SARS-CoV-2-based 5′-UTR sequence derivatives and their applications in enhancing plant gene translation expression

Abstract

The present invention discloses a 5′-UTR sequence derivative based on SARS-CoV-2 and its application in enhancing the translation expression of plant genes, and relates to the technical field of enhanced translation expression of plant genes. The 5′-UTR sequence derivative provided by the present invention is used to enhance the translation expression of target genes in plants; the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2. The present invention applies the 5′-UTR of the SARS-CoV-2 virus to the plant system for the first time, and significantly improves the level of target gene expression protein at the translation level through the translation regulation mediated by the optimized 5′-UTR sequence derivative. The present invention is suitable for a variety of plant varieties such as grasses, crucifers, and solanaceae.

Description

Technical Field

[0001] The present invention relates to the technical field of enhancing the translation expression of plant genes, and in particular to 5′-UTR sequence derivatives based on SARS-CoV-2 and applications thereof in enhancing the translation expression of plant genes. Background Art

[0002] The SARS-CoV-2 virus recognizes angiotensin-converting enzyme 2 (ACE2) receptors on the cell membrane through its spike protein. Under receptor-mediated endocytosis, it enters the cell and replicates new viral particles. Once released, these viral particles can then infect other host cells. To complete replication, SARS-CoV-2 must fully utilize the host cell's protein expression system to translate its own viral proteins, and translation initiation is a critical step. Almost all mRNAs in eukaryotic cells have a 5′-end cap structure, requiring this structure to recruit ribosomes, a process known as cap-dependent translation initiation. However, a small number of mRNAs, in response to stress, can bypass this cap structure and instead utilize internal ribosome entry sites (IRESs) in the 5′-untranslated region (5′-UTR) to directly recruit ribosomes, initiating translation through a cap-independent mechanism.

[0003] To date, the 5′-UTR region of the SARS-CoV-2 virus has been found to have multiple functions, including viral replication and translational regulation. In plants, the translational capacity of mRNA is a key factor in regulating gene expression. Improving the translational capacity of plant mRNA can enhance the expression of target genes and has broad application and potential in plants. Summary of the Invention

[0004] The present invention provides SARS-CoV-2-based 5′-UTR sequence derivatives and their application in enhancing plant gene translation and expression. By screening and optimizing the 5′-UTR sequences of several endogenous transcripts of SARS-CoV-2, the resulting 5′-UTR sequence derivatives are used to construct recombinant vectors and then introduced into plants, significantly improving plant mRNA translation and gene expression. This is achieved specifically through the following technologies.

[0005] In the first aspect of the present invention, a method for enhancing the translation expression of a target gene in a plant is provided. The nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0006] Furthermore, the plant is a grass plant, a cruciferous plant or a solanaceous plant. It should be noted that the plants applicable to the technical solution of the present invention are not limited to these types of plants.

[0007] Furthermore, the plants are rice, corn, Arabidopsis, tobacco, etc. It should be noted that the plants applicable to the technical solution of the present invention are not limited to these species.

[0008] The second aspect of the present invention provides a method for enhancing the translational expression of a target gene in a plant, comprising constructing a recombinant expression vector for expression in a plant using the nucleotide sequence of the 5′-UTR sequence derivative and the target gene sequence, and transferring the recombinant expression vector into the plant.

[0009] The nucleotide sequence of the 5′-UTR sequence derivative is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0010] The third aspect of the present invention provides a recombinant expression vector for enhancing the translation expression of a target gene in a plant, characterized in that the recombinant expression vector contains a target gene fragment and a 5′-UTR sequence derivative.

[0011] In a fourth aspect of the present invention, a nucleic acid expression frame is provided for enhancing the translation expression of a target gene in a plant, wherein the nucleic acid expression frame contains a 5′-UTR sequence derivative or the recombinant expression vector according to claim 5, and the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] In a fifth aspect, the present invention provides an Agrobacterium cell for transfecting a plant, wherein the Agrobacterium cell contains the nucleic acid expression cassette according to claim 6.

