Synsepalum dulcificum protein coding gene endogenous promoter and application thereof

Abstract

The invention discloses a synsepalum dulcificum protein coding gene endogenous promoter and application thereof. The endogenous promoter of the synsepalum dulcificum protein coding gene is any one of proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3 and proSdMIR.1.4, the DNA (deoxyribonucleic acid) nucleotide sequence of the proSdMIR.1 is as shown in SEQ ID NO.3, the DNA nucleotide sequence of the proSdMIR.1.2 is as shown in SEQ ID NO.4, the DNA nucleotide sequence of the proSdMIR.1.3 is as shown in SEQ ID NO.5, and the DNA nucleotide sequence of the proSdMIR.1.4 is as shown in SEQ ID NO.6. The synsepalum dulcificum protein coding gene disclosed by the invention has the advantages that the synsepalum dulcificum protein coding gene can be used for preparing the synsepalum dulcificum protein The miraculin gene promoter provided by the invention has important significance in optimizing a miracle fruit genetic system and creating miracle fruit germplasm resources with excellent characters.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, particularly the field of plant transgenic technology, and specifically relates to an endogenous promoter for a protein-encoding gene of miracle fruit and its application. Background Technology

[0002] Miracle fruit ( Synsepalum dulcificum Daniell, also known as the miracle fruit or the variegated fruit, belongs to the genus *Miracle Fruit* of the family Sapotaceae. Synsepalum Miracle fruit is a perennial, low-growing shrub native to tropical West Africa. Introduced to my country in the 1960s, it is now cultivated in southern regions such as Guangdong, Guangxi, Yunnan, and Hainan. Its various tissues and parts are rich in flavonoids and phenolic compounds, exhibiting significant antioxidant activity. Among these, the signature component of miracle fruit—miraculin—is a natural acidic glycoprotein that requires no chemical synthesis. It possesses unique taste-modifying functions and, after being ingested, can be broken down into amino acids for human absorption and digestion. It has enormous application value and development potential in the food processing industry and the medical and health fields.

[0003] Gene editing technology is a revolutionary biotechnology that, based on a variety of tools, enables scientists to precisely edit and alter specific gene sequences. Applications of gene editing technology in agriculture include improving crop disease resistance and stress tolerance, modifying nutrient composition, increasing yield, and reducing pesticide use. Currently, the CRISPR-Cas system is the most widely used technology in plant gene editing. CRISPR-Cas technology is a gene editing technology developed based on bacterial innate immune mechanisms, achieving targeted modification of the genome through guide RNA precise positioning and Cas protein cleavage of DNA.

[0004] Promoters themselves are not transcribed on the DNA strand; their primary function is to recruit RNA polymerases and transcription factors. Their characteristics were initially identified through mutations that increase or decrease gene transcription rates, and they are mainly classified as constitutive, inducible, and tissue-specific. The promoter region encompasses core elements such as the TATA-box and CAAT-box, as well as several conserved "cis-acting elements" that respond to hormones and environmental signals. This sequence is generally located upstream of the transcription start site of structural genes. Gene expression regulation technologies cover multiple levels: transcription, post-transcriptional, translation, and post-translational regulation. As a "fixed switch" on the DNA strand, the promoter, after binding to trans-acting factors, regulates the expression of genes on that DNA strand. Different promoters have different compositions of cis-acting elements, leading to differential expression of the regulated genes under different growth environmental conditions and growth states, such as temperature, light intensity, and growth period. Endogenous promoter modification and the introduction of exogenous promoters mainly act on the transcription initiation stage and are currently core technologies for transcriptional regulation.

[0005] In current plant genetic engineering and synthetic biology expression systems, the most widely applicable heterologous promoter is the CaMV35S promoter from cauliflower mosaic virus. Previous researchers have used the CaMV35S promoter to link the miraculin-encoding gene into lettuce, strawberry, and other plants, achieving stable inheritance to offspring; however, the miraculin content in the offspring is extremely low. While miraculin can be stably inherited and expressed at high levels in transgenic tomatoes, significant challenges remain in purification. Previous studies have extracted high-purity, non-inactivated recombinant miraculin from transgenic tomatoes using nickel column chromatography combined with ion exchange chromatography, but this method is costly for industrial application and not conducive to large-scale production; further improvements are needed. Regarding endogenous promoters, the endogenous U6 promoter has been used in rice, Arabidopsis thaliana, tobacco, wheat, lettuce, grape, and other species to construct CRISPR / Cas9 editing systems, achieving highly efficient editing.

