Grain specific promoter in early stage of rice filling and application of grain specific promoter

Abstract

The invention discloses a grain specific promoter in the early stage of rice filling and application thereof, the promoter is named as proASP1, and the nucleotide sequence of the promoter is shown as SEQ ID NO: 1. The promoter has highly strict spatio-temporal expression specificity, the activation expression of the promoter is only limited in grains in the early stage of rice filling (4-12 days after flowering), especially the expression of genes is efficiently driven in endosperm and aleurone layers, and the expression is not leaked in other tissues such as roots, stems, leaves, leaf sheaths and glumes and grains in the middle and later stages of filling. The invention provides a basic and precious molecular tool for gene function research, quality improvement (such as protein and starch synthesis regulation) and bioreactor development in a key window period of rice grain development, and has important scientific research and application values.

Description

Technical Field

[0001] This invention belongs to the fields of agricultural genetic engineering and biotechnology, specifically relating to a grain-specific promoter derived from the early grain-filling stage of rice and its application in the construction of transgenic plants and gene function research. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, and its grain-filling process directly determines yield and quality. The pre-grain-filling stage (usually referring to 0–12 days after flowering) is the stage of grain cell division and initial formation of starch bodies, which plays a decisive role in final grain weight and nutrient accumulation. Therefore, precise regulation of the expression of key genes during this stage is of great significance for improving rice yield and quality.

[0003] Promoters are key cis-regulatory elements that regulate the spatiotemporal expression of genes. They are generally located upstream of the gene and can be recognized and bound by RNA polymerases and transcription factors, thereby regulating gene expression. Promoters are generally classified into constitutive promoters, inducible promoters, and tissue-specific promoters. Traditional constitutive promoters, such as 35S, Ubi, and ACTIN, are not limited by plant development or induced by exogenous substances when driving gene expression. This non-specific overexpression of exogenous genes can lead to interference at various growth stages and tissue sites, making it impossible to achieve precise spatiotemporal control of different tissue development.

[0004] Currently, several tissue-specific promoters for rice have been reported, such as the endosperm-specific promoters OsGt1 and OsGluB-1, and the chloroplast-specific promoter OsRbcS. However, most of these promoters are continuously expressed throughout the grain-filling period or in non-grain regions, lacking the ability to precisely regulate the pre-grain-filling stage. There is currently a lack of a promoter element that can efficiently and specifically initiate transcription in rice grains during the pre-grain-filling stage.

[0005] Therefore, developing a promoter that is activated only in grains during the early grain-filling stage and silenced in other tissues or developmental stages is of great application value for achieving precise spatiotemporal gene expression, avoiding off-target effects, and improving transgenic efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a rice-derived grain-specific promoter for the early grain-filling stage and its application. This promoter, named proASP1, has the nucleotide sequence shown in SEQ ID NO:1 (full length 3100 bp). Derived from the 5' upstream regulatory region of Os11g0184800 in the genome of rice varieties (such as Nipponbare), this promoter can efficiently initiate transcription in rice grains during the early grain-filling stage (0–12 days), exhibiting high tissue and developmental stage specificity.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention seeks protection for the use of a rice grain-specific promoter with the nucleotide sequence shown in SEQ ID NO:1 during the early grain-filling stage, in at least one of the following (a1)-(a4):

[0009] (a1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage;

[0010] (a2) Increase rice yield;

[0011] (a3) Improve the nutritional quality of rice grains;

[0012] (a4) Enhance the environmental adaptability of rice.

