Rice endosperm tissue specific promoter pPROLM26 and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly provides a rice endosperm tissue specific promoter pPROLM26 and application thereof. According to the invention, a tissue-specific promoter with a nucleotide sequence as shown in SEQ ID NO.1 is separated from a rice alcohol-soluble protein gene, the tissue-specific promoter is cut off for four times, and then the cut-off promoter fragment with an endosperm-specific promoter function as shown in SEQ ID NO.2-5 is obtained after molecular identification is carried out on the cut-off promoter fragment and an RUBY visual marker; the betaine content of the expression product can reach 2.5% of the dry weight of the seeds through determination, and the method has very high application value in the field of plant synthetic biology. The invention further discloses application of the promoter or the truncated promoter fragment in the aspects of improving crop seed quality, improving crop traits, cultivating new varieties of transgenic plants by using key elements of the promoter or expressing high value-added proteins by using a seed bioreactor and the like.

Description

Technical Field

[0001] The present invention belongs to the field of isolation and application of tissue-specific promoters of plants, and relates to a rice endosperm tissue-specific promoter pPROLM26 and its application. Background Art

[0002] Rice (Oryza sativa. L) is an important food crop, and more than half of the regions in the world take rice as the staple food. By means of genetic transformation, an exogenous DNA fragment sequence, such as a functional gene for insect resistance, disease resistance, etc., is molecularly linked to a specific promoter, so as to achieve the expression of the exogenous functional gene in a host cell, and achieve the purpose of improving plant traits. For some functional genes, the expression level in a specific part can be precisely regulated by using a tissue-specific promoter, so as to achieve fine control of gene expression.

[0003] A promoter, as a DNA sequence specifically recognized and bound by RNA polymerase, is a key cis-acting element, usually located in the 5' domain of a structural gene. In higher plants, the regulation of gene expression mainly occurs at the transcriptional level, and the promoter plays a core role in controlling the starting time and expression level of gene expression, and determines the type of RNA polymerase involved in transcription. Therefore, the promoter is a key element for analyzing gene expression patterns and transcriptional regulation mechanisms, and constitutes the core of the plant gene transcriptional regulation system. According to its transcriptional activity and functional characteristics, promoters can be divided into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. Recent studies have also revealed the existence of bidirectional promoters. Currently, in the practice of plant genetic engineering, constitutive promoters are widely used, and they can drive the high-level expression of exogenous proteins in various tissues of plants. However, such promoters cannot achieve precise regulation of the spatio-temporal expression of target genes, which may lead to excessive consumption of intracellular resources and energy; moreover, the over-expression of some constitutive promoters in specific tissues may interfere with the normal growth and development of plants, thus becoming an obstacle in the process of crop improvement. In contrast, tissue-specific promoters can achieve concentrated expression at specific spatial positions and time points. With the continuous development of transgenic technology and the wide application of transgenic crops, the importance of promoters in crop variety improvement has become increasingly prominent. Especially in the research field of tissue-specific promoters, tissue-specific promoters have received more attention, so they have special significance for molecular improvement and production of crops such as rice. However, at present, the genetic resources of tissue-specific promoters are relatively limited, which to a certain extent restricts the ability to finely improve different traits of crops.

[0004] The gene transcription process regulated by tissue-specific promoters generally occurs only in certain specific tissues or organs. Tissue-specific expression promoters can regulate the expression of heterologous genes more economically and efficiently, specifically acting at specific sites where needed. This can not only increase the expression abundance of heterologous genes but also minimize biological energy consumption, thus not affecting the normal growth of plants.

[0005] Rice is a food crop with a wide planting area and a model crop that has been widely studied. It has a long cultivation history, and its cultivation techniques and genetic transformation techniques are stable and mature. Rice is also a strictly self-pollinating crop with good biosafety. Compared with other species, rice as a host for bioreactors also has some other unique advantages, such as the efficient expression of small molecule polypeptides and a simple protein purification process. Rice endosperm is not only an ideal site for producing recombinant proteins but also an important target component for improving food nutritional value. Developing endosperm-specific highly efficient promoters is the main means to obtain recombinant proteins from endosperm and improve nutritional components. It not only helps to clarify basic theories such as rice morphology, development, and metabolic pathways but also can guide transgenic breeding, creating huge economic and social benefits and better serving human production and life.

