Molecular clone of rice endosperm specific promoter pROLM24 and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly provides molecular cloning of a rice endosperm specific promoter pROLM24 and application of the rice endosperm specific promoter pROLM24. Three tissue-specific promoters with nucleotide sequences as shown in SEQ ID NO.1-3 are separated from a rice alcohol-soluble protein gene, the three tissue-specific promoters are fused with an RUBY visual marker, and a result shows that the content of betacyanin in an expression product is measured to be up to 2.9% of the dry weight of rice seeds. The method has very high application value in the field of plant synthetic biology. The invention further discloses application of the promoter 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] This invention belongs to the field of plant genetic engineering technology and relates to the molecular cloning and application of the rice endosperm-specific promoter pROLM24. Background Technology

[0002] In plant genetic engineering, constitutive promoters are widely used because they can drive the continuous expression of exogenous genes throughout the plant. However, their non-specific expression can lead to a waste of metabolic resources, and overexpression in certain tissues may disrupt the balance of endogenous genes, causing abnormal plant development and thus limiting the effectiveness of genetic improvement. In contrast, tissue-specific promoters can achieve precise spatiotemporal regulation, improving target traits while reducing side effects, making them particularly important in molecular design breeding of crops such as rice. However, the resources of such promoters are still relatively limited, and their functional diversity and tissue coverage are insufficient to support the synergistic improvement of multiple traits, thus restricting the refined application of gene editing in breeding.Furthermore, even with existing initiation systems, the accumulation level of exogenous proteins in seeds remains mostly at the microgram per gram of seed (µg / g) level (Zhu Q, Zeng D, Yu S, Cui C, Li J, Li H, Chen J, Zhang R, Zhao X, Chen L, Liu YG. From GoldenRice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm. Mol Plant. 2018 Dec 3;11(12):1440-1448;Liu X, Li Z, Ying J, Shu Y, Liu W, LiG, Chen L, Luo J, Wang S, Wang Y, Tong X, Huang J, Du H, Zhang J. Multi-geneengineering boosts oil content in rice grains. Plant Commun. 2024 Feb 12;5(2):100736;Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X. Oral immunization with transgenic rice seeds expressing VP2 protein of infectious Bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J. 2007 Sep;5(5):570-8;Ye R, Huang H, Yang Z, Chen T, Liu L, Li X, Chen H, LinY. Development of insect-resistant transgenic rice with Cry1C*-free endosperm. Pest Manag Sci. 2009 Sep;65(9):1015-20; Achieving efficient accumulation at the milligram per gram (mg / g) level is a key goal to overcome expression bottlenecks and promote applications such as seed bioreactors, and it is also a technical challenge that needs to be overcome in current crop genetic engineering.

[0003] Rice, as one of the world's most important food crops, has its grain endosperm as the main storage site for storage substances such as proteins and starches. Endosperm-specific promoters can drive the efficient and specific expression of exogenous genes in the endosperm, and have significant application value in crop quality improvement, nutrient fortification, and the development of plant bioreactors. Therefore, the discovery and identification of highly active endosperm-specific promoters has always been a research hotspot in the fields of plant genetic engineering and synthetic biology.

[0004] Gliadins are a major class of storage proteins in rice endosperm. Their encoding genes are typically highly expressed during the mid-to-late stages of endosperm development; therefore, their promoter regions are considered an important source for cloning endosperm-specific promoters. Existing research has systematically analyzed the rice gliadin gene family using bioinformatics methods. For example, Sun Hongzheng et al. conducted genome-wide identification and expression pattern analysis of the rice storage protein family, identifying several members of the gliadin gene family (such as...) PROLM22 , PROLM24 , PROLM25 , PROLM26 These genes exhibit high transcription levels in the endosperm, suggesting that their promoters may have the potential to serve as highly efficient endosperm-specific promoters (Sun Hongzheng, Guo Kai, Song Ningyuan, et al. Bioinformatics analysis of rice storage protein family [J]. Chinese Rice, 2018, 24(1):6.). However, this type of bioinformatics research is limited to prediction and endogenous expression profiling, exhibiting significant technical limitations: (1) No clear promoter sequence and cloning verification provided: Existing technology only provides gene expression data (RPKM value), without actually isolating or cloning the promoter sequence of the gene, nor experimentally verifying whether it has the activity of driving gene transcription. The function of the promoter must be confirmed by its ability to drive the expression of downstream genes, rather than inferred solely from the expression level of upstream genes.