[0013] Secondly, the present invention seeks protection for the use of biological materials associated with the rice grain-specific promoter with the nucleotide sequence shown in SEQ ID NO:1 during the early grain-filling stage in at least one of the following (a1)-(a4):

[0014] (a1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage;

[0015] (a2) Increase rice yield;

[0016] (a3) Improve the nutritional quality of rice grains;

[0017] (a4) Enhance the environmental adaptability of rice;

[0018] The biomaterials associated with the rice grain-specific promoter during the early grain-filling stage are at least one of the following (b1)-(b9):

[0019] (b1) An expression cassette containing the rice grain-specific promoter for the early grain-filling stage;

[0020] (b2) A recombinant vector containing the rice grain-specific promoter for the early grain-filling stage, or a recombinant vector containing the expression cassette described in (b1);

[0021] (b3) Recombinant microorganisms containing the rice grain-specific promoter in the early grain-filling stage, or recombinant microorganisms containing the expression cassette described in (b1), or recombinant microorganisms containing the recombinant vector described in (b2);

[0022] (b4) A transgenic rice cell line containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice cell line containing the expression cassette described in (b1), or a transgenic rice cell line containing the recombinant vector described in (b2);

[0023] (b5) Transgenic rice tissue containing the grain-specific promoter for the early grain-filling stage of rice, or transgenic rice tissue containing the expression cassette described in (b1), or transgenic rice tissue containing the recombinant vector described in (b2);

[0024] (b6) A transgenic rice organ containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice organ containing the expression cassette described in (b1), or a transgenic rice organ containing the recombinant vector described in (b2);

[0025] (b7) A transgenic rice plant containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice plant containing the expression cassette described in (b1), or a transgenic rice plant containing the recombinant vector described in (b2);

[0026] (b8) Regenerative cells, tissue cultures, or protoplasts derived therefrom of the transgenic rice plant as described in (b7);

[0027] (b9) Propagation material of the transgenic rice plants as described in (b7).

[0028] Furthermore, the aforementioned expression cassette is a grain-specific gene expression cassette for the early grain-filling stage, comprising a rice grain-specific promoter with the nucleotide sequence shown in SEQ ID NO:1, a target gene coding sequence operably linked downstream of the promoter, and a transcription terminator. In practical applications, the target gene can be flexibly replaced according to breeding needs. For example, the target gene can be a reporter gene, a gene related to starch synthesis, a gene related to mineral element enrichment, a gene related to stress resistance, or a gene related to hormone regulation, etc. This allows for precise enhancement of nutrient accumulation or stress resistance in the early grain-filling stage without interfering with the overall metabolism of the plant.

[0029] Furthermore, the scaffold vector of the above-mentioned recombinant vector is the binary vector pBWA(V)HG or its derivative vector.

[0030] Furthermore, the above application is as follows: a plant recombinant expression vector containing the rice grain-specific promoter and the target gene in the early grain-filling stage is introduced into rice to obtain transgenic rice; the specific promoter can drive the target gene operably linked to it to be specifically expressed in the grains of the transgenic rice in the early grain-filling stage.

[0031] Thirdly, the present invention claims protection for a method, which is at least one of the following methods (c1)-(c4):

[0032] (c1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage;

[0033] (c2) Increase rice yield;

[0034] (c3) Improve the nutritional quality of rice grains;

[0035] (c4) Enhance the environmental adaptability of rice;

[0036] A plant recombinant expression vector containing a rice grain-specific promoter with a nucleotide sequence as shown in SEQ ID NO:1 and a target gene is introduced into rice to obtain transgenic rice; the specific promoter can drive the target gene, which is operatively linked to it, to be specifically expressed in the grain-specific early grains of the transgenic rice.

[0037] Furthermore, the method specifically includes the following steps:

[0038] (1) Cloning the rice grain-specific promoter in the early grain-filling stage as shown in SEQ ID NO:1;

[0039] (2) Insert the specific promoter into the plant expression vector to construct a recombinant expression vector containing the proASP1:: target gene;

[0040] (3) Transform Agrobacterium and introduce the recombinant expression vector into rice through rice callus transformation;

[0041] (4) Screening and identification of transgenic rice positive plants;

[0042] (5) Use GUS staining or qRT-PCR to verify the specific expression of the target gene in grains during the early grain-filling stage.

