Modified small nuclear RNA (Ribonucleic Acid) and application of protein complex of modified small nuclear RNA in enhancing photosynthesis of plants

Abstract

The invention discloses application of modified small nuclear RNA (Ribonucleic Acid) and a protein complex thereof in enhancing plant photosynthesis. The invention relates to the technical field of biology, and provides a small nuclear RNA (Ribonucleic Acid) of which the nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2. The invention also provides a small nuclear RNA-protein complex composed of the small nuclear RNA and an Sm protein specifically bound with the small nuclear RNA. The small nuclear RNA and the protein complex thereof disclosed by the invention can be specifically combined with mRNA 3 'UTR regions of a plurality of nuclear coding chloroplast protein genes through base complementary pairing, and the expression of the nuclear coding chloroplast protein is enhanced on the translation level, so that the biosynthesis of chloroplast is promoted, and the photosynthetic efficiency of plants is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a modified small nuclear RNA and its protein complex in enhancing plant photosynthesis. Background Technology

[0002] Improving crop photosynthetic efficiency is one of the key strategies for achieving sustainable agricultural development in the context of global climate change. Efficient biosynthesis and functional regulation of chloroplasts are crucial for enhancing photosynthesis, requiring rapid and precise coordination at the translational level of a large number of chloroplast proteins encoded by the cell nucleus.

[0003] Existing genetic engineering methods to improve photosynthesis mainly focus on the modification of key enzymes and metabolic pathways. For example, photosynthetic efficiency can be improved by overexpressing single rate-limiting enzymes such as Rubisco activator or SBPase, or by introducing exogenous C4 pathways and cyanobacterial concentration mechanisms, or by constructing photorespiration bypasses to reduce energy loss.

[0004] The drawbacks of existing technologies lie in the mismatch between transcription and translation paces, and the imbalance in assembly ratios. Constitutive promoter-driven gene expression is often limited by translation initiation efficiency, resulting in limited conversion efficiency and difficulty in efficiently converting into functional proteins; while overexpression of core factors in the protein translation machine can easily induce protein degradation or cytotoxicity. Furthermore, traditional strategies, lacking responsiveness to environmental signals, can easily impose a severe metabolic burden on plants. Summary of the Invention

[0005] To effectively address the aforementioned technical problems, this invention aims to provide a regulatory element based on RNA-targeted complementary mRNA that synchronously and selectively enhances the expression of multiple nuclear-encoded chloroplast proteins at the translational level, thereby driving efficient chloroplast biosynthesis and ultimately enhancing plant photosynthesis.

[0006] In one respect, the present invention claims protection for a small nuclear RNA.

[0007] The small nuclear RNA claimed in this invention is a modified U5 small nuclear RNA, whose nucleotide sequence is SEQ ID NO:1 (corresponding to U5-M1 in the examples) or SEQ ID NO:2 (corresponding to U5-M2 in the examples).

[0008] The modified U5 small nuclear RNA has a modified Sm binding site that can specifically recognize the 3'UTR region of plant photosynthesis-related gene (such as RPS1 gene and / or LHCB3 gene) mRNA.

[0009] Secondly, this invention claims protection for a DNA molecule.

[0010] The DNA molecule claimed in this invention is a DNA molecule capable of being transcribed to the small nuclear RNA described in the first aspect above.

[0011] Furthermore, the nucleotide sequence of the DNA molecule may be the sequence obtained by replacing uracil in SEQ ID NO:1 with thymine, or the sequence obtained by replacing uracil in SEQ ID NO:2 with thymine.

[0012] Thirdly, the present invention claims protection for expression cassettes, recombinant vectors, or recombinant microorganisms containing the DNA molecules described in the second aspect above.

[0013] The expression cassette includes a promoter and the DNA molecule, and the promoter and the DNA molecule are operatively linked. Further, the expression cassette also includes a terminator, and the promoter, the DNA molecule, and the terminator are operatively linked. The same applies below.

[0014] The term "operable link" refers to the way in which regulatory elements (promoters, terminators) are linked to the target gene (i.e., the DNA molecule) in a way that ensures the regulatory elements can perform normal transcriptional regulation of the target gene and avoids functional failure caused by disordered element arrangement.

[0015] The recombinant vector can be any plasmid, expression vector, or other conventional vector in the art, and is obtained by inserting the DNA molecule or the expression cassette into the vector backbone. The same applies below.

[0016] The recombinant microorganisms include, but are not limited to, bacteria (such as Escherichia coli or Agrobacterium), yeasts, etc., and can be obtained by transforming or transducing the recombinant vector into a host microorganism. The same applies below.

[0017] Fourthly, the present invention claims protection for a small nuclear RNA-protein complex.

[0018] The small nuclear RNA-protein complex claimed in this invention includes (or consists of) the small nuclear RNA described in the first aspect above and the Sm protein that specifically binds to the small nuclear RNA.

[0019] Furthermore, the Sm protein may be selected from any of the following: SmD3a protein, SmD3b protein variant.