[0006] The RUBY reporter system has significant advantages compared with traditional reporter systems (GUS, GFFP, and Luc) and similar visual markers (RFP). First, the RUBY reporter system uses the common amino acid tyrosine in plants as a reaction substrate and achieves color marking by synthesizing red betaine, which can be visually recognized without using a fluorescence microscope, special substrate treatment, or chemical staining. At the same time, RUBY supports in-situ observation without damaging plant tissues, avoiding mechanical damage interfering with gene expression. The red betaine has a significant contrast in plant tissues (especially green leaves), while the green of GFP is easily interfered by chlorophyll, and the blue product of GUS has a lower recognition rate in a complex background. Red betaine is a natural plant product (such as beet and pitaya pigments), which is non-toxic, stable, not easily degraded, or interfere with plant physiology; while the overexpression of fluorescent proteins may inhibit plant growth, and the activity of GUS enzyme is easily affected by the environment. Summary of the Invention

[0007] One of the objectives of the present invention is to provide a DNA molecule with promoter activity, enabling the target gene to be highly specifically expressed in rice endosperm.

[0008] Another objective of the present invention is to provide a tissue-specific promoter isolated from rice or a promoter fragment that still has promoter function after being truncated in segments.

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

[0010] A 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 aspects such as constructing transgenic plants, improving crop traits, using seeds as bioreactors to express foreign proteins, or cultivating new plant varieties with excellent traits.

[0011] To achieve the above object, the present invention provides the following technical means: The first aspect of the present invention provides a DNA molecule, which is any one of the following A1) to A5): A1) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1; A2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.2; A3) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.3; A4) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.4; A5) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.5.

[0012] The DNA molecule as shown in SEQ ID NO.1 is a prolamin gene PROLM26 promoter pPROLM26-1 isolated from rice, which can drive a target gene to specifically and highly express in the endosperm cells of seeds in rice endosperm cells.

[0013] The present invention further gradually deletes part of the sequence of the polynucleotide sequence shown in SEQ ID NO.1 from the 5'-end according to the distribution positions of the endosperm-specific promoter elements CAAT-box, TATA-box, RY-element, O2-site, and GCN4-motif obtained through promoter element analysis to obtain 4 truncated sequences ( Figure 1 ) and respectively connects each truncated sequence to an expression vector with a reporter gene to verify whether the truncated sequence has the function of endosperm specificity; through functional verification experiments, the present invention determines that after truncating the 5'-end nucleotide sequence of SEQ ID NO.1, sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 are obtained, that is, the DNA molecules described in A2)-A5) can all drive the reporter gene to highly express in the rice seed endosperm, indicating that the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 after truncating the 5'-end nucleotide sequence of the sequence shown in SEQ ID NO.1 still have the function of a tissue-specific promoter.

[0014] In the second aspect of the present invention, there is provided a biological material containing the above DNA molecule, which is any one of the following B1) to B4): B1) An expression cassette containing the above DNA molecule; B2) A recombinant vector containing the above DNA molecule or a recombinant vector containing the expression cassette described in B1); B3) A recombinant microorganism containing the above DNA molecule, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2); B4) A transgenic plant cell line containing the above 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).

[0015] In the third aspect of the present invention, there is provided the use of the above DNA molecule as a promoter to specifically express a foreign gene in the seeds of seed plants.

[0016] The technical personnel of the present invention ligated 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 obtained by truncating the 5'-terminal nucleotide sequence of SEQ ID NO.1 to a reporter gene, and found that all 5 promoters or promoter fragments had the function of promoting the expression of the reporter gene in the embryo tissue, and the activity of the promoter did not decrease significantly.

[0017] The above 5 promoters or promoter fragments were obtained by PCR amplification technology.

[0018] During the PCR amplification process, the following 6 pairs of primers were configured for amplification 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: Table 1 Primers for fluorescence quantitative PCR

[0019] In the fourth aspect of the present invention, there is provided the use of the above DNA molecule in cultivating transgenic plants, and the transgenic plants are transgenic seed plants.

[0020] Furthermore, the transgenic seed plants specifically express a foreign gene in the seeds.

[0021] Furthermore, the seeds of the transgenic seed plants are endospermic seeds, and the foreign gene is specifically expressed in the endosperm of the seeds of the transgenic seed plants.

[0022] 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 seeds; in the exogenous gene expression cassette, the above DNA molecule initiates the transcription of the exogenous gene.

[0023] Further, the seeds of the transgenic seed plant are endospermic seeds, and the exogenous gene is specifically expressed in the endosperm of the seeds of the transgenic seed plant.