[0043] The rice grain-specific promoter screened in this invention, named proASP1, has a nucleotide sequence shown in SEQ ID NO:1, with a full length of 3100 bp. This promoter originates from the 5' upstream regulatory region of Os11g0184800 in the genome of rice varieties (such as Nipponbare). Based on this promoter sequence, a plant recombinant expression vector pBWA(V)HG-proASP1::GUS was constructed, and this vector was transformed into rice using Agrobacterium-mediated transformation to obtain stably inherited transgenic lines. GUS histochemical staining and quantitative real-time PCR (qRT-PCR) results showed that proASP1 drives efficient expression of the reporter gene only in rice grains during the early grain-filling stage (0–12 days after flowering), and has almost no activity in roots, stems, leaves, flowers, mid-to-late grain-filling stages (12–24 days), and mature grains, exhibiting excellent spatiotemporal specificity.

[0044] In the above scheme, the proASP1 promoter can be cloned by using rice genomic DNA as a template and designing specific primers at both ends of SEQ ID NO:1 for PCR amplification.

[0045] In the above scheme, the expression of the target gene is strictly limited to the grain tissue in the early grain-filling stage of rice (0–12DAF) to avoid "leakage expression" in non-target tissues, thereby reducing energy waste and potential toxicity, and improving transgenic efficiency and biosafety.

[0046] The early grain-filling stage in rice is a critical window for determining final grain weight and quality. Precise regulation of gene expression during this stage is crucial for increasing yield, improving nutritional quality (such as the enrichment of iron, zinc, and vitamin precursors), and enhancing environmental adaptability. The proASP1 promoter provided in this invention can achieve this goal, offering a powerful regulatory element for molecular design breeding.

[0047] The beneficial effects of this invention are:

[0048] (1) The proASP1 promoter has high tissue specificity (only in seeds) and developmental stage specificity (only 0-12 days after flowering). This characteristic has been confirmed by transcriptome sequencing data (TPM value analysis) and qRT-PCR expression in multiple tissues.

[0049] (2) By double verification by GUS histochemical staining and qRT-PCR, the proASP1 promoter showed a strict spatiotemporal expression pattern in transgenic rice, driving the reporter gene to express efficiently only in the endosperm and aleurone layer of grains in the early grain filling stage (4-12 days), while there was no leakage expression in non-grain tissues such as roots, stems, leaves, leaf sheaths, and glumes, as well as in grains in the middle and late grain filling stages.

[0050] (3) The proASP1 promoter is derived from the endogenous gene regulatory region of rice. As a natural regulatory element, it has good biosafety and provides a reliable spatiotemporally specific expression tool for the functional study of rice grain development-related genes.

[0051] (4) The specific expression pattern of this promoter makes it an ideal regulatory element for gene function research during the critical window period of rice grain filling, and can provide a basic molecular tool for subsequent research on grain development mechanism. Attached Figure Description

[0052] Figure 1 A schematic diagram of the pBWA(V)HG-proASP1::GUS vector.

[0053] Figure 2 The TPM value of OsASP1 during rice grain development is given.

[0054] Figure 3 This is a graph comparing the expression levels of proASP1 in different tissues of rice.

[0055] Figure 4This is a PCR detection diagram of Escherichia coli bacterial culture containing the pBWA(V)HG expression vector with the proASP1 promoter.

[0056] Figure 5 Stereoscopic images taken during the detection of GUS activity in various tissue developmental processes of rice plants transfected with pBWA(V)HG-proASP1::GUS vector and blank control. Detailed Implementation

[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

[0060] I. Analysis of the expression pattern of endogenous OsASP1 gene in different tissues and during grain development

[0061] To assess the expression specificity of the OsASP1 gene, this study combined transcriptome sequencing with quantitative real-time PCR (qRT-PCR) for systematic analysis.