[0020] In some embodiments of the present invention, the Sm protein is the SmD3a protein with the amino acid sequence shown in SEQ ID NO:9.

[0021] In some embodiments of the present invention, the Sm protein is a variant of the SmD3b protein with an amino acid sequence as shown in SEQ ID NO:3 (i.e., the SmD3b-M protein in the examples).

[0022] Fifthly, the present invention claims protection for biological materials related to the small nucleus RNA-protein complex described in the fourth aspect above.

[0023] The biological material claimed in this invention is any one of the following: (A1) DNA molecule assembly; the DNA molecule assembly includes (or consists of) DNA molecule 1 and DNA molecule 2; DNA molecule 1 is a DNA molecule capable of being transcribed to produce the small nuclear RNA; DNA molecule 2 is a DNA molecule capable of expressing the Sm protein; (A2) Expression cassette assembly; the expression cassette assembly includes (or consists of) expression cassette 1 and expression cassette 2; expression cassette 1 contains the DNA molecule 1 described in (A1); expression cassette 2 contains the DNA molecule 2 described in (A1); (A3) Recombinant vector combination; the recombinant vector combination (or composed of the following) includes recombinant vector 1 and recombinant vector 2; the recombinant vector 1 carries the expression cassette 1 in (A2); the recombinant vector 2 carries the expression cassette 2 in (A2); (A4) A recombinant microbial combination; the recombinant microbial combination (or composed of) recombinant microorganism 1 and recombinant microorganism 2; the recombinant microorganism 1 contains the recombinant vector 1 in (A3); the recombinant microorganism 2 contains the recombinant vector 2 in (A3); (A5) Recombinant vector; the recombinant vector simultaneously carries the expression cassette 1 and the expression cassette 2 from (A2); (A6) Recombinant microorganism; wherein the recombinant microorganism contains both the recombinant vector 1 and the recombinant vector 2 in (A3); or, wherein the recombinant microorganism contains the recombinant vector in (A6); (A7) Transgenic plant; the transgenic plant is a plant capable of expressing the small nuclear RNA-protein complex.

[0024] Furthermore, the transgenic plant is a transgenic plant obtained by introducing the DNA molecule combination described in (A1), the expression cassette combination described in (A2), the recombinant vector combination described in (A3), or the recombinant vector described in (A5) into a recipient plant; the photosynthetic activity of the transgenic plant is stronger than that of the recipient plant.

[0025] The "strong photosynthesis" mentioned refers to comparisons made under comparable conditions. "Comparable conditions" are the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly promote or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients.

[0026] Further, in (A2), the promoter for initiating transcription of DNA molecule 1 in expression cassette 1 is the UBQ promoter; the promoter for initiating transcription of DNA molecule 2 in expression cassette 2 is the 35S promoter. More further, the nucleotide sequence of the UBQ promoter is shown in positions 1-636 of SEQ ID NO:4; the nucleotide sequence of the 35S promoter is shown in positions 1-346 of SEQ ID NO:5. In some embodiments of the present invention, the nucleotide sequence of expression cassette 1 is shown in SEQ ID NO:4 (U5-M1 transcription driven by the UBQ promoter); the nucleotide sequence of expression cassette 2 is shown in SEQ ID NO:5 (SmD3b-M protein expression driven by the 35S promoter). In some embodiments of the present invention, the nucleotide sequence of expression cassette 1 is shown in SEQ ID NO:8 (U5-M2 transcription driven by the UBQ promoter); the nucleotide sequence of expression cassette 2 is shown in SEQ ID NO:5 (SmD3b-M protein expression driven by the 35S promoter). In some embodiments of the present invention, the nucleotide sequence of expression cassette 1 is shown in SEQ ID NO:4 (transcription of U5-M1 driven by the UBQ promoter); the nucleotide sequence of expression cassette 2 is shown in SEQ ID NO:6 (expression of SmD3a protein driven by the 35S promoter). In some embodiments of the present invention, the nucleotide sequence of expression cassette 1 is shown in SEQ ID NO:8 (transcription of U5-M2 driven by the UBQ promoter); the nucleotide sequence of expression cassette 2 is shown in SEQ ID NO:6 (expression of SmD3a protein driven by the 35S promoter).

[0027] In some embodiments of the present invention, the recombinant expression vector 1 mentioned in (A3) is a plant expression vector carrying the expression cassette 1 shown in SEQ ID NO:4 (transcribed by U5-M1 driven by the UBQ promoter); the recombinant expression vector 2 is a plant expression vector carrying the expression cassette 2 shown in SEQ ID NO:5 (expressed by SmD3b-M protein driven by the 35S promoter). Specifically, in some embodiments of the present invention, the recombinant expression vector 1 is specifically obtained by inserting the expression cassette 1 shown in SEQ ID NO:4 into the multiple cloning site of the pCAMBIA1300 vector as a backbone; the recombinant expression vector 2 is specifically obtained by inserting the expression cassette 2 shown in SEQ ID NO:5 into the multiple cloning site of the pCAMBIA1300 vector as a backbone.