[0013] In a sixth aspect, the present invention provides a 5′-UTR sequence derivative for enhancing a target gene in a plant. The nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] Compared with the prior art, the present invention is beneficial in that:

[0015] 1. This invention applies the 5′-UTR of the SARS-CoV-2 virus to the plant system for the first time. Through the translational regulation mediated by the optimized 5′-UTR sequence derivative, the level of target gene expression protein is significantly improved at the translation level.

[0016] 2. The optimized 5′-UTR sequence derivatives are suitable for a variety of plant species, including grasses, crucifers, and solanaceae. Commonly applicable plant varieties include rice, corn, Arabidopsis, tobacco, etc., and have very high versatility; the optimized 5′-UTR sequence derivatives will not have a negative impact on the normal physiological activities of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the SARS-CoV-2 genomic RNA of the SARS-CoV-2 virus and the mRNA structure of each subgenomic RNA. Figure 1 A is a schematic diagram of the SARS-CoV-2 genomic RNA. The full-length genomic RNA consists of 29,983 nucleotides. Reading frames ORF1a and ORF1ab encode 16 nonstructural proteins. In addition to the genomic RNA, subgenomic RNA is translated to produce structural proteins (S-spike, E-envelope, M-membrane, and N-nucleocapsid proteins) and accessory proteins (3a, 6, 7a, 7b, and 8). Figure 1 In Figure A, the black rectangle represents the constant region of the structural 5′-UTR. TRS, or transcriptional regulatory sequence, includes TRS-L, TRS-UTR, TRS-B, and TRS-body. The TRS contains a conserved 6-7 nucleotide core sequence (CS) surrounded by discontinuous sequences. Figure 1 B is a schematic diagram of the mRNA structure of each subgenomic RNA, and the transcript abundance of each subgenomic RNA is shown on the left.

[0018] Figure 2 Results of the single-luciferase FLUC assay in tobacco leaves using 5′-UTR sequences from nine different transcripts. FLUC fold changes were normalized to the Ω sequence. NC is the negative control. n = 9, means ± SEM. ** indicates a significant difference of P < 0.01, **** indicates a significant difference of P < 0.0001.

[0019] Figure 3 The results of a single luciferase assay of the 5′-UTR sequence derivative ORF7aΔT are shown. Figure 3 Fold changes in FLUC were normalized to the Ω series. NC is the negative control. n = 9, means ± SEM. ** indicates a significant difference with P < 0.01.

[0020] Figure 4 Schematic diagram of the dual-luciferase reporter system structure and dual-luciferase assay results for the 5′-UTR derivative 6MΔT. Comparative enzyme activity of the 6MΔT cis-acting element relative to the Ω cis-acting element was measured using transient transformation of tobacco leaves. n = 4, means ± SEM.

[0021] Figure 5 Results show the ability of TBF1-5′-UTR, Ω, and 6MΔT to regulate the translation of FLUC in Arabidopsis protoplasts. Arabidopsis protoplasts were transiently transformed with a dual-luciferase vector to measure the translational capacity of 6MΔT relative to Ω and the 5′-UTR of the endogenous Arabidopsis gene TBF1. n = 5, means ± SEM.

[0022] Figure 6 and Figure 7 Results of the assay using rice and maize protoplasts to examine the translational regulation of FLUC by TBF1-5′-UTR, Ω, and 6MΔT. n = 6, means ± SEM. DETAILED DESCRIPTION

[0023] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Enhanced translational expression of plant genes based on SARS-CoV-2 5′-UTR sequence derivatives

[0025] 1. Sequence acquisition

[0026] Refer to "The Architecture of SARS-CoV-2 Transcriptome" (Kim et al., Cell, 2020) to obtain the genome, transcripts, and 5′-UTR sequences of the SARS-CoV-2 virus; Figure 1 Figure A. The SARS-CoV-2 virus has a single-stranded, positive-sense RNA genome of 29,983 nucleotides and exhibits considerable genetic variation between isolates. The reading frames ORF1a and ORF1ab encode 16 nonstructural proteins. In addition to the genomic RNA, subgenomic RNA is translated to produce structural proteins (S, spike; E, envelope; M, membrane; N, nucleocapsid protein) and accessory proteins (3a; 6; 7a; 7b; 8). The genome is encapsidated by the viral nucleocapsid protein (N) to form a large ribonucleoprotein (RNP) complex, which is surrounded by an envelope system containing lipids and the viral proteins S (spike), M, and E.