[0062] First, the transcriptome of rice grains from rice variety W1844 was sequenced at 4, 8, 12, 16, 20, and 24 days after flowering (DAF). Figure 2 The expression levels (TPM) of the OsASP1 gene in rice grains at different grain-filling stages were shown. The results showed that the OsASP1 gene was significantly highly expressed in grains at 4–8 DAF (TPM > 1000), while its expression level was extremely low in the mid-to-late grain-filling stages (10–15 DAF) (TPM < 5), indicating that it has obvious time-specificity in the early stages of grain development.

[0063] To further verify its tissue specificity, roots, stems, leaves, leaf sheaths, and seeds from W1844 plants at various stages were collected. Total RNA was extracted and reverse transcribed into cDNA. qRT-PCR was then performed using the following specific primers:

[0064] Forward primer (qPCR-F): 5′-CGACTCACTCTGCACTGACC-3′

[0065] Reverse primer (qPCR-R): 5′-AGAACCTGTCGATGACGAGC-3′

[0066] OsACTIN forward primer (OsACTIN-F): 5′-CAATCGTGAGAAGATGACCC-3′

[0067] OsACTIN reverse primer (OsACTIN-F): 5′-GTCCATCAGGAAGCTCGTAGC-3′

[0068] Using the rice OsACTIN gene as an internal control, 2 -ΔΔCt The method calculates the relative expression level. Figure 3 The relative expression levels of proASP1 in different rice tissues were shown by qRT-PCR. The results showed that OsASP1 was almost not expressed in vegetative tissues such as roots, stems, leaves, and leaf sheaths (Ct value >30 or undetectable), and was only significantly highly expressed in grains with 4–8 DAF.

[0069] In summary, qRT-PCR and grain transcriptome data together confirm that OsASP1 is a grain-specific high-expression gene in the early grain-filling stage, and its upstream regulatory sequence is very likely capable of driving the specific expression of exogenous genes in this spatiotemporal window, providing a solid basis for the cloning and functional verification of the promoter of this invention.

[0070] II. Cloning of the proASP1 promoter and construction of recombinant Agrobacterium strains

[0071] Based on the above expression analysis, a 3100 bp region upstream of the transcription start site (TSS) of the OsASP1 gene was selected as a candidate promoter. Using W1844 genomic DNA as a template, PCR amplification was performed using the following primers:

[0072] Cloning forward primer (proASP1-F):

[0073] 5′-ACCGCCTGCAGGTCTATAGAgcagagtatctgtatggccgcc-3′

[0074] Cloning the reverse primer (proASP1-R):

[0075] 5′- GATCTACCATGGTCAAGTTGggtgtgatggaggtggagctc-3′

[0076] The amplified products were subjected to 1% agarose gel electrophoresis at 5 V / cm for 20 minutes. The proASP1 (3102 bp) fragment was excised under UV light for gel recovery, following the same procedure as the DNA gel recovery kit (Axygen, product number: AP-GX-250). The proASP1 DNA fragment was dissolved and recovered in 40 μL of water and then recombined with the vector. The binary vector pBWA(V)HG (Biorun, product number: #REC32M) was digested with BsaI / Eco31I (Biorun, product number: #RCA02) to recombinant the proASP1 DNA fragment upstream of the GUS reporter gene in the binary vector pBWA(V)HG, replacing the original promoter, thus constructing the recombinant vector pBWA(V)HG-proASP1::GUS.

[0077] Figure 1 A schematic diagram of the constructed pBWA(V)HG-proASP1::GUS recombinant vector is shown. This vector contains T-DNA left and right boundaries, a hygromycin resistance gene (hpt, used for plant selection), and a kanamycin resistance gene (Kan, used for Agrobacterium selection). The correctly constructed plasmid was electroporated into Agrobacterium strain EHA105 to obtain the recombinant Agrobacterium strain, named EHA105 / pBWA(V)HG-proASP1::GUS. Figure 4 The PCR results of the pBWA(V)HG expression vector containing the proASP1 promoter in *E. coli* are presented. Lanes 1-3 are positive clones, all amplifying specific bands of the expected size (approximately 3100 bp), while the negative control (lane 4) showed no band, demonstrating that the recombinant vector was successfully constructed and stably exists in the *E. coli* host. EHA105 / pBWA(V)HG-proASP1::GUS will be used for subsequent genetic transformation in rice.

