Rice endosperm tissue-specific promoter pPROLM26 and its application

Abstract

The present invention relates to the field of plant genetic engineering technology, specifically providing a rice endosperm tissue-specific promoter, pPROLM26, and its applications. The present invention isolates a tissue-specific promoter with the nucleotide sequence shown in SEQ ID NO. 1 from a rice alcohol-soluble protein gene. This tissue-specific promoter is truncated four times and then molecularly identified with a RUBY visualization marker to obtain truncated promoter fragments shown in SEQ ID NOs. 2 to 5, each with endosperm-specific promoter function. The expression product has been measured to contain up to 2.5% betaine of the seed dry weight, demonstrating its high application value in the field of plant synthetic biology. The present invention further discloses applications of the promoter or truncated promoter fragments in improving crop seed quality, improving crop traits, cultivating new transgenic plant varieties using key elements of the promoter, or expressing high-value-added proteins using seed bioreactors.

Description

Technical Field

[0001] The invention belongs to the field of separation and application of plant tissue specific promoters, and relates to a rice endosperm tissue specific promoter pPROLM26 and application thereof. Background Art

[0002] Rice (Oryza sativa. L.) is an important food crop, a staple food in over half of the world's regions. Transgenics involves linking exogenous DNA sequences, such as functional genes for insect and disease resistance, to specific promoters through molecular means, enabling expression of these exogenous genes in host cells and improving plant traits. For certain functional genes, specific promoters can be used to precisely regulate their expression in specific locations, enabling fine-grained control of gene expression.

[0003] Promoters, DNA sequences specifically recognized and bound by RNA polymerase, are key cis-acting elements typically located in the 5' domain of structural genes. In higher plants, gene expression is primarily regulated at the transcriptional level, with promoters playing a central role in controlling the onset and level of gene expression and determining the type of RNA polymerase involved in transcription. Therefore, promoters are crucial for understanding gene expression patterns and transcriptional regulation mechanisms, forming the core of plant transcriptional regulation. Based on their transcriptional activity and functional properties, promoters can be divided into three major categories: constitutive promoters, tissue-specific promoters, and inducible promoters. Recent research has also revealed the existence of bidirectional promoters. Currently, constitutive promoters are widely used in plant genetic engineering practices, as they can drive efficient expression of exogenous proteins in various plant tissues. However, these promoters cannot precisely regulate the spatiotemporal expression of target genes, potentially leading to excessive consumption of cellular resources and energy. Furthermore, overexpression of certain constitutive promoters in specific tissues can interfere with normal plant growth and development, thus becoming an obstacle to crop improvement. In contrast, specific promoters enable concentrated expression at specific spatial locations and time points. With the continuous development of transgenic technology and the widespread use of transgenic crops, promoters are becoming increasingly important in crop improvement. Research on tissue-specific promoters has garnered particular attention, and therefore holds special significance for the molecular improvement and production of crops like rice. However, the current genetic resources for specific promoters are relatively limited, which, to a certain extent, limits the ability to fine-tune the improvement of different crop traits.

[0004] Tissue-specific promoters regulate downstream gene transcription, which typically occurs only in certain tissues or organs. Tissue-specific promoters can more efficiently and economically regulate heterologous gene expression, specifically targeting specific areas of interest. This not only increases heterologous gene expression but also minimizes bioenergy consumption, thus preserving normal plant growth.

[0005] Rice is a widely cultivated food crop and a widely studied model crop. It has a long history of cultivation, with well-established cultivation and genetic transformation techniques. Rice is also a strictly self-pollinating crop, demonstrating excellent biosafety. Compared to other species, rice offers several unique advantages as a host for bioreactors, including the ability to efficiently express small peptides and simplify protein purification. The rice endosperm is an ideal site for recombinant protein production and a key target for improving the nutritional value of food. Developing efficient endosperm-specific expression vectors is a key approach to obtaining recombinant proteins from the endosperm and improving nutritional composition. These efforts not only help elucidate fundamental theories of rice morphology, development, and metabolic pathways, but also guide transgenic breeding, creating significant economic and social benefits and ultimately serving human production and well-being.

