the role of OsRBP1 in regulating photosynthetic efficiency.

[0100] Example 2: Construction of a binary expression vector for overexpressing the OsRBP1 gene

[0101] The construction of a plant binary expression vector for the OsRBP1 gene includes the following steps:

[0102] A. A 2000bp fragment of the pHsfA2 promoter sequence was amplified by PCR using the fidelity enzyme kod-plus, ligated into the intermediate vector pMD19-T, and sequenced correctly.

[0103] B. Using the SalI / BamHI restriction site, the plant binary expression vector pCAMBIA1300 was ligated into the vector pHsfA2-pCAMBIA1300 to form the vector pHsfA2-pCAMBIA1300.

[0104] C. The OsRBP1 gene coding frame sequence fragment was amplified by PCR using the fidelity enzyme kod-plus, ligated into the intermediate vector pMD19-T, and sequenced correctly.

[0105] D. The BamHI restriction site is used to link the resulting pHsfA2-pCAMBIA1300 to the final vector pHsfA2::OsRBP1-pCAMBIA1300.

[0106] The specific process is as follows:

[0107] 2.1 The 2000bp fragment of the proHsfA2 promoter sequence (SEQ ID NO:3) was amplified by PCR using the fidelity enzyme kod-plus, ligated into the intermediate vector pMD19-T, and sequenced correctly.

[0108] The conditions for RT-PCR cloning of the proHsfA2 promoter sequence were: using the high-fidelity enzyme kod-plus kit (KOD-401, Toyobo (Shanghai) Biotechnology Co., Ltd.).

[0109] Forward primer: pHsfA2-F: ACGCGTCGACCTTTGCCAATTCCTCTGTCCTC,

[0110] Reverse primer: pHsfA2-R: CGGGATCCTTTCGTTGTTTATCTCAAATC.

[0111] PCR conditions: 94℃ for 2 min; 94℃ for 30 s, 60℃ for 30 s, 68℃ for 20 s, 30 cycles; 68℃ for 10 min. Ligation conditions: Using the pMD19-T vector kit (6013, Baori Biotechnology (Beijing) Co., Ltd.), ligation was performed according to the standardized procedures in the instruction manual to construct the vector pMD19-T-proHsfA2.

[0112] 2.2 The SalI / BamHI restriction site was used to ligate the plant binary expression vector pCAMBIA1300 to form the vector proHsfA2-pCAMBIA1300.

[0113] The restriction enzyme digestion conditions were as follows: using the SalI-HF(R3138S) / BamH1-HF(R3136S) restriction endonuclease kit (New England Biotechnology (Beijing) Co., Ltd.), the intermediate vector pMD19-T-proHsfA2 was digested according to the standardized operation of the instruction manual to obtain the proHsfA2 fragment.

[0114] Gel recovery conditions: The proHsfA2 fragment was purified by gel recovery using an agarose gel recovery kit (19101ES70, Yisheng Biotechnology (Shanghai) Co., Ltd.) in accordance with the instructions.

[0115] Ligation conditions: Using the T4 DNA ligase kit (M0202, New England Biotechnology (Beijing) Co., Ltd.), the vector proHsfA2-pCAMBIA1300 was formed by standardized operation according to the instructions.

[0116] 2.3 The OsRBP1 gene coding frame sequence fragment was amplified by PCR using the fidelity enzyme kod-plus, ligated into the intermediate vector pMD19-T, and sequenced correctly.

[0117] The conditions for RT-PCR cloning of the OsRBP1 sequence were: using the high-fidelity enzyme kod-plus kit (KOD-401, Toyobo (Shanghai) Biotechnology Co., Ltd.).

[0118] Forward primer: PBR1-F: CTCACCACTGGCGAAGATGCC,

[0119] Reverse primer: PBR1-R: TCAGCTGCCATTGTGTTCTGTTGTTAC.

[0120] PCR conditions: 94℃ for 2 min; 94℃ for 30 s, 57℃ for 30 s, 68℃ for 20 s, 30 cycles; 68℃ for 10 min.

[0121] Ligation conditions: Using the pMD19-T vector kit (6013, Baori Biotechnology (Beijing) Co., Ltd.), ligation was performed according to the standardized procedures in accordance with the instructions to construct the vector pMD19-T-OsRBP1.