[0028] In some embodiments of the present invention, the recombinant expression vector 1 in (A3) is a plant expression vector carrying the expression cassette 1 shown in SEQ ID NO:8 (transcribed by U5-M2 driven by the UBQ promoter); the recombinant expression vector 2 is a plant expression vector carrying the expression cassette 2 shown in SEQ ID NO:5 (expressed by SmD3b-M protein driven by the 35S promoter). Specifically, in some embodiments of the present invention, the recombinant expression vector 1 is specifically obtained by inserting the expression cassette 1 shown in SEQ ID NO:8 into the multiple cloning site of the pCAMBIA1300 vector as a backbone; the recombinant expression vector 2 is specifically obtained by inserting the expression cassette 2 shown in SEQ ID NO:5 into the multiple cloning site of the pCAMBIA1300 vector as a backbone.

[0029] In some embodiments of the present invention, the recombinant expression vector 1 in (A3) is a plant expression vector carrying the expression cassette 1 shown in SEQ ID NO:4 (transcribed by U5-M1 driven by the UBQ promoter); the recombinant expression vector 2 is a plant expression vector carrying the expression cassette 2 shown in SEQ ID NO:6 (expressed by SmD3a protein driven by the 35S promoter). Specifically, in some embodiments of the present invention, the recombinant expression vector 1 is specifically obtained by inserting the expression cassette 1 shown in SEQ ID NO:4 into the multiple cloning site of the pCAMBIA1300 vector as a backbone; the recombinant expression vector 2 is specifically obtained by inserting the expression cassette 2 shown in SEQ ID NO:6 into the multiple cloning site of the pCAMBIA1300 vector as a backbone.

[0030] In some embodiments of the present invention, the recombinant expression vector 1 in (A3) is a plant expression vector carrying the expression cassette 1 shown in SEQ ID NO:8 (transcribed by U5-M2 driven by the UBQ promoter); the recombinant expression vector 2 is a plant expression vector carrying the expression cassette 2 shown in SEQ ID NO:6 (expressed by SmD3a protein driven by the 35S promoter). Specifically, in some embodiments of the present invention, the recombinant expression vector 1 is specifically obtained by inserting the expression cassette 1 shown in SEQ ID NO:8 into the multiple cloning site of the pCAMBIA1300 vector as a backbone; the recombinant expression vector 2 is specifically obtained by inserting the expression cassette 2 shown in SEQ ID NO:6 into the multiple cloning site of the pCAMBIA1300 vector as a backbone.

[0031] Sixthly, the present invention claims protection for the use of the small nuclear RNA described in the first aspect above, or the DNA molecule described in the second aspect above, or the expression cassette or recombinant vector or recombinant microorganism or recombinant cell described in the third aspect above, or the small nuclear RNA-protein complex described in the fourth aspect above, in any of the following: (B1) Enhances plant photosynthesis; (B2) Improve plant photosynthetic efficiency; (B3) Improve the maximum photochemical efficiency (Fv / Fm) of PSII in plants; (B4) Enhance the expression of photosynthetic proteins at the translational level.

[0032] In some embodiments of the present invention, the photosynthetic protein mentioned in (B4) is RPS1 protein (Nbe.v1.1.chr17g11580) and / or LHCB3 protein (Nbe.v1.1.chr10g29600).

[0033] Seventhly, the present invention claims a method for enhancing plant photosynthesis.

[0034] The method for enhancing plant photosynthesis claimed in this invention includes the following steps: expressing the small nuclear RNA described in the first aspect above and the Sm protein described in the fourth aspect above in the plant, thereby enhancing plant photosynthesis.

[0035] Furthermore, the expression of the small nuclear RNA and the Sm protein in the plant can be achieved by introducing into the plant the DNA molecule combination described in (A1) of the fifth aspect above, or the expression cassette combination described in (A2), or the recombinant vector combination described in (A3), or the recombinant vector described in (A6).

[0036] In some embodiments of the present invention, expression of the small nuclear RNA and the Sm protein in the plant is achieved by introducing recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3b-M-YFP into the plant. The recombinant expression vector pCAMBIA1300-U5-M1 is obtained by inserting expression cassette 1 (transcribed by the UBQ promoter) shown in SEQ ID NO:4 into the multiple cloning site of the pCAMBIA1300 vector. The recombinant expression vector pCAMBIA1300-SmD3b-M-YFP is obtained by inserting expression cassette 2 (expressed by the 35S promoter) shown in SEQ ID NO:5 into the multiple cloning site of the pCAMBIA1300 vector.

[0037] In some embodiments of the present invention, expression of the small nuclear RNA and the Sm protein in the plant is achieved by introducing recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3a-YFP into the plant. The recombinant expression vector pCAMBIA1300-U5-M1 is obtained by inserting expression cassette 1 (transcribed by U5-M1 driven by the UBQ promoter) as shown in SEQ ID NO:4 into the multiple cloning site of the pCAMBIA1300 vector. The recombinant expression vector pCAMBIA1300-SmD3a-YFP is obtained by inserting expression cassette 2 (expressed by the 35S promoter driven by the 35S promoter) as shown in SEQ ID NO:6 into the multiple cloning site of the pCAMBIA1300 vector.