[0006] Currently, there is limited research on the promoter of miracle fruit, which hinders research on gene editing and molecular breeding technologies for miracle fruit. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned problems by providing an endogenous promoter for the miracle fruit protein-encoding gene and its application.

[0008] To achieve its objective, the present invention employs the following technical solution: The first aspect of the present invention provides an endogenous promoter for a miracle fruit protein-encoding gene, wherein the endogenous promoter for the miracle fruit protein-encoding gene is any one of proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4, wherein the DNA nucleotide sequence of proSdMIR.1 is shown in SEQ ID NO.3, the DNA nucleotide sequence of proSdMIR.1.2 is shown in SEQ ID NO.4, the DNA nucleotide sequence of proSdMIR.1.3 is shown in SEQ ID NO.5, and the DNA nucleotide sequence of proSdMIR.1.4 is shown in SEQ ID NO.6.

[0009] A second aspect of the present invention provides an expression box containing the above-described promoter.

[0010] A third aspect of the present invention provides a recombinant vector containing the above-described promoter or expression cassette.

[0011] Preferably, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0012] Preferably, the backbone vector of the recombinant expression vector is the plant binary expression vector pCAMBIA1303, and the backbone vector of the recombinant cloning vector is pMD19-T.

[0013] A fourth aspect of the present invention provides a recombinant bacterium containing the above-described promoter, expression cassette, or recombinant vector.

[0014] The fifth aspect of the present invention provides the use of the above-described endogenous promoter of the miracle fruit protein-encoding gene in any of the following: (1) Application in the construction of expression cassettes, recombinant vectors or recombinant bacteria; (2) Application in the construction of transgenic plants; (3) Application in plant molecular breeding; (4) Application in initiating the expression of target genes in plants; The plant in question is either miracle fruit or Arabidopsis thaliana.

[0015] A sixth aspect of the present invention provides a method for expressing a target nucleic acid molecule in a plant, the method comprising introducing a nucleic acid construct into the plant, the nucleic acid construct containing the aforementioned promoter and a target nucleic acid molecule operatively linked to the promoter, wherein the plant is miracle fruit or Arabidopsis thaliana.

[0016] The beneficial effects of this invention are: This invention is the first to clone the endogenous promoter of the Miraculin gene—proSdMIR.1—from the genomic DNA of Miraculin. Through homologous recombination, it was fused with the GUS gene, and T2 generation transgenic Arabidopsis thaliana was obtained by infecting Arabidopsis thaliana inflorescences using Agrobacterium-mediated transformation. Stable expression of GUS verified that the promoter has transcriptional activity in the rosette leaves, scape leaves, stems, pods, and flowers of transgenic Arabidopsis thaliana, providing key molecular tools to support the future exploration of the stable genetic system of Miraculin. Furthermore, this invention is the first to truncate the Miraculin gene promoter proSdMIR.1, which possesses transcriptional activity, to obtain truncated Miraculin gene promoters proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4. These promoters were then used to construct GUS gene fusion expression vectors for experiments. The results showed that the truncated Miraculin gene promoters still possessed transcriptional activity, and the activities of different truncations differed significantly. The proSdMIR.1.2 promoter, truncated to 1389 bp, exhibited the highest transcriptional activity, exceeding that of the control CaMV 35S promoter. The Miraculin gene promoters provided by this invention are of great significance for optimizing the Miraculin genetic system and creating Miraculin germplasm resources with superior traits. Attached Figure Description

[0017] Figure 1 Agarose gel electrophoresis image of the genome of miracle fruit DNA.

[0018] Figure 2 Agarose gel electrophoresis image of the amplified bands of the proSdMIR.1 promoter.

[0019] Figure 3 Agarose gel electrophoresis image for detecting truncated clones of the proSdMIR.1 promoter.

[0020] Figure 4 Agarose gel electrophoresis image showing PCR-positive identification of the proSdMIR.1 promoter and its truncated promoter in T1 generation transgenic Arabidopsis thaliana.