[0006] The RUBY reporter system offers significant advantages over traditional reporter systems (GUS, GFFP, and Luc) and similar visual markers (RFP). First, the RUBY reporter system uses tyrosine, a common amino acid in plants, as a reaction substrate. It achieves color labeling through the synthesis of red betaine, enabling visual identification without the need for fluorescence microscopy, special substrate treatment, or chemical staining. Furthermore, RUBY supports in situ observation without disrupting plant tissue, thus preventing mechanical damage from interfering with gene expression. Red betaine provides significant contrast in plant tissues (especially green leaves), while the green color of GFP is easily interfered with by chlorophyll, and the blue product of GUS is less recognizable against complex backgrounds. Red betaine is a natural plant product (e.g., beet and pitaya pigments) that is non-toxic, stable, and resistant to degradation or interference with plant physiology. However, overexpression of fluorescent proteins can inhibit plant growth, and GUS enzyme activity is susceptible to environmental influences. Summary of the Invention

[0007] One of the purposes of the present invention is to provide a DNA molecule with promoter activity, so that the target gene can be expressed efficiently and specifically in rice endosperm.

[0008] The second object of the present invention is to provide a tissue-specific promoter isolated from rice or a promoter fragment which still has promoter function after being segmented and truncated.

[0009] A third object of the present invention is to provide a recombinant expression vector containing the above-mentioned tissue-specific promoter or promoter fragment.

[0010] The fourth object of the present invention is to apply the tissue-specific promoter or promoter fragment and the recombinant expression vector containing the tissue-specific promoter or promoter fragment to constructing transgenic plants, improving crop traits, using seeds as bioreactors to express exogenous proteins or cultivating new plant varieties with excellent traits.

[0011] In order to achieve the above-mentioned purpose, the present invention provides the following technical means:

[0012] The first aspect of the present invention provides a DNA molecule, which is any one of the following A1) to A5):

[0013] A1) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1;

[0014] A2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 2;

[0015] A3) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 3;

[0016] A4) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 4;

[0017] A5) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.5.

[0018] The DNA molecule shown in SEQ ID NO. 1 is a prolamin gene PROLM26 promoter pPROLM26-1 isolated from rice, which can drive the target gene to be specifically and efficiently expressed in the endosperm cells of seed tissue in rice endosperm cells.

[0019] The present invention further removes part of the polynucleotide sequence shown in SEQ ID NO.1 from the 5' end according to the distribution position of the endosperm-specific promoter elements CAAT-box, TATA-box, RY-element, O2-site and GCN4-motif obtained by promoter element analysis, and obtains four truncated sequences ( Figure 1), each truncated sequence is connected to an expression vector carrying a reporter gene to verify whether the truncated sequence has endosperm-specific function; the present invention has determined through functional verification experiments that the sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 obtained by truncating the 5'-end nucleotide sequence of SEQ ID NO.1, i.e., the DNA molecules described in A2) to A5), can all drive the reporter gene to be efficiently expressed in the endosperm of rice seeds, indicating that the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, which are obtained by truncating the 5'-end nucleotide sequence of the sequence shown in SEQ ID NO.1, still have the function of a tissue-specific promoter.

[0020] The second aspect of the present invention provides a biological material containing the above-mentioned DNA molecule, which is any one of the following B1) to B4):

[0021] B1) an expression cassette containing the above-mentioned DNA molecule;

[0022] B2) a recombinant vector containing the aforementioned DNA molecule or a recombinant vector containing the expression cassette described in B1);

[0023] B3) a recombinant microorganism containing the aforementioned DNA molecule, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2);

[0024] B4) A transgenic plant cell line containing the aforementioned DNA molecule, or a transgenic plant cell line containing the expression cassette described in B1), or a transgenic plant cell line containing the recombinant vector described in B2).

[0025] The third aspect of the present invention provides the use of the above-mentioned DNA molecule as a promoter to initiate the specific expression of exogenous genes in seeds of seed plants.

[0026] The present technicians connected the nucleotide sequence of SEQ ID NO.1 and the nucleotide sequences of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, which are truncated from the 5' end of SEQ ID NO.1, to the reporter gene and found that the five promoters or promoter fragments all had the function of promoting the expression of the reporter gene in embryonic tissue, and the promoter activity did not decrease significantly.

[0027] The five promoters or promoter fragments are obtained by PCR amplification technology.

[0028] During the PCR amplification process, the following six pairs of primers were configured according to the DNA nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 for amplification:

[0029] Table 1 Primers used for fluorescence quantitative PCR

[0030]

[0031] The fourth aspect of the present invention provides the use of the DNA molecule in cultivating transgenic plants, wherein the transgenic plants are transgenic seed plants.

[0032] Furthermore, the transgenic seed plant specifically expresses exogenous genes in seeds.

[0033] Furthermore, the seeds of the transgenic seed plant are endosperm seeds, and the exogenous gene is specifically expressed in the endosperm of the seeds of the transgenic seed plant.