[0078] III. Obtaining Transgenic Rice Plants

[0079] Wuhan Boyuan Biotechnology Co., Ltd. used Agrobacterium-mediated callus transformation to genetically transform the aforementioned recombinant Agrobacterium strain into the rice variety W1844. The specific procedure included: inducing embryogenic callus from mature seeds, co-culturing with Agrobacterium, and then transferring the callus to a selection medium containing hygromycin (50 mg / L) for resistant callus selection. After differentiation and rooting, T0 generation transgenic plants were obtained. After sowing T1 generation seeds, T-DNA integration was detected by PCR, and multiple homozygous positive lines were screened for subsequent GUS expression analysis.

[0080] IV. GUS histochemical staining to verify the spatiotemporal specificity of the proASP1 promoter

[0081] Tissue samples from T1 generation positive transgenic plants (including roots, stems, leaves, leaf sheaths, and seeds from DAFs 0, 4, 8, 12, 16, 20, and 24) were collected and stained using the Solarbio GUS staining kit (catalog number: G3060) according to the manufacturer's instructions. The specific procedure was as follows: Tissue samples were immersed in pre-chilled GUS staining solution and incubated at 37°C in the dark for 24 hours. After staining, the staining solution was discarded, and anhydrous ethanol was added. The samples were then destained in an 80°C water bath, with the anhydrous ethanol replaced 2-3 times until the background was completely removed, retaining the specific blue signal.

[0082] Figure 5 The image shows the GUS histochemical staining results of pBWA(V)HG-proASP1::GUS vector-positive rice and a blank control at different developmental stages. The following can be clearly observed from the image:

[0083] (1) The transgenic plants showed a strong blue reaction in the grains of 4-12 DAF, indicating that the proASP1 promoter efficiently drives GUS gene expression at this stage.

[0084] (2) In kernels with 0 DAF and 16-24 DAF, the blue reaction was significantly reduced or completely disappeared;

[0085] (3) No blue reaction was observed in any developmental stage of the non-seed tissues such as roots, stems, leaves, leaf sheaths, and glumes of the transgenic plants;

[0086] (4) The negative control (non-transgenic rice) showed no GUS activity in all tissues and developmental stages.

[0087] These results directly confirm that the proASP1 promoter has a strict grain-specific expression pattern in the early grain-filling stage and that there is no leakage expression.

[0088] The results showed that a clear blue precipitate was observed only in grains aged 4-12 DAF, mainly distributed in the endosperm and aleurone layer; while no staining was observed in roots, stems, leaves, leaf sheaths, glumes, and mature grains after 12 DAF. This result is highly consistent with the qRT-PCR expression profile of the OsASP1 gene, fully demonstrating that the proASP1 promoter has a strictly specific grain-specific driving ability in the early grain-filling stage and exhibits no leakage expression in non-target tissues.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It should be understood that those skilled in the art can make various modifications, substitutions, or improvements to the above embodiments without departing from the technical concept of the present invention, and these equivalent or equivalent technical solutions should all be covered within the scope of protection of the present invention.

[0090] sequence list

[0091] SEQ ID NO:1

[0092]

Claims

1. The application of the rice grain-specific promoter with the nucleotide sequence shown in SEQ ID NO:1 during the early grain-filling stage in at least one of the following (a1)-(a4): (a1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage; (a2) Increase rice yield; (a3) Improve the nutritional quality of rice grains; (a4) Enhance the environmental adaptability of rice.