[0024] In the embodiments of the present invention, the DR5-RUBY plasmid vector sequence is used as a vector, and a DNA double strand of a rice endosperm-specific promoter or promoter fragment - reporter gene is constructed through restriction enzyme digestion and ligation reactions, and the target plant is transformed with this plant expression vector to complete genetic transformation.

[0025] Any of the above plants may be C1) or C2) or C3): C1) Monocotyledonous plants; C2) Gramineous plants; C3) Rice.

[0026] The advantages of the technical solution provided by the present invention compared with the prior art are as follows: The five rice endosperm-specific promoters or promoter fragments provided by the present invention can utilize or operate the rice endosperm-specific promoter or promoter fragment to drive the target gene to be expressed in the rice endosperm tissue cells, so as to achieve the purpose of expressing the target gene in specific rice endosperm tissue cells. In the molecular biology and synthetic biology level breeding in agricultural production, the rice endosperm tissue cells can be used as a bioreactor in this way for transgenic and gene transformation to efficiently produce heterologous proteins in protein bodies or enhance the expression of their own proteins and other nutrients, which are commonly used in the production of products such as medicinal proteins, antigens, and vaccines, or in cultivating varieties with excellent traits such as high nutritional value and high yield. Description of the Drawings

[0027] Figure 1 It shows the distribution of promoter elements on 5 promoters or promoter fragments.

[0028] Figure 2 It is the map of the recombinant vector with the pPROLM26-1 promoter linked to the reporter gene RUBY: pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY, and pPROLM26-5-RUBY are all digested and ligated in this way to form recombinant vectors.

[0029] Figure 3 It is the double digestion verification of 5 recombinant vectors.

[0030] Figure 4 Plant phenotypes of wild type and five transgenic rice lines: Figure 4 In (a), (b), (c), (d), and (e) of [figure], they are the plant phenotypes of transgenic rice lines of five recombinant vectors, namely pPROLM26-1-RUBY, pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY, and pPROLM26-5-RUBY, and wild type rice plants (the leftmost one is the wild type, and the white scale bar is 20 cm).

[0031] Figure 5 Seed phenotypes of the reporter gene in five transgenic rice lines: Figure 5 In (a), (b), (c), (d), (e), and (f) of [figure], they are the seed phenotypes of transgenic rice lines of six recombinant vectors, namely pPROLM26-1-RUBY, pPROLM26-2-RUBY, pPROLM26-3-RUBY, pPROLM26-4-RUBY, pPROLM26-5-RUBY, and pGt13A-RUBY, and wild type rice seeds.

[0032] Figure 6 Comparative determination of betaine content in seeds of six transgenic rice lines. Detailed implementation manners

[0033] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions created using the concept of the present invention are within the scope of protection.

[0034] Example 1: Molecular cloning of rice endosperm-specific promoters or promoter fragments pPROLM26-1, pPROLM26-2, pPROLM26-3, pPROLM26-4, and pPROLM26-5 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 sequences of rice endosperm-specific promoters pPROLM26-1, pPROLM26-2, pPROLM26-3, pPROLM26-4, and pPROLM26-5 respectively; 2. Perform DNA nucleotide sequence amplification according to the primer sequences shown in Table 1 to obtain five promoter fragments; 3. Using the genomic DNA of rice variety Xiushui 11 as a template, the target promoter or promoter fragment was amplified by high-fidelity DNA polymerase KOD. The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter pPROLM26-1 are 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. The program of steps 2-4 was cycled 33 times, and then 68 °C for 10 min.

[0035] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-2 are 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. The program of steps 2-4 was partially cycled 33 times, and then 68 °C for 10 min.

[0036] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-3 are 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. The program of steps 2-4 was partially cycled 33 times, and then 68 °C for 10 min.

[0037] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-4 are 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. The program of steps 2-4 was partially cycled 33 times, and then 68 °C for 10 min.

[0038] The PCR amplification conditions for the nucleotide sequence of the rice endosperm-specific promoter fragment pPROLM26-5 are 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. The program of steps 2-4 was partially cycled 33 times, and then 68 °C for 10 min.