[0034] The fifth aspect of the present invention provides a method for cultivating transgenic plants, which is to introduce an exogenous gene expression cassette into a recipient seed plant to obtain a transgenic seed plant that specifically expresses the exogenous gene in the seed; in the exogenous gene expression cassette, the above-mentioned DNA molecule initiates the transcription of the exogenous gene.

[0035] Furthermore, the seeds of the transgenic seed plant are endosperm seeds, and the exogenous gene is specifically expressed in the endosperm of the seeds of the transgenic seed plant.

[0036] In an embodiment of the present invention, the DR5-RUBY plasmid vector sequence is used as a vector, and a rice endosperm-specific promoter or promoter fragment-reporter gene DNA double strand is constructed through enzyme digestion and ligation reactions. The plant expression vector is used to transform the target plant to complete genetic transformation.

[0037] Any of the above plants may be C1) or C2) or C3):

[0038] C1) Monocots;

[0039] C2) Grasses;

[0040] C3) Rice.

[0041] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0042] The five rice endosperm-specific promoters or promoter fragments provided by the present invention can be used or operated to drive the expression of target genes in rice endosperm tissue cells, thereby achieving the purpose of expressing target genes in specific rice endosperm tissue cells. In the molecular biology and synthetic biology breeding of agricultural production, this method can be used to use rice endosperm tissue cells as bioreactors for genetic modification and gene transformation to efficiently produce heterologous proteins in protein bodies or enhance the expression of self-proteins and other nutrients. They are often used in the production of products such as pharmaceutical proteins, antigens and vaccines, or in the cultivation of varieties with excellent traits such as high nutritional value and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The distribution of promoter elements on 5 promoters or promoter fragments is shown.

[0044] Figure 2 This is a map of the recombinant vector in which the pPROLM26-1 promoter is connected to the reporter gene RUBY: pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY and pPROLM26-5-RUBY were all digested and connected in this way to form recombinant vectors.

[0045] Figure 3 Double enzyme digestion verification of 5 recombinant vectors.

[0046] Figure 4 The plant phenotypes of wild type and five transgenic rice families are as follows: Figure 4 (a), (b), (c), (d), and (e) are the phenotypes of transgenic rice lines carrying five recombinant vectors, pPROLM26-1-RUBY, pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY, and pPROLM26-5-RUBY, respectively, and wild-type rice plants (the wild type is on the left, and the white scale is 20 cm).

[0047] Figure 5 Seed phenotypes of the reporter gene in five transgenic rice families: Figure 5 (a), (b), (c), (d), (e), and (f) are the phenotypes of seeds of transgenic rice families harbouring six recombinant vectors, namely pPROLM26-1-RUBY, pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY, pPROLM26-5-RUBY, and pGt13A-RUBY, and those of wild-type rice.

[0048] Figure 6This study was used to compare the betaine content in seeds of six transgenic rice families. DETAILED DESCRIPTION

[0049] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0050] Example 1: Molecular cloning of rice endosperm-specific promoters or promoter fragments pPROLM26-1, pPROLM26-2, pPROLM26-3, pPROLM26-4 and pPROLM26-5

[0051] 1. Select the DNA nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 as the rice endosperm-specific promoter pPROLM26-1, pPROLM26-2, pPROLM26-3, pPROLM26-4 and pPROLM26-5 sequences respectively;

[0052] 2. DNA nucleotide sequence amplification was performed according to the primer sequences shown in Table 1 to obtain five promoter fragments;

[0053] 3. Using the genomic DNA of rice variety Xiushui 11 as a template, obtain the target promoter or promoter fragment by amplification using the high-fidelity DNA polymerase KOD;

[0054] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter pPROLM26-1 were as follows: 1. 98°C for 5 min, 2. 98°C for 10 sec, 3. 59.2°C for 2 min, 4. 68°C for 45 sec, program 2-4 for 33 cycles, and then 68°C for 10 min.

[0055] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-2 were as follows: 1. 98°C for 5 min, 2. 98°C for 10 sec, 3. 55.9°C for 2 min, 4. 68°C for 45 sec, 33 cycles of parts 2-4, and then 68°C for 10 min.

[0056] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-3 were as follows: 1. 98°C for 5 min, 2. 98°C for 10 sec, 3. 57.5°C for 90 sec, 4. 68°C for 45 sec, 33 cycles of parts 2-4, and then 68°C for 10 min.

[0057] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-4 were as follows: 1. 98°C for 5 min, 2. 98°C for 10 sec, 3. 61°C for 90 sec, 4. 68°C for 45 sec, 33 cycles of parts 2-4, and then 68°C for 10 min.