2. Application of biomaterials associated with the rice grain-specific promoter with the nucleotide sequence shown in SEQ ID NO:1 during the early grain-filling stage in rice, in at least one of the following (a1)-(a4): (a1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage; (a2) Increase rice yield; (a3) Improve the nutritional quality of rice grains; (a4) Enhance the environmental adaptability of rice; The biomaterials associated with the rice grain-specific promoter during the early grain-filling stage are at least one of the following (b1)-(b9): (b1) An expression cassette containing the rice grain-specific promoter for the early grain-filling stage; (b2) A recombinant vector containing the rice grain-specific promoter for the early grain-filling stage, or a recombinant vector containing the expression cassette described in (b1); (b3) Recombinant microorganisms containing the rice grain-specific promoter in the early grain-filling stage, or recombinant microorganisms containing the expression cassette described in (b1), or recombinant microorganisms containing the recombinant vector described in (b2); (b4) A transgenic rice cell line containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice cell line containing the expression cassette described in (b1), or a transgenic rice cell line containing the recombinant vector described in (b2); (b5) Transgenic rice tissue containing the grain-specific promoter for the early grain-filling stage of rice, or transgenic rice tissue containing the expression cassette described in (b1), or transgenic rice tissue containing the recombinant vector described in (b2); (b6) A transgenic rice organ containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice organ containing the expression cassette described in (b1), or a transgenic rice organ containing the recombinant vector described in (b2); (b7) A transgenic rice plant containing the grain-specific promoter for the early grain-filling stage of rice, or a transgenic rice plant containing the expression cassette described in (b1), or a transgenic rice plant containing the recombinant vector described in (b2); (b8) Regenerative cells, tissue cultures, or protoplasts derived therefrom of the transgenic rice plant as described in (b7); (b9) Propagation material of the transgenic rice plants as described in (b7).

3. The application according to claim 2, characterized in that, The expression cassette is a grain-specific gene expression cassette for the early grain-filling stage, comprising a rice grain-specific promoter with a nucleotide sequence as shown in SEQ ID NO:1, a target gene coding sequence operably linked downstream of the promoter, and a transcription terminator.

4. The application according to claim 3, characterized in that, The target gene is a reporter gene, a gene related to starch synthesis, a gene related to mineral element enrichment, a gene related to stress resistance, or a gene related to hormone regulation.

5. The application according to claim 2, characterized in that, The scaffold vector of the recombinant vector is the binary vector pBWA(V)HG or its derivative vector.

6. The application according to claim 1 or 2, characterized in that, A plant recombinant expression vector containing a rice grain-specific promoter and a target gene in the early grain-filling stage is introduced into rice to obtain transgenic rice; the specific promoter can drive the target gene operably linked to it to be specifically expressed in the early grain-filling stage grains of the transgenic rice.

7. A method, characterized in that, The method is at least one of the following (c1)-(c4): (c1) Drives the specific expression of the target gene in rice grains during the early grain-filling stage; (c2) Increase rice yield; (c3) Improve the nutritional quality of rice grains; (c4) Enhance the environmental adaptability of rice; Transgenic rice was obtained by introducing a plant recombinant expression vector containing a grain-specific promoter for the early grain-filling stage of rice, as shown in SEQ ID NO:1, and the target gene. The specific promoter can drive the specific expression of the target gene operatively linked to it in the early grain-filling stage of the transgenic rice.

8. The method according to claim 7, characterized in that, The method specifically includes the following steps: (1) Cloning the rice grain-specific promoter in the early grain-filling stage as shown in SEQ ID NO:1; (2) Insert the specific promoter into the plant expression vector to construct a recombinant expression vector containing the proASP1:: target gene; (3) Transform Agrobacterium and introduce the recombinant expression vector into rice through rice callus transformation; (4) Screening and identification of transgenic rice positive plants; (5) Use GUS staining or qRT-PCR to verify the specific expression of the target gene in grains during the early grain-filling stage.

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