[0027] After the SARS-CoV-2 virus genome enters the cell, it acts directly as messenger RNA (mRNA). The SARS-CoV-2 virus uses its own genomic RNA (gRNA) as a template for replication and transcription. The SARS-CoV-2 virus mainly encodes four structural proteins (S, E, M, and N) and five other auxiliary proteins (3a, 6, 7a, 7b, and 8). Different transcripts have various sequence forms in the host, and the 5′-UTR sequence and length of different transcripts are variable, such as Figure 1 B shows the highest copy numbers of different transcripts and their corresponding 5′-UTR structures.

[0028] 2. Plant expression vector construction and transient transformation

[0029] In this example, the gene encoding firefly luciferase was selected as the target gene. The enzymatic activity of firefly luciferase was detected to evaluate the regulatory effects of different 5′-UTR sequences and 5′-UTR sequence derivatives on the expression level of the target gene.

[0030] This example evaluates the effect of different 5′-UTR sequences of endogenous transcripts on the expression of the luciferase FLUC reporter gene in plants. Figure 1 Based on the analysis results, we selected 9 5′-UTR sequences of endogenous transcripts with different translation potentials (as shown in Table 1) and cloned them into plant expression vectors. The following is a detailed description of the experimental design and steps:

[0031] 1. Vector Construction

[0032] Select a plant expression vector (basic vector) that contains a strong promoter (such as the CaMV 35S promoter) and a multiple cloning site (MCS) for inserting different 5′-UTR sequences.

[0033] The 5′-UTR sequences of the nine endogenous transcripts were cloned downstream of the 35S promoter of the basic vector using restriction endonucleases, ensuring that the 5′-UTR sequences were located upstream of the FLUC reporter gene to obtain a recombinant expression vector.

[0034] 2. Sequence Verification

[0035] The correctness of the 5′-UTR sequence and the accuracy of the reading frame of each clone were verified by DNA sequencing.

[0036] 3. Agrobacterium-mediated infection of plant leaves:

[0037] The constructed recombinant expression vector was transformed into GV3101 Agrobacterium tumefaciens strains. These Agrobacterium strains were used to infect tobacco leaves to achieve transient expression of the FLUC reporter gene.

[0038] Table 1 5′-UTR sequences corresponding to different transcripts

[0039]

[0040] Given that the promoter sequences used by different expression vectors are consistent, and their differences are primarily reflected in the nucleotide sequence of the 5′-UTR, it can be inferred that the 5′-UTR sequence has no role in gene transcription and its primary function is to regulate the translation of mRNA. This conclusion is based on the molecular mechanisms of transcription and translation, namely that the promoter is responsible for recruiting RNA polymerase to initiate transcription, while the 5′-UTR regulates the translation process by affecting the recognition and binding of ribosomes.

[0041] The constructed recombinant expression vector is transferred into GV3101 Agrobacterium tumefaciens, and the Agrobacterium suspension containing the recombinant plasmid is then injected into tobacco leaves using a needle-free syringe. This transient expression system allows for rapid assessment of luciferase reporter gene activity in plant cells. Alternatively, the recombinant expression vector can be directly transformed into plant protoplasts. Protoplasts are plant cells with their cell walls removed that can take up and express foreign DNA. After transformation, the protoplasts are lysed and the activity of FLuc and RLuc is measured using a luciferase assay kit.

[0042] 3. Translation ability test

[0043] In this example, we used the plant universal translation-enhancing cis-acting element Omega (Ω) as a positive control to evaluate its ability to enhance translation efficiency in plant systems. We also used a native expression vector containing the gene ccdB as a negative control (NC) to determine basal translation levels. By infecting tobacco leaves with Agrobacterium GV3101 containing each of these vectors, we were able to compare the effects of different 5′-UTRs on translational capacity in a controlled experimental system.