[0038] In some embodiments of the present invention, expression of the small nuclear RNA and the Sm protein in the plant is achieved by introducing recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3b-M-YFP into the plant. The recombinant expression vector pCAMBIA1300-U5-M2 is obtained by inserting expression cassette 1 (transcribed by U5-M2 driven by the UBQ promoter) as shown in SEQ ID NO:8 into the multiple cloning site of the pCAMBIA1300 vector. The recombinant expression vector pCAMBIA1300-SmD3b-M-YFP is obtained by inserting expression cassette 2 (expressed by the 35S promoter driven by the 35S promoter) as shown in SEQ ID NO:5 into the multiple cloning site of the pCAMBIA1300 vector.

[0039] In some embodiments of the present invention, expression of the small nuclear RNA and the Sm protein in the plant is achieved by introducing recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3a-YFP into the plant. The recombinant expression vector pCAMBIA1300-U5-M2 is obtained by inserting expression cassette 1 (transcribed by U5-M2 driven by the UBQ promoter) as shown in SEQ ID NO:8 into the multiple cloning site of the pCAMBIA1300 vector. The recombinant expression vector pCAMBIA1300-SmD3a-YFP is obtained by inserting expression cassette 2 (expressed by the 35S promoter driven by the 35S promoter) as shown in SEQ ID NO:6 into the multiple cloning site of the pCAMBIA1300 vector.

[0040] Eighthly, the present invention claims protection for the use of the small nuclear RNA described in the first aspect above, or the DNA molecule described in the second aspect above, or the expression cassette or recombinant vector or recombinant microorganism or recombinant cell described in the third aspect above, or the small nuclear RNA-protein complex described in the fourth aspect above, or the biological material described in the fifth aspect above, or the method described in the seventh aspect above, in any of the following: (C1) Increase plant biomass; (C2) Increase plant yield; (C3) Plant breeding.

[0041] Furthermore, the biomass mentioned in (C1) refers to aboveground biomass.

[0042] Furthermore, the plant breeding described in (C3) can be for breeding plant varieties with enhanced photosynthesis, or for breeding plant varieties with increased biomass (such as aboveground biomass), or for breeding plant varieties with increased yield.

[0043] Ninthly, the present invention claims protection for a plant.

[0044] The plant claimed in this invention expresses the small nuclear RNA described in the first aspect above and the Sm protein described in the fourth aspect above; the plant has at least one of the following characteristics: enhanced photosynthesis, improved photosynthetic efficiency, increased maximum photochemical efficiency of PSII (Fv / Fm), increased biomass, and increased yield.

[0045] In the aforementioned related aspects, the plants may be various groups of crops, such as rice, soybeans, tobacco, tomatoes, etc.

[0046] The beneficial effects of this invention are: This invention constructs a protein translation enhancement system based on a plant small nuclear RNA (snRNA) complex. This system exhibits photothermal signal responsiveness and can efficiently drive the synthesis of multiple chloroplast proteins at the translational level, thereby significantly increasing plant photosynthetic efficiency to over 20%. Since the increased photosynthetic efficiency directly drives the synthesis and accumulation of plant photosynthetic products, it effectively promotes the increase of aboveground biomass in the recipient plant and lays the material foundation for increased yield per plant. This technology breaks through the limitations of traditional single-gene manipulation for the first time and has significant application value as a universal regulatory tool in crop breeding and plant synthetic biology. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the U5 snRNA variant design of the present invention. A shows the base pairing design of U5 snRNA variant M1 with tobacco NbeRPS1; B shows the base pairing design of U5 snRNA variant M2 with tobacco NbeLHCB3.

[0048] Figure 2 Subcellular localization features of the fusion proteins SmD3a-YFP, SmD3b-YFP, and the variant SmD3b-M-YFP in leaves of *Nicotiana benthamiana* were displayed using confocal microscopy. Scale bar: 20 μm.

[0049] Figure 3 This study aimed to investigate the modified U5 snRNA variant and its synergistic effect with SmD3 protein in promoting the accumulation of photosynthetic proteins. In Figure A, RT-qPCR was used to detect the expression level of U5 snRNA in transgenic Nicotiana benthamiana (U5, U5-M1, U5-M2), with EF1α as an internal control gene. ns indicates no significant difference in expression levels between groups after a two-tailed t-test. Figures B and C show the abundance of photosynthetic proteins RPS1 and LHCB3 as detected by Western blot. Figure B shows that expressing U5-M1 or U5-M2 alone can increase the protein levels of RPS1 and LHCB3, respectively. Figure C shows that co-expression of U5-M1 / U5-M2 and SmD3a / SmD3b-M further significantly promoted the accumulation of RPS1 and LHCB3 compared to the control group (U5+SmD3a). PEPC was used as an internal control protein.