[0122] 2.4 The BamHI restriction site was used to ligate into pHsfA2-pCAMBIA1300 described in C to form the final vector pHsfA2::OsRBP1-pCAMBIA1300.

[0123] The restriction enzyme digestion conditions were as follows: The intermediate vector pMD19-T-OsRBP1 was digested using the BamHI-HF (R3136S) restriction endonuclease kit (New England Biotechnology (Beijing) Co., Ltd.) in accordance with the instructions and standardized procedures to obtain the OsRBP1 fragment.

[0124] Gel recovery conditions: The OsRBP1 fragment was purified by gel recovery using an agarose gel recovery kit (19101ES70, Yisheng Biotechnology (Shanghai) Co., Ltd.) in accordance with the instructions.

[0125] Ligation conditions: Using the T4 DNA ligase kit (M0202, New England Biotechnology (Beijing) Co., Ltd.), the vector proHsfA2::OsRBP1-pCAMBIA1300 was formed following standardized procedures according to the manufacturer's instructions. See the plasmid structure diagram below. Figure 6 a.

[0126] Example 3: Construction of transgenic rice with overexpression of gene OsRBP1

[0127] The binary expression vector pHsfA2::OsRBP1-pCAMBIA1300 constructed in Example 2 was sent to Wuhan Boyuan Biotechnology Co., Ltd., and the DJ-background rice genetic transformation service was selected.

[0128] Transgenic rice seedlings overexpressing the OsRBP1 gene, provided by Wuhan Boyuan Biotechnology Co., Ltd., were planted in the Shanghai Songjiang field of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (N30°56′36″, E121°07′26″). Based on phenotypic observation, three transgenic lines, OE1, OE12, and OE21, were selected for subsequent experiments.

[0129] Example 4: Construction of OsRBP1 gene knockout mutant

[0130] By constructing CRISPR-Cas9 system plasmids and using customized rice genetic transformation services with ZH11 background from Wuhan Boyuan Biotechnology Co., Ltd., OsRBP1 knockout mutants (osrbp1-1 and osrbp1-2) were obtained through subsequent planting and identification.

[0131] The specific steps are as follows:

[0132] 4.1 The knockout target was designed and evaluated using an online website (http: / / skl.scau.edu.cn / ), and the OsRBP1 knockout target sequence was determined to be: TGGCGTGCTCGGCCGCACGCTGAG.

[0133] 4.2 Primer Design:

[0134] OsU31-sgF:TGGCGTGGCGTGCTCGGCCGCACGCTGAG,

[0135] OsU31-sgR: AAACCTCAGCGTGCGGCCGAGCACGCCAC.

[0136] PCR conditions: Mix equal amounts of the two primers OsU31-sgF and OsU31-sgR, heat to 98°C, and after 5 minutes, cool to room temperature to obtain the double-stranded DNA template of OsU31-sgRNA.

[0137] The enzyme digestion conditions are: using The v2(R3733S) restriction endonuclease kit (New England Biotechnology (Beijing) Co., Ltd.) was used to digest the double-stranded DNA template of the vectors PC2-OsU3 and OsU31-sgRNA according to the standardized operation of the instructions.

[0138] Gel recovery conditions: Agarose gel recovery kit (19101ES70, Yisheng Biotechnology (Shanghai) Co., Ltd.) was used, and standardized procedures were followed according to the instructions for gel recovery and purification.

[0139] Ligation conditions: Using the T4 DNA ligase kit (M0202, New England Biotechnology (Beijing) Co., Ltd.), the vector PC2-OsU3-OsRBP1-sgRNA was formed by following the standardized procedures in accordance with the instructions.

[0140] Transformation conditions: The correctly sequenced plasmid PC2-OsU3-OsRBP1-sgRNA was sent to Wuhan Boyuan Biotechnology Co., Ltd. for rice genetic transformation.

[0141] Example 5: Cultivation and Gene Identification of WT and Transgenic Plants

[0142] 1. Plant cultivation

[0143] It was cultivated and planted in the Shanghai Songjiang field (N30°56′36″, E121°07′26″) of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0144] 2. Identification at the rice genome level

[0145] Leaves from wild-type WT (ZH11 or DJ) and transgenic plants (osrbp1-1, osrbp1-2, OE1, OE12, OE21) were collected, ground into powder using liquid nitrogen, and rice genome was extracted using a plant genomic DNA extraction kit (DP305) (Tiangen Biotech (Beijing) Co., Ltd.). RT-PCR amplification was performed using primers PBR1-F and PBR1-R with the extracted knockout transgenic lines as templates; RT-PCR amplification was performed using primers q-PBR1-F and MYC-qRT-R with the extracted overexpression lines as templates.