[0044] On day 3 after infection, we measured the fluorescence intensity of FLUC using a luciferase assay system as an indicator of translation activity. Figure 2 As shown, the 5′-UTR of the endogenous transcript M exhibited the highest FLUC enzyme activity, indicating that it has stronger translation capacity than the 5′-UTRs of the Ω element and other tested transcripts.

[0045] This result not only confirms the reliability of the Ω element as a positive control but also highlights the potential of the 5′-UTR of the endogenous transcript M in translational regulation. Our findings provide important information for further exploring the function of 5′-UTRs in plants and their role in translational regulation, and offer new strategies for optimizing gene expression in plant systems. These results also offer new insights into the complexity of translational regulation in plants in response to diverse environmental cues.

[0046] 4. Translational ability testing of 5′-UTR sequence derivatives

[0047] The luciferase assay was mediated by the 5′-UTR of the endogenous transcript ORF7a and its derivative ORF7aΔT (shown in SEQ ID NO. 1). Figure 3 As shown, the translation ability of ORF7a was significantly enhanced after deleting two T bases from its 5′-UTR.

[0048] based on Figure 2 and Figure 3 The results showed that both the 5′-UTR sequence of transcript M and its 5′-UTR derivative, ORF7aΔT, exhibited strong translational enhancement. Therefore, the present invention integrated the sequence characteristics of both, deleting two T bases from the 5′-UTR sequence of transcript M and naming it 6MΔT. The sequence information is shown in Table 2.

[0049] Table 2 5′-UTR sequence derivatives

[0050]

[0051]

[0052] In order to detect the translation ability of the optimized 5′-UTR sequence derivative 6MΔT, the present invention constructed a dual luciferase reporter system containing FLUC and RLUC (Renilla luciferase), such as Figure 4 As shown in A. Agrobacterium containing Ω and 5′-UTR sequence derivative 6MΔT sequence was used to infect tobacco leaves, and leaf proteins were extracted after infection; enzyme activities of FLUC and RLUC were detected using a microplate reader. Figure 4 As shown in Figure 2, when the enzyme activity of RLUC was used as an internal reference, the enzyme activity intensity of FLUC produced by the 5′-UTR sequence derivative 6MΔT was 2.6 times that of Ω, indicating that the 5′-UTR sequence derivative 6MΔT had stronger translation ability.

[0053] Example 2: Verification of the 5′-UTR sequence derivative 6MΔT in different plant species to enhance translation of plant genes

[0054] In order to test the universality of the translation enhancement effect of the 5′-UTR sequence derivative 6MΔT, the present invention selected several plants (Arabidopsis thaliana, rice and corn) in addition to tobacco for verification experiments.

[0055] like Figure 5 As shown in Figure 2, the dual-luciferase recombinant expression vectors carrying TBF1-5′-UTR (endogenous disease resistance gene of Arabidopsis thaliana), Ω or 5′-UTR sequence derivative 6MΔT were transformed into Arabidopsis thaliana protoplasts. Figure 5 As shown, the translational abilities of TBF1-5′-UTR, Ω, and 6MΔT in regulating FLUC increased in sequence.

[0056] In this example, the dual luciferase recombinant expression vectors of TBF1-5′-UTR, Ω or 6MΔT were transformed into rice protoplasts and corn protoplasts respectively. Figure 6 and Figure 7 As shown, in rice and maize protoplasts, the ability of TBF1-5′-UTR, Ω, and 6MΔT to regulate FLUC translation increased in that order. This result is consistent with the previous conclusions, indicating that the 5′-UTR sequence derivative 6MΔT has universal application in enhancing the translation of plant genes and can be used to enhance the translational expression of target genes in a variety of plant species.

[0057] The 5′-UTR of the SARS-CoV-2 virus may have evolved to enable high expression of viral proteins, enabling efficient replication in host organisms. The experimental results of the present invention demonstrate that the SARS-CoV-2 5′-UTR sequence derivative 6MΔT can enhance the translational expression of plant genes, and its enhancing effect is significantly stronger than that of the translation enhancing cis-acting element Ω. This sequence can serve as a functional 5′-UTR cis-acting element to increase the level of gene expression proteins in plants.