[0050] Figure 4This study analyzed the maximum photochemical efficiency (Fv / Fm) of PSII in *Nicotiana benthamiana*. The Fv / Fm values ​​of co-expressed U5-M1 / U5-M2 and different proteins (SmD3a, SmD3b-M), as well as the control groups (YFP, SmD3a), were measured in *Nicotiana benthamiana* leaves. The percentages above the bars indicate the improvement of each experimental group compared to the control group (NC+YFP). Each scatter point represents an independent biological replicate; error bars represent mean ± standard deviation (Mean ± SD); an asterisk (*) indicates a significant difference between groups after a two-tailed t-test (P < 0.05), an asterisk (**) indicates an extremely significant difference between groups after a two-tailed t-test (P < 0.01), and an asterisk (***) indicates an extremely significant difference between groups after a two-tailed t-test (P < 0.001). The statistical analysis results in the first row from the top of the figure are relative to the NC+YFP group, and the statistical analysis results in the second row from the top are relative to the NC+SmD3a group. Detailed Implementation

[0051] This invention provides a novel approach to improving crop photosynthetic efficiency using a plant small nucleus RNA-protein complex module. Through the synergistic effect of an artificially modified SmD3 variant and sequence-optimized snRNA (using U5 snRNA as an example), multi-gene regulation is achieved at the translational level. Specific implementation steps and methods are as follows: (1) Design of SmD3 translation enhancement variants. Based on the artificial intelligence model, SmD3 protein in the target crop was rationally designed. By replacing amino acids at the C-terminus of the protein to increase the content of arginine-glycine and disrupt the interaction of the stable helical structure, the modified protein (SmD3b-M) was made to obtain cytoplasmic localization characteristics similar to Arabidopsis SmD3a.

[0052] (2) Based on the sequences in the 3'UTR of key photosynthetic genes (such as RPS1, LHCB3, etc.) mRNA in the target crop that specifically bind to U5 snRNA, the U5 snRNA sequence is optimized. By achieving "complete complementarity" of the base sequence, the limitation that the base pairing between endogenous U5 snRNA and target gene is not optimal is overcome.

[0053] (3) Codon optimization was performed on the coding gene sequence of the SmD3b-M protein to ensure its expression stability in the crop system. The optimized SmD3b-M coding gene sequence and the U5 snRNA variant coding sequence were co-constructed into a plant expression vector.

[0054] (4) Plant transformation of the U5 translation regulation system. The target crop was transformed using Agrobacterium-mediated transformation, and the protein content levels of the transformed plants were measured (Western Blot) to confirm whether the expression level of the U5 target protein was increased. Finally, superior lines with significantly improved photosynthetic efficiency were screened by measuring photosynthetic rate.

[0055] Small nuclear RNA (snRNA) is a class of RNA molecules, 100-300 bases in length, that participate in RNA processing within the cell nucleus. Sm proteins are the core structural proteins that specifically bind to and assemble into the functional ribonucleoprotein complex (snRNP). RPS1 and LHCB3 are key factors driving chloroplast protein translation and optimizing light capture, respectively; increasing their protein content can enhance plant photosynthetic efficiency.

[0056] The working principle of this invention is as follows: by constructing an artificial snRNP protein complex module with enhanced stability and activity, the protein synthesis efficiency of target gene mRNA is precisely optimized, thereby increasing the accumulation of key photosynthetic proteins in crops.

[0057] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Example 1: Construction of modified U5 snRNA and SmD3 expression vector 1. Design of U5 snRNA variants Using the Arabidopsis U5-4 gene as a backbone, the Sm binding site was sequenced using overlap extension PCR technology to enable it to specifically and complementaryly recognize Nicotiana benthamiana. NbeRPS1 and NbeLHCB3 UBE element in the 3' UTR region of the gene. Figure 1 This is a schematic diagram of the U5 snRNA variant design of the present invention. The diagram shows the base pairing design of the U5 snRNA variants (M1 and M2) with tobacco NbeRPS1 and NbeLHCB3. The two U5 snRNA variants obtained are named U5-M1 and U5-M2, respectively. The nucleotide sequence of U5-M1 is shown in SEQ ID NO:1; the nucleotide sequence of U5-M2 is shown in SEQ ID NO:2.

[0060] 2. Design of SmD3 variants Based on an artificial intelligence model, the SmD3 protein in the target crop (Nicotiana benthamiana) was rationally designed. By replacing amino acids at the C-terminus of the protein to increase the arginine-glycine content and disrupt the interaction of the stable helical structure, a modified protein was obtained and named SmD3b-M. The amino acid sequence of the SmD3b-M protein is shown in SEQ ID NO:3. Figure 2 The confocal microscopy images shown reveal the subcellular localization characteristics of the fusion proteins SmD3a-YFP, SmD3b-YFP, and the variant SmD3b-M-YFP in leaves of *Nicotiana benthamiana*. These results indicate that the modified protein SmD3b-M (SEQ ID NO:3) of this invention possesses cytoplasmic localization characteristics similar to those of the *Arabidopsis thaliana* SmD3a protein (SEQ ID NO:9).

