WO2019190130A1 - Hevea brasiliensis-derived, lactiferous tissue-specific pep16 gene promtor (ppep16) and use thereof - Google Patents

Abstract

The present invention relates to a Hevea brasiliensis-derived, lactiferous tissue-specific PEP16 gene promoter and a use thereof. The use of the Hevea brasiliensis)-derived PEP16 gene promoter of the present invention allows the attainment of a transgenic plant that can constitutively express a foreign gene specifically in the lactiferous tissue, thereby controlling biosynthesis processes and production yields of natural rubber in plants. When used to express a foreign gene in a lactiferous tissue-specific manner, a recombinant vector including the promoter of the present invention is expected to effectively produce valuable materials in addition to natural rubber.

Description

Latex-secreting tissue-specific PEP16 gene promoter derived from Para-rubber and its use

The present invention relates to latex secretory tissue specific PEP16 gene promoter (p PEP16 ) derived from para-rubber and its use.

A promoter is a genome region that is linked upstream of a gene, and regulates the transcription of the structural gene linked to mRNA. The expression of foreign genes (ie transgenes) introduced into the plant is greatly influenced by transcriptional, post-transcriptional, translational and posttranslational factors. In particular, promoters belonging to the transcriptional factor are the most important factors that can directly affect the transcription of foreign genes, resulting in altered levels of expression and altering the stage at which foreign genes are expressed, tissue or cell specificity. . To date, numerous promoters have been isolated from various plants for expression of foreign genes, but only a few of them are actually used for plant transformation.

Tissue specific promoters are promoters that allow genes to be expressed only in specific cells and can be used to limit gene expression to specific tissues or cells. Plant tissue specific expression of foreign useful genes is of great value in the research and horticultural industry because they can allow them to exhibit altered properties in plants.

Hevea brasiliensis , commonly known as the rubber tree (rubber tree), belongs to the family Euphorbiaceae and is the most economically important tree in the genus Hebea. Para-rubber is capable of producing large quantities of latex (latex), which is a major raw material of natural rubber, and is currently known as the only natural rubber resource available industrially. More than 90% of natural rubber is produced in Southeast Asia, including Malaysia, Indonesia, Thailand, and Myanmar. Korea uses more than 200,000 tons of natural rubber annually, depending on imports.

Synthesis of proteins is initiated through a transcription process in which genetic information encoded in DNA is transferred to mRNA. The transcription begins by binding an RNA polymerase to a promoter located upstream of the gene. All promoters have a consensus sequence at a certain position from the start of transcription, which is known to be important for promoter recognition and binding of RNA polymerase. Such promoters are one of the important factors in determining the production efficiency of recombinant protein.

Meanwhile, Korean Patent No. 1281068 discloses a latex secretion tissue-specific SRPP promoter and its use derived from Paragomu tree, and Korean Patent Application Publication No. 2016-0131964 discloses 'natural rubber polymerase gene and its use'. Although disclosed, the latex secretion tissue-specific PEP16 gene promoter (p PEP16 ) and its use as described in the present invention is not known at all.

The present invention is derived from the above requirements, and the present invention is to newly isolate the promoter (p PEP16 ) of the rubber polymerase candidate gene ( PEP16 ) expressing latex tissue specific expression in the para-rubber ( Hevea brasiliensis ). It is characterized by constant high expression, similar to the conventional SRPP gene promoter (p SRPP ) is a latex secretion tissue-specific promoter of para-rubber, p SRPP is usually expressed at low temperature but high expression at low temperature 5, the p PEP16 of the present invention is usually expressed high but low expression at low temperature, the two promoters are opposite in character, so it was strategically developed to be selectively usefully applied as needed.

In order to solve the above problems, the present invention provides a PEP16 gene promoter (p PEP16 ) derived from Para-rubber ( Hevea brasiliensis ), consisting of the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a recombinant plant expression vector comprising the promoter.

The present invention also provides a dicotyledonous plant and seed thereof transformed with the recombinant plant expression vector.

In addition, the present invention provides a method for expressing latex secretion tissue-specifically in a transgenic dicotyledonous plant comprising a foreign gene recombination in the recombinant plant expression vector.