[0021] Figure 5 GUS staining images of leaves from T2 generation transgenic Arabidopsis plants with the proSdMIR.1 promoter and its truncated promoter GUS fusion expression vector.

[0022] Figure 6 The figure shows the quantitative results of the GUS gene in the T2 generation transgenic Arabidopsis plants of the proSdMIR.1 promoter and its truncated promoter GUS fusion expression vector.

[0023] Figure 7GUS staining images of various tissues of T2 generation transgenic Arabidopsis plants with the proSdMIR.1 promoter and its truncated promoter GUS fusion expression vector. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Unless otherwise specified, the methods described in the following examples are conventional methods.

[0026] Example 1: Obtaining the endogenous promoter proSdMIR.1 sequence of the Miraculin gene from Miracle Fruit Our previous research established a miracle fruit transcriptome database, and this research team is studying miracle fruit proteins. Chr10G0299340 The gene sequence is completely identical to the reported miracle fruit protein gene sequence (GenBank: BAH84844.1). Our research team's transcriptome study discovered the miracle fruit protein gene (…). MIR,Chr10G0299340 ) is the gene with the highest expression level in the miracle fruit, and further research shows that... MIR The expression levels of the gene in six tissues and three stages of miracle fruit were studied. It was found that the gene was almost not expressed in roots, stems, and leaves; it showed some expression in seeds and flowers; and its expression level increased throughout fruit development, reaching its highest point in mature fruit. This gene was also the most highly expressed gene in the transcriptome data of the six tissues and three stages of miracle fruit (refer to the doctoral dissertation "Study on Genome Assembly, Functional Evolution of Miracle Protein and Molecular Basis of Anthocyanin Synthesis in Miracle Fruit," Yang Zhuang, 2022, Hainan University). Miracle fruit genome sequencing data has been stored in the CNGBdb CNGB sequence archive, accession number CNP0002330 (https: / / db.cngb.org / data_resources / project / CNP0002330). Transcriptome sequencing data has been stored in the NCBI Sequence Read Archive database, accession number PRJNA778426 (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA778426 / ).

[0027] Based on previous research results, we hypothesize that the Miraculin gene promoter has a very high activation efficiency, possibly even higher than that of the 35S promoter, and therefore we have developed this invention.

[0028] Based on the gene sequence of miracle fruit protein ( MIR,Chr10G0299340The upstream reference sequence of this gene was obtained. Primer pairs JT-Miraculin-sF and JT-Miraculin-sR were designed using the primer design tool Primer Premier 5 to amplify the Miraculin gene promoter (named proSdMIR.1) plus a 51 bp Miraculin gene sequence including the start codon. The nucleotide sequences of primers JT-Miraculin-sF and JT-Miraculin-sR are as follows: JT-Miraculin-sF (SEQ ID NO.1): 5'-TATTTCGGGTTTACAAAATTTTTGAA-3'; JT-Miraculin-sR (SEQ ID NO.2): 5'-CAATGCAGAGACGAAGAAGAACG-3'.

[0029] 100 mg of fresh miracle fruit pulp was selected, flash-frozen in liquid nitrogen, and then ground into a fine powder. Genomic DNA was extracted from the miracle fruit pulp using the CATB method. The DNA concentration was determined by spectrophotometry, and the DNA quality was assessed by agarose gel electrophoresis. The results are as follows: Figure 1 As shown. Using miracle fruit pulp genomic DNA as a template, PCR amplification was performed using PrimeSTAR® Max DNA Polymerase high-fidelity polymerase from Baori Biotechnology (Beijing) Co., Ltd. The reaction system consisted of 1 μL each of 10 μM JT-Miraculin-sF / JT-Miraculin-sR primers, 200-500 ng of miracle fruit genomic DNA, 25 μL of PrimeSTAR® Max DNA Polymerase, and sterile water to a final volume of 50 μL. The reaction program was: 95℃ pre-denaturation for 3 min; then 95℃ for 15 s, 54℃ for 2 min, 72℃ for 30 s, 35 cycles; 72℃ for 5 min. The amplification results are shown below. Figure 2 As shown, the amplified band is 1872 bp, which consists of a 1821 bp promoter sequence and a 51 bp gene sequence.