[0061] 3. Construction of modified U5 snRNA and SmD3 expression vectors Based on tobacco codon bias, the SmD3a and SmD3b-M gene sequences were optimized and artificially synthesized. Then, the pre- and post-modification U5 snRNA gene sequences, as well as the optimized SmD3a and SmD3b-M genes fused with YFP tags, were cloned into the plant binary vector pCAMBIA1300. BamH I or Kpn Site I. After successful sequencing and identification, the following five recombinant expression vectors were obtained: Recombinant expression vector pCAMBIA1300-SmD3b-M-YFP: In the pCAMBIA1300 vector BamH The recombinant plasmid obtained by inserting the expression cassette shown in SEQ ID NO:5 at site I (positions 1-346 are the 35S promoter, positions 374-766 are the optimized SmD3b-M gene, and positions 803-1519 are the YFP coding sequence) is obtained by inserting the expression cassette shown in SEQ ID NO:5 at site I.

[0062] Recombinant expression vector pCAMBIA1300-SmD3a-YFP: In the pCAMBIA1300 vector BamH The recombinant plasmid obtained by inserting the expression cassette shown in SEQ ID NO:6 at site I (positions 1-346 are the 35S promoter, positions 374-757 are the optimized SmD3a gene, and positions 794-1510 are the YFP coding sequence) is obtained by inserting the expression cassette shown in SEQ ID NO:6 at site I.

[0063] Recombinant expression vector pCAMBIA1300-U5: In the pCAMBIA1300 vector KpnThe recombinant plasmid obtained by inserting the expression cassette shown in SEQ ID NO:7 (positions 1-636 are the UBQ promoter and positions 660-786 are the U5 coding sequence) at site I.

[0064] Recombinant expression vector pCAMBIA1300-U5-M1: In the pCAMBIA1300 vector Kpn The recombinant plasmid obtained by inserting the expression cassette shown in SEQ ID NO:4 (positions 1-636 are the UBQ promoter and positions 660-786 are the U5-M1 gene sequence) at position I.

[0065] Recombinant expression vector pCAMBIA1300-U5-M2: In the pCAMBIA1300 vector Kpn The recombinant plasmid obtained by inserting the expression cassette shown in SEQ ID NO:8 (positions 1-636 are the UBQ promoter and positions 660-786 are the U5-M2 gene sequence) at position I.

[0066] Example 2: Obtaining and Molecular Identification of Transgenic Tobacco Plants Transgenic lines of *Nicotiana benthamiana* containing the recombinant expression vectors described in Example 1 were obtained using Agrobacterium-mediated genetic transformation. These included: (1) Single U5 control group: The recombinant expression vector pCAMBIA1300-U5 obtained in Example 1 was introduced into Tobacco Benzoinus.

[0067] (2) Single transgenic U5-M1 experimental group: The recombinant expression vector pCAMBIA1300-U5-M1 obtained in Example 1 was introduced into Tobacco Benedictine.

[0068] (3) Single transgenic U5-M2 experimental group: The recombinant expression vector pCAMBIA1300-U5-M2 obtained in Example 1 was introduced into Tobacco Benedictine.

[0069] (4) Co-expression of U5 and SmD3a (U5+SmD3a) experimental group: The recombinant expression vectors pCAMBIA1300-U5 and pCAMBIA1300-SmD3a-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0070] (5) Co-expression of U5-M1 and SmD3a (U5-M1+SmD3a) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3a-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0071] (6) Co-expression of U5-M1 and SmD3b-M (U5-M1+SmD3b-M) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3b-M-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0072] (7) Co-expression of U5-M2 and SmD3a (U5-M2+SmD3a) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3a-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0073] (8) Co-expression of U5-M2 and SmD3b-M (U5-M2+SmD3b-M) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3b-M-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0074] 1. Real-time quantitative PCR (RT-qPCR) detection Total RNA was extracted from tobacco leaves using TRIzol reagent (Invitrogen). Following the kit instructions, 1 μg of total RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Amplification reactions were performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) and detected on a 7500 Fast Real-Time PCR System (Applied Biosystems). Each sample was tested in triplicate, with the relative expression levels of the target gene adjusted according to 2-1. -ΔΔCt The calculation was performed. Finally, a two-tailed Student's t-test was used to analyze the significant differences in data between groups. Internal reference genes were used. EF1α (Elongation factor 1-alpha) was used as the calibration benchmark. The detection genes and their primer sequences involved in this experiment are shown in Table 1.