[0058] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-5 were as follows: 1. 98°C for 5 min, 2. 98°C for 10 sec, 3. 60.2°C for 70 sec, 4. 68°C for 45 sec, 33 cycles of parts 2-4, and then 68°C for 10 min.

[0059] Example 2: Experiment on the specific expression of RUBY reporter gene driven by candidate promoter or promoter fragment in rice endosperm

[0060] Plant expression vector construction: In order to verify whether the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, which are truncated from the 5' end of SEQ ID NO.1, have tissue-specific promoter function, the five promoters or promoter fragments were combined with the modified DR5-RUBY plant expression vector to verify their promoter function ( Figure 2 To facilitate cloning, this experiment modified the DR5-RUBY plant expression vector. The DR5-RUBY plant expression vector and five candidate promoters or promoter fragments were double-digested with BamHⅠ and PstⅠ. The double-digested promoters or promoter fragments were then ligated with the DR5-RUBY plant expression vector to obtain a new recombinant expression vector. The newly obtained recombinant plant expression vector was double-digested and verified using the BamHⅠ and PstⅠ restriction sites ( Figure 3 The new plant expression vector was transformed into DH5α competent Escherichia coli, sequenced, and transformed into a rice variety (Zhonghua 11) via Agrobacterium-mediated plant genetic transformation vector to obtain a stably expressed transgenic line.

[0061] Identification of endosperm-specific promoter function in stably transformed rice: The obtained stably transformed T0 rice plants were used to identify transgenic components using RUBY-F / R primers. The identification primers are as follows:

[0062] RUBY-F: CCACATCCTCCACATTC (SEQ ID NO. 16);

[0063] RUBY-R: CGCCGTTCATCATCTT (SEQ ID NO. 17).

[0064] Transgenic plants that tested positive were planted individually in the experimental field at the Fuyang base of the China National Rice Research Institute. Rows were spaced 19.8 cm apart and plant spacing was 16.5 cm. All field trials were managed in accordance with the same production methods as those in the field. After generations of planting, the phenotypes of wild-type and different transgenic lines were examined. (For phenotypes, see [see phenotypes]). Figure 4 ), the results showed that compared with the wild type, the phenotype of the five transgenic families was the same as that of the wild type at maturity, but the endosperm appeared purple or bright red ( Figure 5 ).

[0065] Example 3: Determination of betaine content in RUBY reporter gene synthesis product

[0066] Transgenic lines with strict endosperm-specific expression were selected. After generations of cultivation, the betaine content of the wild type and five transgenic rice lines was determined.

[0067] Specifically, about 0.1 g of mature transgenic rice seeds were taken, completely crushed, and then 1.5 ml of ultrapure water was added to fully dissolve them. The seeds were centrifuged at 12,000 rpm at room temperature for 5 minutes, and the supernatant was filtered through a 0.22 mm sterile filter. 200 ml of the supernatant was transferred to an ELISA plate, and the OD value of each sample at a wavelength of 538 nm was measured using an ELISA reader. Betaine standard solutions with concentrations of 0.00625 mg / ml, 0.0125 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, and 0.4 mg / ml were prepared, the OD values ​​were measured at a wavelength of 538 nm, and a standard curve was drawn to calculate the betaine content per kilogram of rice.

[0068] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0069] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for initiating the specific expression of a foreign gene in rice endosperm, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.4, and the exogenous gene is RUBY Reporter gene.

2. A method for using a DNA molecule biomaterial to activate the specific expression of exogenous genes in rice endosperm, characterized in that: The exogenous gene is RUBY The reporter gene, the biological material of the DNA molecule, is characterized by being any one of the following B1) to B4): B1) an expression cassette containing the DNA molecule according to claim 1; B2) a recombinant vector containing the DNA molecule according to claim 1 or a recombinant vector containing the expression cassette according to B1); B3) a recombinant microorganism containing the DNA molecule of claim 1, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2); B4) A transgenic plant cell line containing the DNA molecule of claim 1, or a transgenic plant cell line containing the expression cassette of B1), or a transgenic plant cell line containing the recombinant vector of B2).

3. An application of a DNA molecule in cultivating transgenic rice, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.4; the transgenic rice specifically expresses the exogenous gene in the endosperm, and the exogenous gene is RUBY Reporter gene.

4. A method for cultivating transgenic rice, comprising introducing an exogenous gene expression cassette into rice to obtain transgenic rice that specifically expresses the exogenous gene in the endosperm; wherein the exogenous gene expression cassette comprises a DNA molecule as claimed in claim 1 that initiates transcription of the exogenous gene, wherein the exogenous gene is RUBY Reporter gene.

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