[0005] (2) Lack of experimental evidence for the ability of exogenous genes to drive expression: Existing studies focus on the expression patterns of endogenous genes during natural development, without using reporter gene systems (such as GUS, GFP, or RUBY) to verify whether these hypothetical promoter fragments can drive the specific and efficient expression of exogenous genes in the endosperm. This is a crucial step in assessing whether a promoter element can be applied to genetic engineering.

[0006] (3) No promoter structure and function optimization: Even if a gene's upstream region may contain a promoter, its core functional region, minimum effective length, and whether it can still maintain activity after truncation optimization are all unknown. The length of the promoter and the arrangement of cis-acting elements directly affect its activity and specificity, which must be determined through systematic truncation mutations and functional experiments, and cannot be directly obtained through bioinformatics prediction.

[0007] It is worth noting that, based on the same family genes ( PROLM26 Previous research on the promoter of [the gene] was limited to the aforementioned bioinformatics prediction stage. Subsequently, through the inventive efforts of the inventors' team, the pPROLM26 promoter was successfully cloned experimentally. Further truncation and optimization yielded a fragment with higher activity and easier manipulation. Ultimately, the RUBY reporter system verified that it could drive the exogenous gene to achieve a high expression level of 2.307% of seed dry weight in the endosperm, thus obtaining patent rights (CN120350009B). This successful case precisely demonstrates the significant technological gap between "bioinformatics prediction" and "experimentally verified functional elements," requiring non-obvious inventive effort.

[0008] In this context, other members of the prolysin family (such as...) PROLM22 , PROLM24 , PROLM25 Existing technologies have only provided clues about the high endogenous expression levels of these genes, but have not provided any experimentally validated, sequence-defined promoter elements, nor have they demonstrated their ability to drive exogenous genes. Therefore, there is still an urgent need in the field to isolate highly efficient endosperm-specific promoters from these genes that have been experimentally cloned, sequenced, and functionally validated by exogenous reporter systems. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for obtaining rice gliadin genes. PROLM22 , PROLM24 and PROLM25 A novel promoter with a well-defined sequence was isolated from rice and its ability to drive efficient and specific expression of exogenous genes in rice endosperm was demonstrated using the RUBY reporter system, providing a new key tool for rice molecular breeding and the development of seed bioreactors.

[0010] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides three promoters isolated from rice: PROLM22 promoter pROLM22, PROLM24 promoter pROLM24, and PROLM25 promoter pROLM25, with nucleotide sequences of SEQ ID NO. 1 (2061 bp), SEQ ID NO. 2 (2079 bp), and SEQ ID NO. 3 (2062 bp), respectively. These rice endosperm-specific promoters can drive the specific and efficient expression of target genes in seed tissue endosperm cells within rice endosperm cells (see appendix). Figure 1 ).

[0011] The present invention further linked the nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 with reporter genes and found that all three promoters have the function of initiating reporter gene expression in embryonic tissues, and the activity of the promoters did not decrease significantly.

[0012] A second aspect of the present invention provides a biological material containing the above-described promoter, which is any one of A1) to A4) below: A1) Expression cassettes containing promoter pROLM22, promoter pROLM24, or promoter pROLM25; A2) A recombinant vector containing promoter pROLM22 or promoter pROLM24 or promoter pROLM25 or a recombinant vector containing the expression cassette described in A1); A3) Recombinant microorganisms containing promoter pROLM22 or promoter pROLM24 or promoter pROLM25, or recombinant microorganisms containing the expression cassette described in A1), or recombinant microorganisms containing the recombinant vector described in A2); A4) A transgenic plant cell line containing promoter pROLM22 or promoter pROLM24 or promoter pROLM25, or a transgenic plant cell line containing the expression cassette described in A1), or a transgenic plant cell line containing the recombinant vector described in A2).

[0013] The three promoters or promoter fragments were obtained by PCR amplification.

[0014] During the PCR amplification process, the following three pairs of primers were prepared according to the DNA nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 for amplification: Table 1. Amplification Primer Sequence List

[0015] The third aspect of the present invention provides any of the following applications of the above-mentioned three promoters, expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm; The exogenous gene is RUBY Reporter genes.