[0058] This study reveals the critical role of the 5′-UTR of the SARS-CoV-2 virus in regulating gene translation in plants and its potential applications in bioproduction. The knowledge gained from this study not only contributes to our understanding of SARS-CoV-2 but also offers practical implications for developing more effective strategies to address the ongoing threat of the virus. Further exploration of the 5′-UTR and its applications in biotechnology holds great potential for protein regulation, production, and synthetic biology.

[0059] This invention provides a novel strategy to enhance gene translation in plants. By utilizing a SARS-CoV-2 5′-UTR-derived sequence (6MΔT), it not only expands new avenues for genetic research but also provides a new and powerful tool for studying gene expression in plants. Through implementation of this invention, the translation-enhancing cis-acting element 6MΔT can achieve more stable and efficient translational expression of target genes and is expected to play an important role in fields such as genetic breeding and synthetic biology.

[0060] In summary, after the above experiments and analysis, the methods provided by the present invention are applicable to plant species including but not limited to grasses, crucifers, or solanaceae. Almost all plant species can be used to enhance the translational expression of target genes in plants using the methods of the present invention.

[0061] Specifically, applicable plants also include but are not limited to rice, corn, Arabidopsis, tobacco and the like.

[0062] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. An application of a 5′-UTR sequence derivative based on SARS-CoV-2, characterized in that: The 5′-UTR sequence derivative is used to enhance the translation expression of a target gene in a plant; the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2; the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.1 is a Solanaceae plant, and the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.2 is a Poaceae plant, a Cruciferae plant, or a Solanaceae plant.

2. The use of the 5′-UTR sequence derivative based on SARS-CoV-2 according to claim 1, characterized in that The grass plant is rice or corn, the cruciferous plant is Arabidopsis thaliana, and the solanaceous plant is tobacco; the 5′-UTR sequence derivative of SEQ ID NO.1 corresponds to tobacco, and SEQ ID NO.2 corresponds to rice, corn, Arabidopsis thaliana or tobacco.

3. A method for enhancing the translational expression of a target gene in a plant, characterized in that: A recombinant expression vector for plant expression is constructed using the nucleotide sequence of the 5′-UTR sequence derivative and the target gene sequence, and the recombinant expression vector is transferred into the plant; the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2; the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.1 is a Solanaceae plant, and the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.2 is a Poaceae plant, a Cruciferae plant, or a Solanaceae plant; the 5′-UTR sequence derivative is located downstream of the 35S promoter in the recombinant expression vector and upstream of the target gene.

4. A recombinant expression vector for enhancing the translational expression of a target gene in a plant, characterized in that: The recombinant expression vector contains a target gene fragment and a 5′-UTR sequence derivative; the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2; the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.1 is a Solanaceae plant, and the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.2 is a Poaceae plant, a Cruciferae plant or a Solanaceae plant; the 5′-UTR sequence derivative is located downstream of the 35S promoter in the recombinant expression vector and upstream of the target gene.

5. A nucleic acid expression cassette for enhancing the translational expression of a target gene in a plant, characterized in that: The nucleic acid expression frame contains a 5′-UTR sequence derivative or the recombinant expression vector according to claim 4, and the nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2; the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.1 is a Solanaceae plant, and the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.2 is a Poaceae plant, a Cruciferae plant or a Solanaceae plant; the 5′-UTR sequence derivative is located downstream of the 35S promoter in the nucleic acid expression frame and upstream of the target gene.

6. An Agrobacterium cell for transfecting a plant, characterized in that The Agrobacterium cells contain the nucleic acid expression cassette according to claim 5.

7. A 5′-UTR sequence derivative for enhancing the expression of a target gene in a plant, characterized in that: The nucleotide sequence of the 5′-UTR sequence derivative is shown in SEQ ID NO.1 or SEQ ID NO.2; the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.1 is a Solanaceae plant, and the plant corresponding to the 5′-UTR sequence derivative shown in SEQ ID NO.2 is a Poaceae plant, a Cruciferae plant or a Solanaceae plant.

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