[0030] 40 μL of PCR product was added to 9 μL of 2× Taq Master Mix DNA polymerase from Nanjing Novizan Biotechnology Co., Ltd., and reacted at 72℃ for 20 min. An A-tail was added, and the concentration was measured. The vector was constructed using the pMDm19-T Vector Cloning Kit (TaKaRa, Japan). The ligation system was: Solution I 5 μL, pMD 19-T Vector 1 μL, 800 ng of PCR product with A-tail, and sterile water to a final volume of 10 μL. The mixture was incubated overnight at 16℃ and ligated into the T vector pMD 19-T. The resulting fragment was then chemically transformed into E. coli DH5α. After plating, bacterial culture was selected for colony PCR identification. After confirming the Sanger sequencing results were correct, the fragment was named p19T-proSdMIR.1. The sequence of the inserted amplified fragment, as shown in SEQ ID NO.12, is the proSdMIR.1 promoter sequence shown in SEQ ID NO.3 plus a 51 bp Miraculin gene sequence.

[0031] Example 2: Construction of the Miraculin Gene Promoter GUS Fusion Expression Vector from Miraculin Fruit The endogenous promoter proSdMIR.1 (1821 bp, nucleotide sequence as shown in SEQ ID NO. 3) from Example 1 was truncated at the 5' end to obtain three different truncated versions: the first truncated version was 1389 bp, named proSdMIR.1.2 (nucleotide sequence as shown in SEQ ID NO. 4); the second truncated version was 785 bp, named proSdMIR.1.3 (nucleotide sequence as shown in SEQ ID NO. 5); and the third truncated version was 542 bp, named proSdMIR.1.4 (nucleotide sequence as shown in SEQ ID NO. 6). Primers for amplification of the truncated fragments were designed, and a 19 bp vector homologous arm sequence was added to the 5' end of the primers.

[0032] Using the p19T-proSdMIR.1 bacterial culture from Example 1 as a template, PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (TaKaRa, Japan). The reaction system was 50 μL: 1 μL each of 10 μM upstream and downstream primers, 1 μL of p19T-proSdMIR.1 bacterial culture, 25 μL of PrimeSTAR® Max DNA Polymerase, and sterile water to a final volume of 50 μL. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 65℃ for 45 s, 72℃ for 90 s, 35 cycles, 72℃ for 5 min. The amplification primer sequences for proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 are as follows (where the underlined lowercase bases are vector homologous arm sequences): The primer pair for PCR amplification of the proSdMIR.1 promoter is JT-Miraculin-s-1 F / JT-Miraculin-s-1 R: JT-Miraculin-s-1 F (SEQ ID NO.7): 5'- agctcggtacccggggatc TATTTCGGGTTTACAAAATTTTTGAA-3'; JT-Miraculin-s-1 R (SEQ ID NO.8): 5'- actagtcagatctaccatg TGTTGTAGAGACTATAGGGCTGTTGAT-3'; The primer pair for PCR amplification of the proSdMIR.1.2 promoter is JT-Miraculin-s-2 F / JT-Miraculin-s-1R: JT-Miraculin-s-2 F (SEQ ID NO.9): 5'- agctcggtacccggggatc CAAAATTGGGGTTGCAAATGTAAT-3'; The primer pair for PCR amplification of the proSdMIR.1.3 promoter is JT-Miraculin-s-3 F / JT-Miraculin-s-1R: JT-Miraculin-s-3 F (SEQ ID NO.10): 5'- agctcggtacccggggatc TATATCAAAAGCAATATCCCCTTTTGA-3'; The primer pair for PCR amplification of the proSdMIR.1.4 promoter is JT-Miraculin-s-4 F / JT-Miraculin-s-1R: JT-Miraculin-s-4 F (SEQ ID NO.11): 5'- agctcggtacccggggatc AATAAGAGAAGAGAGGTGATTGGCA.

[0033] The downstream amplification primers for promoters proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 are all JT-Miraculin-s-1 R (SEQ ID NO.8).