[0016] The application described in the preparation of genetically modified rice is to... RUBY Gene expression cassettes were introduced into rice to obtain transgenic rice; RUBYIn the gene expression cassette, the aforementioned promoter pROLM22, promoter pROLM24, or promoter pROLM25 drives the expression of the gene. RUBY Gene transcription.

[0017] The application involves using or manipulating the three promoters to drive the expression of target genes in rice endosperm tissue cells, thereby using endosperm cells as bioreactors to synthesize related proteins at the molecular biology or synthetic biology level.

[0018] The specific process of the application involves genetic manipulation of rice seeds, using the DR5-RUBY plasmid vector sequence as a vector, constructing a double-stranded DNA of the rice endosperm-specific promoter-reporter gene through enzyme digestion and ligation reactions, and then using this plant expression vector to transform the target plant to complete the genetic transformation.

[0019] The advantages of the technical solution provided by this invention compared with the prior art are as follows: (1) "Ultra-high efficiency expression": This is the most significant advancement. Experiments have demonstrated that the final product of the RUBY gene driven by pROLM24 is betaine at 2.9 mg / g, while the betaine obtained by driving RUBY with the existing technology pPROLM26-4 is 2.307 mg / g (CN120350009B), representing a 25.7% increase in transcription efficiency. The pROLM24 promoter can push the product accumulation of the exogenous reporter system to an extremely high level of 2.9% of the seed dry weight, solving the technical problem of insufficient promoter driving efficiency in existing technologies.

[0020] (2) "Driven by a multi-gene complex system": This reflects the complexity of the function. When verifying its function, this invention drives not a single gene, but the complex metabolic pathway of RUBY. This indicates that the promoter provided by this invention has a sufficiently strong driving ability to coordinate the operation of the entire biosynthetic pathway, solving the efficiency problem of driving complex genetic pathways.

[0021] (3) The three rice endosperm-specific promoters provided by this invention can be used or manipulated 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 to carry out transgenic and gene transformation to efficiently produce heterologous proteins or enhance the expression of self-proteins and other nutrients in protein body I. It is often used for the production of pharmaceutical proteins, antigens and vaccines or to cultivate varieties with high nutritional value, high yield and other excellent traits. Attached Figure Description

[0022] Figure 1 The cis-acting elements are distributed across the three promoters; Figure 2 A schematic diagram of a recombinant vector with three promoters and the reporter gene RUBY; Figure 3 Here are the enzyme digestion verification diagrams for the three recombinant vectors; Figure 4 Spider phenotypes of wild-type and three transgenic rice families: a, b, and c are the plant phenotypes of transgenic rice families with three recombinant vectors pPROLM22-RUBY, pPROLM24-RUBY, and pPROLM25-RUBY, respectively, and wild-type rice plants (the wild-type is on the far left, and the white scale bar is 20 cm). Figure 5 Seed phenotypes of the reporter gene in three transgenic rice families: a, b, c, and d are the seed phenotypes of four recombinant vector transgenic rice families (pPROLM22-RUBY, pPROLM24-RUBY, pPROLM25-RUBY, and pGt13A-RUBY) and wild-type rice seeds, respectively. Figure 6 The content of betalains in seeds from four transgenic rice families was determined by comparison. Detailed Implementation

[0023] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0024] Example 1: Molecular cloning of rice endosperm-specific promoters or promoter fragments pPROLM22, pPROLM24, and pPROLM25 1. Select the DNA nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 as the rice endosperm-specific promoter sequences pPROLM22, pPROLM24 and pPROLM25, respectively; 2. Amplify the DNA nucleotide sequence according to the primer sequences in Table 1; 3. Using the genomic DNA of rice variety Xiushui 11 as a template, the target promoter or promoter fragment was obtained by amplification with high-fidelity DNA polymerase KOD. The PCR amplification conditions for the rice endosperm-specific and heterologous promoter pPROLM22 nucleotide sequence were as follows: 1. 98 ℃ for 5 min, 2. 98 ℃ for 10 sec, 3. 58.2 ℃ for 2 min, 4. 68 ℃ for 45 sec, with 33 cycles of program 2-4, followed by 68 ℃ for 10 min. The PCR amplification conditions for the rice endosperm-specific and heterologous promoter fragment pPROLM24 nucleotide sequence were as follows: 1. 98 ℃ for 5 min, 98 ℃ for 10 sec, 3. 60.0 ℃ for 2 min, 4. 68 ℃ for 45 sec, with 33 cycles of program 2-4, followed by 68 ℃ for 10 min. The PCR amplification conditions for the rice endosperm-specific promoter fragment pPROLM25 nucleotide sequence were as follows: 1. 98 ℃ for 5 min, 98 ℃ for 10 sec, 3.5-5.5 ℃ for 90 sec, 4.68 ℃ for 45 sec, 33 cycles of parts 2-4, followed by 68 ℃ for 10 min.