[0146] Enzymes: PCR SuperMix (+dye) (TransGen Biotech, Catalog No.: AS112-11)

[0147] PCR conditions: 94℃ for 2 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 20 s, 30 cycles; 72℃ for 10 min.

[0148] Genome-level identification and sequencing verification confirmed that all transgenic plants (osrbp1-1, osrbp1-2, OE1, OE12, OE21) were positive plants.

[0149] Example 6: Investigating the effects of OsRBP1 gene knockout on carbon dioxide assimilation rate and growth and development in rice.

[0150] The carbon dioxide assimilation rate of ZH11 wild-type and OsRBP1 knockout mutants osrbp1-1 and osrbp1-2 was measured during the grain-filling stage of rice (n=6, measurement leaf position: flag leaf).

[0151] See Figure 1 The photos show that the OsRBP1 knockout mutants exhibited weaker growth than the wild type. During the seedling stage, mutants osrbp1-1 and osrbp1-2 were stunted with yellowing new leaves. In the mature seedling stage, both mutants osrbp1-1 and osrbp1-2 produced fewer tillers and were shorter. Their carbon dioxide assimilation rate was lower than the wild type, indicating that the OsRBP1 knockout mutants showed weaker growth and lower photosynthetic efficiency.

[0152] Example 7: Investigating the localization of OsRBP1 protein

[0153] The steps for constructing the 35S::OsRBP1-GFP plasmid are as follows:

[0154] 7.1 The OsRBP1 gene coding frame sequence fragment was amplified by PCR using the fidelity enzyme kod-plus, ligated into the intermediate vector pMD19-T, and sequenced correctly.

[0155] The conditions for RT-PCR cloning of the OsRBP1 sequence were: using the high-fidelity enzyme kod-plus kit (KOD-401, Toyobo (Shanghai) Biotechnology Co., Ltd.).

[0156] Forward primer: PBR1-F: CTCACCACTGGCGAAGATGCC,

[0157] Reverse primer: PBR1-R: TCAGCTGCCATTGTGTTCTGTTGTTAC.

[0158] PCR conditions: 94℃ for 2 min; 94℃ for 30 s, 57℃ for 30 s, 68℃ for 20 s, 30 cycles; 68℃ for 10 min.

[0159] Ligation conditions: Using the pMD19-T vector kit (6013, Baori Biotechnology (Beijing) Co., Ltd.), ligation was performed according to the standardized procedures in accordance with the instructions to construct the vector pMD19-T-OsRBP1.

[0160] 7.2 The salI and speI restriction sites were used to ligate into 35S::GFP-pCAMBIA1300 to form the final vector 35S::OsRBP1-GFP-pCAMBIA1300.

[0161] The restriction enzyme digestion conditions were as follows: using the salI-HF (R3138S) and speI-HF (R3133S) restriction endonuclease kit (New England Biotechnology (Beijing) Co., Ltd.), the intermediate vector pMD19-T-OsRBP1 was digested according to the standardized operation of the instructions to obtain the OsRBP1 fragment.

[0162] Gel recovery conditions: The OsRBP1 fragment was purified by gel recovery using an agarose gel recovery kit (19101ES70, Yisheng Biotechnology (Shanghai) Co., Ltd.) in accordance with the instructions.

[0163] Ligation conditions: Using the T4 DNA ligase kit (M0202, New England Biotechnology (Beijing) Co., Ltd.), the vector 35S::OsRBP1-GFP-pCAMBIA1300 was formed by standardized operation according to the instructions.

[0164] 7.3 Take 1 μg of the plasmid 35S::OsRBP1-GFP-pCAMBIA1300 constructed in Example 2 (with correct sequencing) and Agrobacterium GV3101 competent cells (Shanghai Weidi Biotechnology Co., Ltd.). Incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, heat shock at 37℃ for 5 min, place on ice for 5 min, add 1 mL of LB medium, and incubate at 28℃ on a shaker for 4 hours. Spread all bacterial cells onto kanamycin-resistant and rifampicin-resistant LB agar plates and incubate at 28℃ for 2-4 days to obtain engineered Agrobacterium bacteria. Pick single colonies from kanamycin- and rifampicin-resistant LB agar plates and incubate in LB (Kan+Rif) tubes for 48 h until OD600 ≥ 2.0. Incubate overnight (12 h) at 28℃. After collecting the bacterial cells, they were incubated with MMAbuffer (10mM MES, 10mM MgCl2 and 100μM acetylsyl syringone) for 3h, and then injected into tobacco leaves with a needleless syringe. After 48h, the epidermis was peeled off for observation.