[0034] PCR products were detected and identified by agarose gel electrophoresis (e.g.) Figure 3 As shown in the figure, the PCR product was purified and recovered, and sequenced. The sequencing results showed that the amplified band sequence was the corresponding promoter sequence (shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6) plus the vector homologous arm sequences at both ends. BamHI and Nco The plant binary expression vector pCAMBIA1303 (selected for the hygromycin gene) was digested overnight with restriction enzyme I, and the linearized vector was purified and recovered. The promoter and linearized vector were ligated using the ClonExpress II One Step Cloning Kit (Novozymes, China). The ligation mixture consisted of 2 μL of 5*CE II Buffer, 200 ng of linearized vector, 50 ng of promoter fragment, and sterile water to a final volume of 10 μL. Homologous recombination was performed at 37°C for 30 min to obtain the recombinant product. The recombinant product was transformed into E. coli DH5α competent cells, and bacterial PCR identification was performed. Four Miraculin gene promoter GUS fusion expression vectors were successfully obtained: pCAMBIA1303-proSdMIR.1::GUS expression vector, pCAMBIA1303-proSdMIR.1.2::GUS expression vector, pCAMBIA1303-proSdMIR.1.3::GUS expression vector, and pCAMBIA1303-proSdMIR.1.4::GUS expression vector.

[0035] Example 3: Genetic transformation of the Miraculin gene promoter GUS fusion expression vector from Miraculin fruit The Miraculin gene promoter GUS fusion expression vector obtained in Example 2 was transformed into GV3101 Agrobacterium competent cells. Using wild-type Arabidopsis thaliana (col) as recipients, the pCAMBIA1303-proSdMIR.1::GUS expression vectors, pCAMBIA1303-proSdMIR.1.2::GUS expression vectors, pCAMBIA1303-proSdMIR.1.3::GUS expression vectors, and pCAMBIA1303-proSdMIR.1.4::GUS expression vectors were transformed into Arabidopsis thaliana via Agrobacterium infection.

[0036] Seeds from Arabidopsis thaliana plants transfected with the Miraculin gene promoter GUS fusion expression vector were collected. After disinfection with 75% ethanol and sodium hypochlorite, the seeds were sown on 1 / 2 MS solid medium containing 60 mg / L hygromycin for antibiotic selection. Once the plants developed true leaves and roots, they were transplanted into soil. Five plants were selected from each vector, and genomic DNA was extracted from the rosette leaves of the transgenic Arabidopsis thaliana for PCR identification of positive plants. The PCR amplification primers for each promoter were those specified in Example 2. PCR was performed using 2× Taq Master Mix DNA polymerase. The PCR reaction system consisted of 1 μL each of 10 μM upstream and downstream primers, 100-200 ng of transgenic Arabidopsis rosette leaf genomic DNA, 5 μL of 2× Taq Master Mix, and sterile water to a final volume of 10 μL. The reaction program was 95℃ for 3 min; 95℃ for 30 s, 65℃ for 30 s, 72℃ for 1 min, for 35 cycles; 72℃ for 5 min. Five positive seedlings from each recombinant vector were selected for testing. Figure 4 As shown, the amplification bands of each promoter in the Arabidopsis transgenic plants of each recombinant vector are of the correct size.

[0037] Example 4: Functional Verification of the Endogenous Promoters proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 of Miracle Fruit Seeds from the positive T1 generation of transgenic Arabidopsis thaliana plants using the Miraculin gene promoter GUS fusion expression vector from Example 3 were collected. After sterilization with 75% alcohol, the seeds were sown on 1 / 2 MS medium containing 60 mg / L hygromycin for antibiotic selection. The Arabidopsis seedlings that had developed true leaves and roots were transplanted into soil for further cultivation. T2 generation transgenic Arabidopsis thaliana plants using the Miraculin gene promoter GUS fusion expression vector were obtained.