[0075] Table 1. Primer sequences for RT-qPCR detection of genes

[0076] 2. Western Blot Detection Western blotting was used to analyze the expression of target proteins NbeRPS1 and NbeLHCB3. The specific steps were as follows: 0.1 g of tobacco leaves were weighed, ground in liquid nitrogen, and then RIPA lysis buffer was added. Lysis was performed on ice for 10 minutes. The mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. An equal volume of protein sample was subjected to 10% SDS-PAGE electrophoresis, followed by semi-dry transfer to a PVDF membrane. The transferred PVDF membrane was blocked in 5% skim milk PBST buffer at room temperature for 1 hour. The corresponding primary antibodies were added, and the membrane was incubated overnight at 4°C. Primary antibodies included: Anti-GFP rabbit polyclonal antibody (Abcam, Cat. #ab290, 1:1000 dilution), Rabbit polyclonal anti-RPS1 (PhytoAB, Cat. #PhytoAB-PHY0424A, 1:1000 dilution), Rabbit polyclonal anti-LHCB3 (PhytoAB, Cat. #PhytoAB-PHY2561A, 1:1000 dilution), and internal control antibody Anti-PEPC (Agrisera, AS09 458, 1:5000 dilution). The membrane was washed three times with PBST for 5 minutes each time. Then, goat anti-rabbit HRP-labeled secondary antibody (Sigma-Aldrich, Cat. #A0545, 1:10000 dilution) was added, and the membrane was incubated at room temperature for 1 hour. After washing, chemiluminescence detection was performed using ECL ultrasensitive chemiluminescence buffer, and imaging was performed using the ChemiDoc system. Finally, the band grayscale values ​​were analyzed using ImageJ software.

[0077] 3. Results and Analysis Molecular experiments showed that the abundance of U5 snRNA was basically consistent across groups. Compared with the control group expressing U5 alone, the abundance of U5 snRNA in the transgenic U5-M1 and U5-M2 experimental groups was significantly higher. NbeRPS1 and NbeLHCB3 The mRNA level did not change significantly, but the corresponding protein level increased significantly. Figure 3 Compared with the U5+SmD3a group, the protein levels of NbeRPS1 and NbeLHCB3 in the U5-M1+SmD3a, U5-M1+SmD3b-M, U5-M2+SmD3a, and U5-M2+SmD3b-M experimental groups were significantly increased. Figure 3 (C). This result verifies that the system of the present invention can significantly increase the expression of target photosynthetic proteins (NbeRPS1 and NbeLHCB3) at the translational level while maintaining stability at the mRNA level.

[0078] Example 3: Detection of the photosynthetic efficiency enhancement effect of various transgenic plants The recombinant expression vectors involved in the embodiments of this invention are as follows: the recombinant expression vectors pCAMBIA1300-U5-M1, pCAMBIA1300-U5-M2, pCAMBIA1300-SmD3a-YFP, and pCAMBIA1300-SmD3b-M-YFP obtained in Example 1, and the recombinant expression vectors pCAMBIA1300-UBQ and pCAMBIA1300-YFP as controls. The recombinant expression vector pCAMBIA1300-UBQ is a vector derived from pCAMBIA1300. Kpn The recombinant plasmid obtained by inserting the sequence “SEQ ID NO:4, positions 1-636, are the UBQ promoter sequence” at site I. The recombinant expression vector pCAMBIA1300-YFP is a plasmid obtained by inserting the sequence “SEQ ID NO:4, positions 1-636, are the UBQ promoter sequence” into the pCAMBIA1300 vector. BamH The recombinant plasmid obtained by inserting "the 35S promoter shown in positions 1-346 of SEQ ID NO:6 + the YFP coding sequence shown in positions 794-1510 of SEQ ID NO:6" at site I.

[0079] The aforementioned recombinant expression vectors were introduced into *Nicotiana benthamiana* using Agrobacterium-mediated genetic transformation. These included: (1) Control group (NC+YFP): Recombinant expression vectors pCAMBIA1300-UBQ and pCAMBIA1300-YFP were introduced into Nicotiana benthamiana.

[0080] (2) Transgenic SmD3a (NC+SmD3a): Recombinant expression vector pCAMBIA1300-UBQ and recombinant expression vector pCAMBIA1300-SmD3a-YFP obtained in Example 1 were introduced into Nicotiana benthamiana.

[0081] (3) Co-expression of U5-M1 and SmD3a (U5-M1+SmD3a) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3a-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0082] (4) Co-expression of U5-M1 and SmD3b-M (U5-M1+SmD3b-M) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M1 and pCAMBIA1300-SmD3b-M-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0083] (5) Co-expression of U5-M2 and SmD3a (U5-M2+SmD3a) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3a-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0084] (6) Co-expression of U5-M2 and SmD3b-M (U5-M2+SmD3b-M) experimental group: The recombinant expression vectors pCAMBIA1300-U5-M2 and pCAMBIA1300-SmD3b-M-YFP obtained in Example 1 were transferred into Tobacco Benzoinus.

[0085] The maximum photochemical efficiency (Fv / Fm) of photosystem II in leaves of each plant line was measured using a Closed FluorCam FC 800 chlorophyll fluorometer (Photon Systems Instruments, Czech Republic). Before measurement, the plants were placed in complete darkness for 20-30 minutes to ensure the PSII reaction centers in the leaves were fully open. After dark adaptation, the leaves were laid flat and fixed on the measuring platform. A weak light was first applied to record the initial fluorescence value (F0) under dark adaptation, followed by a saturated pulse of light to record the maximum fluorescence value (Fm) under dark adaptation. After measurement, the target parameter was calculated using the formula Fv / Fm = (Fm - F0) / Fm. At least five independent representative plants from each treatment group or line were selected for repeated measurements. A two-tailed Student's t-test was used to analyze the significance of differences between groups.