In addition, the present invention provides a transformed dicotyledonous plant and seeds thereof, wherein the foreign gene produced by the above method is specifically expressed in latex secretory tissue.

By using the PEP16 gene promoter (p PEP16 ) derived from the para-rubber ( Hevea brasiliensis ) of the present invention, it is possible to obtain a transgenic plant that expresses foreign genes specifically in latex secretion tissues. The process and productivity increase and decrease can be controlled, which is very useful for the industry concerned. In addition, it is expected that the recombinant vector including the promoter of the present invention may be useful for producing useful substances other than natural rubber by expressing foreign genes specifically for latex tissue.

Figure 1 shows the nucleotide sequence of the PEP16 gene promoter (p PEP16 ) and the estimated cis-acting factor.

Figure 2 shows the results of confirming that the genes were stably inserted into genomic DNA in the Taraxacum koksaghyz plant lines transformed with the pGA3383 vector inserted with the p PEP16 :: GUS gene. (A) schematically shows the structure of the pGA3383 vector into which the p PEP16 :: GUS gene was used, which was used to prepare a Russian dandelion transformant. (B) p Genomic DNA was extracted from plants transformed with PEP16 :: GUS gene and PCR amplification of GUS and Hygromycin ( Hpt ) genes. (C) p Extracted RNA from T. koksaghyz plants transformed with PEP16 :: GUS gene and shows the result of RT-PCR analysis. Wild Russian Dandelion (Wild type; W) was used as a control.

3 shows tissue specific expression of GUS induced by the PEP16 gene promoter (p PEP16 ) in T. koksaghyz plants transformed with the p PEP16 :: GUS gene. (A) is a vertical section of root tissue, wild type and p PEP16 :: GUS transgenic Russian dandelion (p PEP16 :: GUS ), (B) wild type and p PEP16 :: The horizontal cross section of the root of the Russian dandelion plant (p PEP16 :: GUS ) transformed with the GUS gene, and (C) shows the GUS staining of latex secretions collected from the roots, wild type and p PEP16 :: Russian dandelion plants (p PEP16 :: GUS ) transformed with the GUS gene. Scale bars in (A) and (B) were 1.0 mm and 0.5 mm, respectively. sx, secondary xylem; sp, secondary phloem.

Figure 4 shows the results of observing the root tissue specific expression of GUS induced by the PEP16 gene promoter (p PEP16 ) in Russian Dandelion plants transformed with p PEP16 :: GUS gene under environmental stress conditions. (A) wild type, (B) p PEP16 :: GUS transgenic Russian dandelion control (Control), (C) salt stress treatment (NaCl), (D) 4 ℃ cold treatment (cold) , (E) shows that GUS expression changes after dark condition treatment (Dark) and after light treatment (Light). Scale bar = 1 mm. sx, secondary xylem; sp, secondary phloem; ck, cork; p, pith.

5 shows the results of observing the root tissue specific expression of GUS induced by the SRPP gene promoter (p SRPP ) in Russian dandelion plants transformed with p SRPP :: GUS gene under stress conditions. (A) GUS-stained images are shown in root cross section after light-treated (middle panel) and tapping-treated (right panel). Control (left panel) and tapping-treatment (right panel) were performed in dark conditions. Scale bar = 1 mm. (B) GUS-dyed image shows GUS-dyed image in cold treated root (right panel) cross section. Control (left panel) and cold treated plants were maintained at 12 h photoperiod. Scale bar = 1 mm. sp (secondary phloem), sx (secondary xylem), ck (cork), p (pith).

In order to achieve the object of the present invention, the present invention provides a PEP16 gene promoter (p PEP16 ) derived from the para rubber ( Hevea brasiliensis ) consisting of the nucleotide sequence of SEQ ID NO: 1. Preferably, the PEP16 gene promoter (p PEP16 ) may specifically express a specific gene in latex secreted tissue.

While CaMV35S promoter derived from Cauliflower mosaic virus, which is widely used, expresses genes introduced in whole tissues, the latex secretory tissue specific expression promoters of the present invention are specific for latex secreted tissues. Can be expressed as.