[0165] See Figure 2 The strong fluorescence signal appeared as dots and largely overlapped with the fluorescence of chloroplasts. Subsequent observation of protoplasts from the transiently transformed tobacco leaves yielded results largely consistent with those observed in the epidermis and stomata: strong fluorescence signals appeared as dots and largely overlapped with the fluorescence of chloroplasts. Fluorescence data indicated that the OsRBP1 protein is located in the chloroplasts.

[0166] Example 8: Effects of OsRBP1 gene knockout on rice chloroplast development

[0167] Ultrathin sections were prepared from leaves of 26-day-old wild-type and osrbp1-1 seedlings. Electron microscopy revealed that the wild-type thylakoid stacks were uniform, with good continuity between different stacks and no breaks, and the starch granules were completely consumed or transferred at night. In contrast, the chloroplasts of the mutant osrbp1-1 showed many defects, with blurred chloroplast membrane boundaries compared to the wild-type. Notably, the thylakoid stacks were disordered, discontinuous between different stacks, and some stacks were dense, hindering normal photosynthesis. Furthermore, after nighttime consumption, the starch granules in the chloroplasts were not completely consumed or transferred.

[0168] Example 8: Effects of OsRBP1 gene knockout on some photocomplex proteins in rice

[0169] 8.1 The thylakoid membrane was extracted and isolated from plant leaves using the method described in the following literature: Peng, L. Wetal., LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell, 18, 955-969 (2006).

[0170] Fresh leaves (0.8g each) from 26-day-old wild-type and osrbp1-1 seedlings were harvested, ground, and thylakoid membrane proteins were extracted. After chlorophyll quantification of both samples, the thylakoid membrane protein complexes were separated by blue natural polyacrylamide gel electrophoresis (PAGE). Figure 4 As shown in Figure a, we found that the mutant osrbp1-1 contained less of the five thylakoid membrane protein complexes—PSII super complex, PSI+PSIIcore dimer, PSII monomer, and CP43-less-PSII—than the wild-type, and all four thylakoid membrane protein complexes were associated with PSII. This result suggests that the OsRBP1 gene selectively regulates photocomplex biosynthesis.

[0171] 8.2 The extraction of PSII core protein and related procedures for Western blotting are as follows:

[0172] The chloroplast thylakoid membranes extracted in step 8.1 were heated in a 98°C water bath for 10 minutes, centrifuged at 12500g for 30 minutes, and the supernatant protein was collected for routine Western blotting. The following materials were used for Western blotting: D1 antibody (Agrisera, Sweden, catalog number: AS05 084); D2 antibody (Agrisera, Sweden, catalog number: AS06 146); CP47 antibody (Agrisera, Sweden, catalog number: AS04 038); PVDF membrane (thermofish, catalog number: 88518); goat anti-rabbit: Sigma (catalog number: AP132); Immobilon ECL Ultra Western HRP Substrate (catalog number: WBULS0500). Figure 4 As shown in Figure b, after serial dilution electrophoresis of the two samples, the content of the internal control ATPβ was found to be basically the same in both samples. However, compared with the wild type, the contents of the three core subunits of the PSII complex, D1, D2, and CP47, decreased significantly. This indicates that the OsRBP1 mutation has an adverse effect on the accumulation of PSII core protein content.

[0173] Example 9: Effect of OsRBP1 gene knockout on photochemical efficiency in rice

[0174] For methods to assess the photochemical efficiency of PSII, please refer to the following literature:

[0175] Peng,LWet al.,LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana.Plant Cell 18,955-969(2006).

[0176] Lu Y,Hall DA,Last RL(2011),Asmall zinc finger thylakoid protein playsarole in maintenance of photosystem II in Arabidopsis thaliana.Plant Cell.23:1861–1875.