[0038] GUS histochemical staining was performed on the obtained T2 generation transgenic Arabidopsis plants. The procedure was as follows: wild-type Arabidopsis plants were used as negative controls, and transgenic Arabidopsis plants transformed with the plant binary expression vector pCAMBIA1303 (containing the CaMV 35S promoter) were used as positive controls to detect the GUS gene expression pattern and relative activity. Rosette leaves, scape leaves, stems, pods, and flowers of transgenic and wild-type plants were immersed in freshly prepared GUS staining solution (200 mL: 0.7444 g EDTA-2Na + 0.3292 g potassium ferricyanide + 0.4224 g potassium ferrocyanide + 190 mL sodium phosphate buffer (pH 7.0) + 1 mL 10 mMMX-Gluc) and stained at 37°C for 48 h. Remove the GUS staining solution and rinse it with ultrapure water. Place it in 75% alcohol and repeatedly decolorize it to remove chlorophyll until the green color fades. Use absorbent paper to dry the alcohol on the surface of the leaf and lay it flat under a stereomicroscope for observation and photography.

[0039] Leaf tissues from T2 generation transgenic Arabidopsis and wild-type Arabidopsis were harvested, flash-frozen in liquid nitrogen, and ground into powder. GUS protein was extracted using a GUS reporter gene quantification kit (Shanghai Bosen, China) according to the kit's instructions. Protein content was determined using a Bradford protein assay kit (Shanghai Bosen, China) according to the kit's instructions, and a protein standard curve was constructed. A 4-MUG standard curve was also constructed and 4-MUG fluorescence values ​​were measured according to the GUS reporter gene quantification kit's instructions.

[0040] GUS histochemical staining results as follows Figure 5 As shown, no blue change was observed in wild-type Arabidopsis thaliana. However, distinct differences in blue color were observed in the leaf veins and petioles of transgenic Arabidopsis thaliana with the CaMV35S, proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 promoters. Among them, the blue color of CaMV 35S and proSdMIR.1.2 was darker, while the blue color of proSdMIR.1, proSdMIR.1.3, and proSdMIR.1.4 was lighter.

[0041] GUS reporter gene quantification results as follows Figure 6As shown, the proSdMIR.1.2 promoter exhibits the highest GUS activity at 17463 pmol / min / mg; followed by the proSdMIR.1.3 promoter at 15045 pmol / min / mg; CaMV 35S at 14731 pmol / min / mg; proSdMIR.1 at 13382 pmol / min / mg; and the proSdMIR.1.4 promoter shows the lowest GUS activity at 13105 pmol / min / mg.

[0042] GUS staining and quantitative detection of GUS genes in transgenic Arabidopsis leaves showed that all three promoters of the miracle fruit proSdMIR.1 promoter were truncated and had strong transcriptional activity in Arabidopsis. The proSdMIR.1.2 promoter, which was truncated to 1389 bp, had the highest transcriptional activity.

[0043] GUS histochemical staining results of various tissues from T2 generation transgenic Arabidopsis thaliana are as follows: Figure 7 As shown, no blue staining was observed in the rosette leaves, stems, pods, and flowers of wild-type Arabidopsis plants. GUS staining was detected in the rosette leaves, stems, pods, and flowers of transgenic Arabidopsis plants with CaMV 35S, proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 promoters, indicating that the proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.4 promoters possess transcriptional activity in the rosette leaves, stems, pods, and flowers.

[0044] The nucleotide sequences of the four promoters and the amplified fragments of JT-Miraculin-sF / JT-Miraculin-sR in this invention are as follows: 1. The sequence of the promoter proSdMIR.1 (SEQ ID NO.3) is as follows:

[0045] 2. The sequence of promoter proSdMIR.1.2 (SEQ ID NO.4) is as follows:

[0046] 3. The sequence of promoter proSdMIR.1.3 (SEQ ID NO.5) is as follows: TATATCAAAAGCAATATCCCCTTTTGAATCGTAGGCGACCCAAACAATTAGGTCGGGATTTTTTTTAATTTTTTTATGTAAGCTATTGAAATATCATCAGTCAATTGCCGTGGAGATAAATTAGGTCGGGATTGCAACATTTTTTTTTTTAATACCATTAAAATGTCGGTCATTTGTTGTGGATATAGTTTAGGCCGGGTTTTATGGTTTTTATTATTATTTTTTTAATACCATTAGAATATTAATAAGAGAAGAGAGGTGATTGGCAAAAAAAACTTGGGTTAGTTTGGTTTGAAAAAAAAAAAAAAAAAACTTTAAAAAACTCTACAATTTTCTAAGACTTATCTCACTGTACGTATTACGAACTCTCACTTTTTCTCATTTTCTCTCGTTTCAAATACTACCAAAATCTTTACATGAGACTGTTAGGCCCGACATCAATTAACAAGTGACAAACTCGAAGCCCCTAATGAAAGCTTTAGGCTTAGTTTGGAATGAAAAAATAGTAATTTTTTAACAAATTATTAAAATATTTAAAAAAAAAAAAGTTAATTTTTGTCAATTTAACATTTGTACCATTTATAATGAAAAGAATTAAAATATAAGTGATTAAATTTGATGAATTGAGAAAAAGTTAAAAAAAATTGAAATTGCCTAAATCTTACTCTCCCAAACGAAGCCTTAGCTTTCAACTCAGTGCTACGTGGCATTCGTATCTCTTCGTATCTATAAATAATTGTGAAGTGCCCACGTTGGTGATCAACAGCCCTATAGTCTCTACAACA。