In addition, homologues of such promoter sequences are included within the scope of the present invention. Homologues are base sequences that vary in base sequence but have similar functional properties to the base sequence of SEQ ID NO: 1. Specifically, the promoter sequence has a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence of SEQ ID NO: 1 It may include.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant plant expression vector comprising the PEP16 gene promoter (p PEP16 ). Preferably, the recombinant plant expression vector may be prepared by operably linking a gene of interest encoding a protein of interest downstream of the PEP16 gene promoter (p PEP16 ). In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to heterologous DNA encoding a protein promotes the production of functional mRNA corresponding to the heterologous DNA.

The latex secretion tissue-specific expression vector of the present invention can be used as a transient expression vector capable of temporarily expressing in a plant into which a foreign gene has been introduced, and as a plant expression vector capable of permanently expressing a foreign gene in a introduced plant. Can be.

Binary vectors that can be used in the present invention can be any binary vector containing the right border (RB) and left border (LB) of the T-DNA capable of transforming plants in the presence of Ti plasmid of Agrobacterium tumefaciens . However, it is preferable to use pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA, pGA vector, and the like, which are frequently used in the art.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, and the like. It doesn't happen.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Terminator of the octopine gene, but is not limited thereto. With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The present invention also provides a dicotyledonous plant and seeds thereof transformed with the recombinant plant expression vector.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The "plant cells" used for plant transformation may be any plant cells. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, fruits, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

The present invention also specifically expresses the latex secretion tissue-specific in the transgenic dicotyledonous plant comprising the step of recombining the foreign gene into the recombinant plant expression vector and transforming the dicotyledonous plant with the recombinant plant expression vector. It provides a method to make it. Preferably, the foreign gene is a rubber polymerase, rubber biosynthesis related enzymes, interleukin, interferon, platelet induced growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin. It may encode a protein selected from the group consisting of, but is not limited thereto.

The foreign gene may be any gene desired to be expressed in a plant latex secretory tissue, and may be located after the promoter in the latex secretory tissue-specific expression vector of the present invention and may be expressed by fusion with a reporter gene as necessary. The recombinant latex secretion tissue specific expression vector may be transformed into a dicotyledonous plant as described above.

The present invention also provides a transformed dicotyledonous plant and seeds thereof, wherein the foreign gene produced by the above method is specifically expressed in latex secretory tissue. Preferably, the plant is one ssangjayeop plants such ssangjayeop plants in, for example, rubber tree (Hevea) is in a dandelion belongs to the counter electrode and (family Euphorbiaceae) plant or the Russian dandelion belongs (Taraxacum) with latex tissue May be, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 From Pararub Leaves PEP16  Gene promoter (p PEP16 ) Separation

Isolation of the PEP16 gene promoter was performed using the inverse PCR (iPCR) method (Ochman et al., 1988, Genetics 120: 621-623). Genomic DNA is extracted from the leaf tissue of the para-rubber ( Hevea brasiliensis ) according to a conventional method, and the extracted genomic DNA is treated with HincII, and then fragmented, followed by ligase to the fragmented genomic DNA. ) To self-ligation. Then, in the first intron of the first exon (exon) in the upstream region of Hinc II in the (intron) of PEP16 made the primer were carried out in both directions and then PCR. The primary PCR contains 5'-GATGATATCGAATGCAGAAGC-3 '(SEQ ID NO: 2), 5'-CAAGACATCCTTCGCCATGT-3' (SEQ ID NO: 3) and 5'-GATACTGCACCTTATCAACAC-3 '(SEQ ID NO: 4), 5'-CAGCATGGATTCGAAGCAAG-3' (SEQ ID NO: 5) A primary PCR product was prepared by combining primers. The diluted solution was used as a template, and the second PCR was performed again (5'-CTGAAAGTAATCAATCTGCAGC-3 '(SEQ ID NO: 6) and 5'-GGATCACTATGTTCATCATAG-3' (SEQ ID NO: 7)). The nucleotide sequence was confirmed by sequencing the second PCR product (FIG. 1).

Example 2. PEP16  Gene promoter (p PEP16 Construction of GUS Gene Binding Transfection Vector

Example 1 was designed primers for the nucleotide sequence obtained in the PEP16 gene promoter based (5'- GGATCC GTTATATCGAGGAATATGC-3 '(SEQ ID NO: 8), 5'-TAGTGGTAAGGTGTATAATATTTATC-3 ' ( SEQ ID NO: 9)), pGA3383 A recombinant vector (pGA3383-p PEP16 :: GUS ) was constructed to induce the expression of the marker gene GUS by inserting the PEP16 gene promoter into BamH1 / HpaI of the vector (FIG. 2A).