[0177] The light reactivity of the flag leaves of mutant and wild-type plants was measured using the PAM2500 instrument. Figure 5 a and b: We found that under a fixed light intensity, the Fv / Fm of the mutant and the actual electron yield (Φ) of photorecombination system II were... PSII All were significantly lower than the wild type. Figure 5 c and d: We measured the numerical trends of PSII quantum yield and electron transport rate under progressively increasing light intensities, from... Figure 5 Analysis reveals that the actual electron yield and electron transfer efficiency of the photorecombinant system II are significantly lower than those of the wild type.

[0178] Example 11: Effects of OsRBP1 gene overexpression on rice growth and development and detection of overexpression levels

[0179] Three lines (OE1, OE12, and OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice were observed in the field at 24 and 75 days of growth; the expression level of the OsRBP1 gene in the three lines (OE1, OE12, and OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice was detected at the grain-filling stage.

[0180] 11.1 Three lines (OE1, OE12, OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice were observed in the field after 24 and 75 days of growth. (See below) Figure 6 In the case of strain b, overexpression of all three strains resulted in stronger seedlings and more tillers in the mature seedling stage.

[0181] 11.2 Real-time fluorescence quantitative detection of the OsRBP1 gene, the specific operation is as follows:

[0182] RNA was extracted from wild-type and transgenic plants in the field. Leaf samples from the flag leaf of each line were collected, ground into powder using liquid nitrogen, and total RNA was extracted using a plant RNA extraction kit (DP432, Tiangen Biotech (Beijing) Co., Ltd.). After reverse transcription into cDNA, qRT-PCR amplification was performed using primers q-PBR1-F and q-PBR1-R to detect OsRBP1 transcription levels. Reverse transcription kit: PrimeScript. TM RT reagent kit (Perfect Real Time) (Takara, catalog number: RR037Q) PCR kit: TB Premix Ex TaqTMII (Tli RNaseH Plus) (Takara, catalog number: RR820Q). Two-step PCR amplification standard procedure: Stage 1: 95℃ 30s pre-denaturation, Stage 2: PCR reaction: 95℃ 5s, 60℃ 30-34s, 40 cycles. Figure 6 As shown in Figure c, the expression level of the OsRBP1 gene in the three lines (OE1, OE12, and OE21) of pHsfA2::OsRBP1 transgenic rice was higher than that of the wild type, with OE12 and OE21 showing particularly significant differences compared to the wild type.

[0183] Example 12: Effects of OsRBP1 overexpression on carbon dioxide assimilation rate and biomass in rice

[0184] Carbon dioxide assimilation rates (n=6, leaf position: flag leaf) were measured in three lines (OE1, OE12, OE21) of DJ wild-type and pHsfA2::OsRBP1 transgenic rice during the grain-filling stage. See also... Figure 7a) The carbon dioxide assimilation rate of the three lines (OE1, OE12, and OE21) of pHsfA2::OsRBP1 transgenic rice was higher than that of the wild type. b) Aboveground biomass (b, n=3) of wild-type rice and transgenic lines (OE1, OE12, and OE21) grown in the Shanghai Songjiang field (N30°56′36″, E121°07′26″) of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, in 2024. The OsRBP1 biomass of the three lines (OE1, OE12, and OE21) of pHsfA2::OsRBP1 transgenic rice was higher than that of the wild type, with OE12 and OE21 showing particularly significant differences compared to the wild type.

[0185] Example 13: Investigating the effect of OsRBP1 gene overexpression on rice heat stress resistance

[0186] Heat treatment materials and conditions: Flag leaves of wild-type and pHsfA2::OsRBP1 transgenic rice were selected for heat treatment (55℃ for 2h). After recovery at room temperature for 0.5h, the light reaction efficiency of transgenic rice and wild-type flag leaves was measured using a PAM2500 instrument.

[0187] like Figure 8 As shown in a and b, we found that after heat stress treatment, under a fixed light intensity, the Fv / Fm ratio and the actual electron yield (Φ) of the pHsfA2::OsRBP1 transgenic lines (OE12, OE21) were significantly different. PSII Both were significantly higher than the wild type. For example... Figure 8 As shown in Figures c and d, we measured the numerical trends of PSII quantum yield and electron transport rate under progressively increasing light intensity. Analysis of the figures reveals that after heat stress treatment, the actual electron yield and electron transport efficiency of the photorecombinant system II in the pHsfA2::OsRBP1 transgenic lines (OE12, OE21) were higher than those in the wild type.