[0047] 4. The sequence of promoter proSdMIR.1.4 (SEQ ID NO.6) is as follows: AATAAGAGAAGAGAGGTGATTGGCAAAAAAAACTTGGGTTAGTTTGGTTTGAAAAAAAAAAAAAAAAAACTTTAAAAAACTCTACAATTTTCTAAGACTTATCTCACTGTACGTATTACGAACTCTCACTTTTTCTCATTTTCTCTCGTTTCAAATACTACCAAAATCTTTACATGAGACTGTTAGGCCCGACATCAATTAACAAGTGACAAACTCGAAGCCCCTAATGAAAGCTTTAGGCTTAGTTTGGAATGAAAAAATAGTAATTTTTTAACAAATTATTAAAATATTTAAAAAAAAAAAAGTTAATTTTTGTCAATTTAACATTTGTACCATTTATAATGAAAAGAATTAAAATATAAGTGATTAAATTTGATGAATTGAGAAAAAGTTAAAAAAAATTGAAATTGCCTAAATCTTACTCTCCCAAACGAAGCCTTAGCTTTCAACTCAGTGCTACGTGGCATTCGTATCTCTTCGTATCTATAAATAATTGTGAAGTGCCCACGTTGGTGATCAACAGCCCTATAGTCTCTACAACA。

[0048] 5. The amplified fragment (SEQ ID NO.12) sequence of JT-Miraculin-s-F / JT-Miraculin-s-R is as follows:

Claims

1. An endogenous promoter for a miracle fruit protein-encoding gene, characterized in that: The endogenous promoter of the miracle fruit protein encoding gene is any one of proSdMIR.1, proSdMIR.1.2, proSdMIR.1.3, and proSdMIR.1.

4. The DNA nucleotide sequence of proSdMIR.1 is shown in SEQ ID NO.3, the DNA nucleotide sequence of proSdMIR.1.2 is shown in SEQ ID NO.4, the DNA nucleotide sequence of proSdMIR.1.3 is shown in SEQ ID NO.5, and the DNA nucleotide sequence of proSdMIR.1.4 is shown in SEQ ID NO.

6.

2. An expression box containing the promoter of claim 1.

3. A recombinant vector containing the promoter of claim 1 or the expression cassette of claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is a recombinant expression vector or a recombinant cloning vector.

5. The recombinant vector according to claim 4, characterized in that: The backbone vector of the recombinant expression vector is the plant binary expression vector pCAMBIA1303, and the backbone vector of the recombinant cloning vector is pMD19-T.

6. A recombinant bacterium containing the promoter of claim 1, the expression cassette of claim 2, or the recombinant vector of claim 3.

7. The use of the endogenous promoter of the miracle fruit protein-encoding gene as described in claim 1 in any of the following: (1) Application in the construction of expression cassettes, recombinant vectors or recombinant bacteria; (2) Application in the construction of transgenic plants; (3) Application in plant molecular breeding; (4) Application in initiating the expression of target genes in plants; The plant in question is either miracle fruit or Arabidopsis thaliana.

8. A method for expressing a target nucleic acid molecule in a plant, characterized in that: The method includes introducing a nucleic acid construct into a plant, the nucleic acid construct containing the promoter of claim 1 and a target nucleic acid molecule operatively linked to the promoter, wherein the plant is miracle fruit or Arabidopsis thaliana.

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