[0086] Chlorophyll fluorescence kinetics analysis showed that, compared with the transgenic plant NC+YFP, the maximum photochemical efficiency (Fv / Fm) of PSII in all other transgenic plants was significantly improved; compared with the transgenic plant NC+SmD3a, the maximum photochemical efficiency (Fv / Fm) of PSII in transgenic plants U5-M1+SmD3a, U5-M2+SmD3a, U5-M1+SmD3b-M and U5-M2+SmD3b-M were all significantly improved. The maximum photochemical efficiency (Fv / Fm) of PSII in transgenic plants U5-M1+SmD3a and U5-M1+SmD3b-M was increased by 27.3% and 31.1% respectively compared with the control group (NC+YFP); the maximum photochemical efficiency (Fv / Fm) of PSII in transgenic plants U5-M2+SmD3a and U5-M2+SmD3b-M was increased by 29.6% and 33.3% respectively compared with the control group (NC+YFP). Figure 4 This indicates a substantial enhancement in photosynthetic performance, suggesting a significant potential for yield and biomass growth.

[0087] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A small nuclear RNA, characterized in that: The nucleotide sequence of the small nuclear RNA is SEQ ID NO:1 or SEQ ID NO:

2.

2. A DNA molecule, characterized in that: The DNA molecule is a DNA molecule capable of being transcribed to yield the small nuclear RNA of claim 1.

3. An expression cassette, recombinant vector, or recombinant microorganism containing the DNA molecule of claim 2.

4. A small nuclear RNA-protein complex, characterized in that: The small nuclear RNA-protein complex comprises the small nuclear RNA of claim 1 and the Sm protein that specifically binds to the small nuclear RNA.

5. The small nuclear RNA-protein complex according to claim 4, characterized in that: The Sm protein is a variant of the SmD3a or SmD3b protein. The amino acid sequence of the SmD3b protein variant is shown in SEQ ID NO:3; The amino acid sequence of the SmD3a protein is shown in SEQ ID NO:

9.

6. A biomaterial relating to the small nuclear RNA-protein complex of claim 4 or 5, characterized in that: The biomaterial is any one of the following: (A1) DNA molecule assembly; the DNA molecule assembly includes DNA molecule 1 and DNA molecule 2; DNA molecule 1 is a DNA molecule capable of being transcribed to produce the small nuclear RNA; DNA molecule 2 is a DNA molecule capable of expressing the Sm protein; (A2) Expression cassette assembly; the expression cassette assembly includes expression cassette 1 and expression cassette 2; expression cassette 1 contains DNA molecule 1 as described in (A1); expression cassette 2 contains DNA molecule 2 as described in (A1); (A3) Recombinant vector combination; the recombinant vector combination includes recombinant vector 1 and recombinant vector 2; the recombinant vector 1 carries the expression cassette 1 in (A2); the recombinant vector 2 carries the expression cassette 2 in (A2); (A4) A recombinant microbial combination; the recombinant microbial combination includes recombinant microorganism 1 and recombinant microorganism 2; the recombinant microorganism 1 contains the recombinant vector 1 in (A3); the recombinant microorganism 2 contains the recombinant vector 2 in (A3); (A5) Recombinant vector; the recombinant vector simultaneously carries the expression cassette 1 and the expression cassette 2 from (A2); (A6) Recombinant microorganism; wherein the recombinant microorganism contains both the recombinant vector 1 and the recombinant vector 2 in (A3); or, wherein the recombinant microorganism contains the recombinant vector in (A5); (A7) Genetically modified tobacco; the genetically modified tobacco is tobacco capable of expressing the small nuclear RNA-protein complex.

7. The biomaterial according to claim 6, characterized in that: The transgenic tobacco is a transgenic tobacco obtained by introducing the DNA molecule combination described in (A1) of claim 6, the expression cassette combination described in (A2), the recombinant vector combination described in (A3), or the recombinant vector described in (A5) into the recipient tobacco; the photosynthetic activity of the transgenic tobacco is stronger than that of the recipient tobacco.

8. The use of the small nuclear RNA of claim 1, the DNA molecule of claim 2, the expression cassette or recombinant vector or recombinant microorganism of claim 3, or the small nuclear RNA-protein complex of claim 4 or 5 in enhancing tobacco photosynthesis.

9. A method for enhancing tobacco photosynthesis, comprising the steps of: expressing the small nuclear RNA of claim 1 and the Sm protein of claim 4 or 5 in tobacco cells, thereby enhancing tobacco photosynthesis.

10. The application of the small nuclear RNA of claim 1, or the DNA molecule of claim 2, or the expression cassette, recombinant vector, or recombinant microorganism of claim 3, or the small nuclear RNA-protein complex of claim 4 or 5, or the method of claim 9 in tobacco breeding; The tobacco breeding mentioned refers to the development of tobacco varieties with enhanced photosynthesis.

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