[0039] Example 2: Experiment on the specific expression of the RUBY reporter gene driven by the candidate promoter or promoter fragment in rice endosperm Construction of plant expression vector: 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 the 5'-terminal nucleotide sequences of SEQ ID NO.1 truncated, have the function of tissue-specific promoters, the 5 promoters or promoter fragments were used to verify their promoter functions with the modified DR5-RUBY plant expression vector (Figure 2 ). For the convenience of cloning, in this experiment, the DR5-RUBY plant expression vector was modified. The DR5-RUBY plant expression vector and 5 candidate promoters or promoter fragments were double digested with BamHⅠ and PstⅠ, and then the digested promoters or promoter fragments and the DR5-RUBY plant expression vector were ligated to obtain a new recombinant expression vector. The newly obtained recombinant plant expression vector was verified by double digestion using the BamHⅠ and PstⅠ restriction sites ( Figure 3 ). The obtained new plant expression vector was transformed into competent DH5α Escherichia coli, sequenced, and a stably expressed transgenic family was obtained by Agrobacterium-mediated plant genetic transformation of the rice variety (Zhonghua 11).

[0040] Identification of the function of the endosperm-specific promoter in stably transformed rice: The stably transformed T0 generation rice plants were identified for transgenic components using the RUBY-F / R primers. The identification primers are as follows: RUBY-F: CCACATCCTCCACATTC (SEQ ID NO.16); RUBY-R: CGCCGTTCATCATCTT (SEQ ID NO.17).

[0041] The transgenic plants with positive identification results obtained from the experimental field of the China National Rice Research Institute in Fuyang were planted individually with a row spacing of 19.8 cm and a plant spacing of 16.5 cm. All field trials were uniformly managed following the production methods of the large field. After generations of planting, the phenotypes of the wild type and different transgenic families were investigated. (See the phenotype in Figure 4 ). The results showed that compared with the wild type, the phenotypes of the 5 transgenic family plants were the same as those of the wild type at the mature stage, but the endosperm showed purple-red or bright red ( Figure 5 ).

[0042] Example 3: Determination of the betaine content of the RUBY reporter gene synthesis product Select transgenic lines with strict endosperm-specific expression. After generations of planting, the betaine content of the wild type and 5 transgenic rice families was determined.

[0043] Specifically, take about 0.1 g of mature transgenic rice seeds, completely pulverize them, add 1.5 ml of ultrapure water and fully dissolve, centrifuge at 12,000 rpm for 5 min at room temperature, and filter the supernatant with a 0.22 mm sterile filter. Take 200 μl of the supernatant into an ELISA plate, and use an ELISA reader to measure the OD value of each sample at a wavelength of 538 nm. Prepare 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 respectively, measure the OD value at a wavelength of 538 nm and draw a standard curve, and then calculate the betaine content per kilogram of rice.

[0044] The various embodiments in this specification are described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0045] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a DNA molecule in initiating specific expression of a foreign gene in rice endosperm, characterized in that, The DNA molecule is any one of the following A1) to A5): A1) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.1; A2) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.2; A3) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3; A4) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.4; A5) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.5; Wherein, the DNA molecule shown in SEQ ID NO.2 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.3 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.4 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.5 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.

1.

2. Use of a biological material of a DNA molecule in initiating specific expression of a foreign gene in rice endosperm, characterized in that, The biological material of the DNA molecule is characterized in that it is any one of the following B1) to B4): B1) An expression cassette containing the DNA molecule recited in claim 1; B2) A recombinant vector containing the DNA molecule recited in claim 1 or a recombinant vector containing the expression cassette recited in B1); B3) A recombinant microorganism containing the DNA molecule recited in claim 1 or a recombinant microorganism containing the expression cassette recited in B1), or a recombinant microorganism containing the recombinant vector recited in B2); B4) A transgenic plant cell line containing the DNA molecule recited in claim 1, or a transgenic plant cell line containing the expression cassette recited in B1), or a transgenic plant cell line containing the recombinant vector recited in B2).

3. Use of a DNA molecule in cultivating transgenic rice, characterized in that, The DNA molecule is any one of the following A1) to A5): A1) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.1; A2) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.2; A3) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.3; A4) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.4; A5) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.5; Wherein, the DNA molecule shown in SEQ ID NO.2 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.3 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.4 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.1; The DNA molecule shown in SEQ ID NO.5 is a DNA fragment obtained by truncating the DNA molecule shown in SEQ ID NO.

1.

4. The application according to claim 3, wherein The transgenic rice specifically expresses foreign genes in the endosperm.

5. A method for cultivating transgenic rice, which comprises introducing a foreign gene expression cassette into rice to obtain transgenic rice that specifically expresses the foreign gene in the endosperm; in the foreign gene expression cassette, the DNA molecule described in claim 1 initiates the transcription of the foreign gene.

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