Example 3 Transformation of Latex Secretion Model Plants (Rushamine)

Para-rubber isolated from the PEP16 gene promoter (p PEP16 ) is not only difficult to transform but also requires several years to obtain results because it is an arborescent tree, so it is relatively easy to transform and has a short growth period. Russian dandelion was used for transformation. p PEP16 :: GUS vector was introduced into Agrobacterium strain LBA4404 and transformation of Russian dandelion was performed using the method described by Bae et al. (2005, Plant Cell, Tissue and Organ Culture 80: 51-57). p PEP16 :: GUS transformants were selected in selection medium and grown for 2-3 months in soil medium. Genomic DNA and total RNA were extracted and assayed again for transformation by PCR / RT-PCR analysis (FIGS. 2B and 2C). As a result, it was confirmed that the GUS gene is stably inserted into the genome and expressed in the transformant.

Example 4 Tissue Specific Expression of GUS in Transgenic Russian Dandelion

Expression of the marker gene GUS was assayed by blue staining produced by the activity of the GUS protein. Histological GUS (β-glucuronidase) staining was performed according to the previously described method (Jefferson et al., 1987, Plant Mol. Biol. Rep. 5: 387-405). While the activity of the GUS protein was clearly visible in the vertical cross-section of the roots of many latex secretion tissues of the P PEP16 :: GUS transformant (Fig. 3A right panel), the GUS activity was detected in the wild-type unmodified Russian Russian (Figure 3A left panel). The same result was obtained in root horizontal section and latex secretion, respectively (FIGS. 3B and 3C).

Example 5 Changes in GUS Expression According to Environmental Stress in Transgenic Russian Dandelion

The degree of blue staining indicates the degree of enzymatic activity of the GUS protein, and the degree of enzymatic activity of the GUS protein is a parameter indicating the degree of GUS gene expression. Changes in the degree of blue staining, ie GUS gene expression, in response to environmental stresses or signals were investigated. Compared to Russian dandelion wild root tissue (FIG. 4A), high GUS gene expression was observed in the root tissue of p PEP16 :: GUS transformed Russian dandelion (FIG. 4B). On the other hand, when the salt (NaCl), cold (Cold), or cancer treatment (Gark) GUS gene expression was significantly reduced (Fig. 4C-4E). GUS gene expression increased slightly when lighted again after cancer treatment (Figure 4E right panel). Also, p SRPP :: the observed results of the root tissue-specific expression of GUS driven by the SRPP gene promoter (p SRPP) from Russian Dandelion plants transformed with the GUS gene under stress conditions, p SRPP is the usual expression has mimihana The high expression at low temperature was confirmed (FIG. 5) (Tata et al., 2012 Industrial Crops and Products 40; 219-224).

Claims (9)

1. A latex secretion tissue-specific PEP16 gene promoter derived from Hevea brasiliensis , consisting of the nucleotide sequence of SEQ ID NO: 1.
2. A recombinant plant expression vector comprising the PEP16 gene promoter of claim 1.
3. The recombinant plant expression vector of claim 2, wherein the recombinant plant expression vector is prepared by operably linking a gene of interest encoding a protein of interest downstream of the promoter.
4. A dicotyledonous plant transformed with the recombinant plant expression vector of claim 3.
5. Transformed seeds of the dicotyledonous plant according to claim 4.
6. Recombining the foreign gene into the recombinant plant expression vector of claim 2; And

   A method for expressing a latex secretion tissue-specific expression in a transgenic dicotyledonous plant comprising the step of transforming the dicotyledonous plant with the recombinant plant expression vector.
7. The method of claim 6, wherein the foreign gene is a rubber polymerase, a rubber biosynthesis-related enzyme, interleukin, interferon, platelet-induced growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin A method for encoding a protein selected from the group consisting of.
8. A transgenic dicotyledonous plant in which a foreign gene produced by the method of claim 6 is specifically expressed in latex secretory tissue.
9. Transformed seeds of the dicotyledonous plant according to claim 8.

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