[0025] Example 2: Experiment on the specific expression of the RUBY reporter gene in rice endosperm driven by candidate promoters or promoter fragments Construction of plant expression vector: To verify whether the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 possess tissue-specific promoter functions, the three promoters were coupled with the modified DR5-RUBY plant expression vector to verify their promoter functions (see appendix). Figure 2 To facilitate cloning, the DR5-RUBY plant expression vector was modified in this experiment. The DR5-RUBY plant expression vector and three candidate promoters were digested with HindIII. The digested promoters were then ligated into the DR5-RUBY plant expression vector to obtain a new recombinant expression vector. The newly obtained recombinant plant expression vector was verified by HindIII restriction site digestion (see attached). Figure 3 The obtained new plant expression vector was transformed into DH5α competent Escherichia coli cells, sequenced, and then transformed into rice variety (Zhonghua 11) using Agrobacterium-mediated plant genetic transformation vector to obtain a transgenic family with stable expression.

[0026] Identification of endosperm-specific promoter function in stably transformed rice: The obtained stably transformed T0 generation rice plants were used to identify transgenic components using RUBY-F / R primers. The primers for identification are as follows: RUBY-F: CCACATCCTCCACATTC (SEQ ID NO.4); RUBY-R: CGCCGTTCATCATCTT (SEQ ID NO. 5).

[0027] Transgenic plants that tested positive were planted in experimental fields at the Fuyang base of the China National Rice Research Institute. They were planted individually with a row spacing of 19.8 cm and a plant spacing of 16.5 cm. All field trials followed the same standardized management practices as large-scale field production. After generations of planting, phenotypic analysis was conducted on wild-type and different transgenic families. (See appendix for phenotypic details.) Figure 4 The results showed that, compared with the wild type, the phenotypes of the three transgenic families were no different from those of the wild type at maturity; however, the endosperm was purplish-red or bright red (see attached image). Figure 5 ).

[0028] Example 3: Determination of betalain content, a synthetic product of the RUBY reporter gene. Transgenic rice lines that strictly express endosperm-specific expression were selected, and after generations of planting, the betalain content of wild-type and three transgenic rice families was determined.

[0029] Specifically, about 0.1 g of mature transgenic rice seeds were completely crushed and dissolved in 1.5 ml of ultrapure water. The mixture was centrifuged at 12,000 rpm for 5 min at room temperature, 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. Standard solutions of betaine 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 plotted. The betaine content per kilogram of rice was then calculated.

[0030] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0031] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the rice endosperm-specific promoter pROLM24, characterized in that, The application is any one of the following: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm, wherein the exogenous gene is RUBY Reporter genes; The nucleotide sequence of the promoter pROLM24 is shown in SEQ ID NO.

2.

2. The application of an expression cassette containing the promoter pROLM24 as described in claim 1, characterized in that, The application is any one of the following: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm, wherein the exogenous gene is RUBY Reporter genes.

3. The use of a recombinant vector containing the promoter pROLM24 of claim 1 or the expression cassette of claim 2, characterized in that, The application is any one of the following: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm, wherein the exogenous gene is RUBY Reporter genes.

4. The use of a recombinant microorganism containing the promoter pROLM24 of claim 1, the expression cassette of claim 2, or the recombinant vector of claim 3, characterized in that, The application is any one of the following: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm, wherein the exogenous gene is RUBY Reporter genes.

5. The use of a transgenic plant cell line containing the promoter pROLM24 of claim 1, the expression cassette of claim 2, or the recombinant vector of claim 3, characterized in that, The application is any one of the following: 1) Application in the preparation of transgenic rice; 2) Application of driving the specific expression of exogenous genes in rice endosperm, wherein the exogenous gene is RUBY Reporter genes.

6. A method for cultivating transgenic rice, characterized in that, The method involves 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, wherein the foreign gene is... RUBY Reporter genes.

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