[0188] In summary, the OsRBP1 protein plays an important role in regulating rice growth and development, photosynthetic carbon assimilation efficiency, and the formation of biomass traits. OsRBP1 gene expression in response to heat stress is beneficial for improving the photosynthetic efficiency and biomass of transgenic rice. Furthermore, OsRBP1 gene expression in response to heat stress is beneficial for improving the resistance of transgenic rice to high-temperature stress, and has the potential to be applied to food production and the solution of the greenhouse effect problem.

[0189] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A polypeptide, said polypeptide being selected from the group consisting of: (a) A polypeptide with the amino acid sequence shown in SEQ ID NO:1, which is protein OsRBP1; (b) A conserved variant polypeptide derived from (a) having the polypeptide function of (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:

1. (c) A conserved variant polypeptide derived from (a) that has more than 95% homology to the polypeptide sequence defined in (a) and has the function of the polypeptide in (a); (d) A derivative polypeptide containing the polypeptide sequence described in (a), (b), or (c). The aforementioned function refers to the ability to bind to chloroplast RNA and promote an increase in the content of the chloroplast photocomplex PSII. The photocomplex PSII is the photosystem I (PSII) complex reported in the patent document with publication number CN113462715A.

2. A polynucleotide, said polynucleotide being selected from the group consisting of: (A) A polynucleotide encoding the polypeptide of claim 1; (B) A polynucleotide encoding a polypeptide with the amino acid sequence shown in SEQ ID NO:1; (C) The nucleotide sequence is a polynucleotide as shown in SEQ ID NO:2, which is the CDS region sequence of the gene encoding protein OsRBP1; (D) Polynucleotides with ≥95% homology to the nucleotide sequence shown in SEQ ID NO:2; (E) A nucleotide sequence complementary to any of the nucleotide sequences described in (A)-(D).

3. A DNA molecule, characterized in that, It contains the polynucleotide as described in claim 2.

4. The DNA molecule as described in claim 3, characterized in that, It contains the OsRBP1 encoding gene with the nucleotide sequence SEQ ID NO:2 and the upstream pHsfA2 promoter, Ubi promoter or 35S promoter, which is the expression cassette / expression box of the protein OsRBP1. The pHsfA2 promoter is the heat-responsive pHsfA2 promoter reported in the patent document with publication number CN113462715A (derived from the HsfA2 gene of wild-type Arabidopsis thaliana Col-0, whose NCBI accession number is AT2G26150), and its nucleotide sequence is SEQ ID NO:

3.

5. A recombinant plasmid, characterized in that, It contains the DNA molecule as described in claim 3 or 4.

6. A type of engineered microbial bacterium, characterized in that, The engineered microbial bacteria are transformants containing the recombinant plasmid as described in claim 5.

7. The engineered microbial strain as described in claim 5, characterized in that, The microbial host is Agrobacterium.

8. The application of the polypeptide of claim 1, the polynucleotide of claim 2, the DNA molecule of claim 3, the recombinant plasmid of claim 4, or the microbial engineered bacteria of claim 6 in improving the photosynthetic efficiency of plants such as rice, in increasing the biomass of plants such as rice, in improving the heat tolerance of plants such as rice, i.e., resistance to heat stress, or in cultivating new varieties of plants such as rice with high photosynthetic efficiency and / or high temperature tolerance.

9. The application as described in claim 8, characterized in that, The plant is a grass crop, preferably rice.

10. The application as described in claim 8, characterized in that, Overexpression of the polypeptide as described in claim 1, such as OsRBP1, in plants using the following method: (i) The gene encoding the polynucleotide, such as protein OsRBP1 as described in claim 2, is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely OsRBP1 overexpression vector, and plants are transformed by Agrobacterium-mediated transformation to obtain transgenic plants that overexpress the polypeptide, such as OsRBP1 as described in claim 1. (ii) By using gene editing technology, the gene encoding the polynucleotide, such as the protein OsRBP1 as described in claim 2, is cloned into a plant chromosome to obtain a transgenic plant that overexpresses the gene encoding the polypeptide, such as OsRBP1 as described in claim 1. (iii) Place the existing OsRBP1 coding gene in the plant genome under the regulation of a functionally enhanced promoter, such as the pHsfA2 promoter, the 35S promoter, or the Ubi promoter.

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