KR102703473B1 - A promoter for transgenic expression in alfalfa - Google Patents

Abstract

The present invention relates to a promoter for leaf-specific expression in plants, a vector comprising the promoter, and a transformant and a transformation method comprising the vector. The Arabidopsis thaliana promoter according to the present invention was proven to be a promoter capable of expression not only in Arabidopsis thaliana but also in alfalfa, a useful forage crop, and was also confirmed to be expressed in tobacco, another crop. This can be usefully used to regulate the expression of genes that are beneficial to the growth and development of plants or to enhance resistance to various environmental stresses by inducing expression specifically in the leaves of plants. Therefore, a promoter that is specifically expressed in leaves and the promoter can be usefully used to breed new high-value-added varieties having excellent quality or yield-increasing effects.

Description

{A PROMOTER FOR TRANSGENIC EXPRESSION IN ALFALFA}

The present invention relates to a leaf-specific promoter, and more specifically, to a promoter base sequence of the At5G13590 gene derived from Arabidopsis thaliana , a vector containing the promoter, and the ability of the promoter to be transformed and expressed in alfalfa and tobacco.

Crop molecular breeding can use all kinds of genes as materials, and it is a core technology that will lead the next generation of agriculture in that it can finely control the infinite breeding effects by reducing the existing breeding technology that was only possible at the genome level to the gene level. The cultivation area and quantity of genetically modified crops using this crop molecular breeding technology are continuously increasing worldwide, and the global market size of commercialized genetically modified new seeds increased from 4 billion dollars in 2002 to 10 billion dollars in 2010 (including seeds and technology fees), accounting for 15% of the total global seed market size of 30 billion dollars. Soybeans, corn, cotton, and rapeseed form the main markets. In order to maximize the effects of genetically modified crops, the expression of externally inserted genes must be finely controlled according to the developmental stage and tissue of the plant, and for this purpose, various promoters must be developed.

Accordingly, research on promoters that control the expression of plant genes has been conducted since the early 1980s. It was suggested that the promoter of the cauliflower mosaic virus could induce strong expression in all tissues of the plant, and the base sequence of the promoter was revealed, and strong expression in the plant was proven. After that, the CaMV35S (patent number: JP1993192172-A1) promoter became the most widely used universal promoter in plants.

Although the CaMV35S promoter is generally used for systemic expression, there are cases where tissue-specific or expression-time-specific promoters must be used depending on the functionality of the gene and the characteristics of the target trait. Accordingly, research on the expression of tissue-specific promoters that induce expression in a specific tissue of plants has been continuously conducted, and specifically, flower tissue-specific promoters (van der Meer et al., 1990, Plant Mol. Biol. 15: 95-109; Ruiz-Rivero and Prat, 1998, Plant Mol. Biol. 36: 639-648; Chiou and Y도, 2008, Plant Molecular Biology, 66: 379-388; Verdonk et al., 2008, Plant Biotechnology Journal 6: 694-701; Liu et al., 2011, Plant Cell Reports 30: 2187-2194), anther-specific promoters (van Tunen et al., 1990, Plant Cell 2: 393-401; Kato et al., 2010, Plant Molecular Biology Reports 28: 381-387), seed-specific promoters (Bustos et al., 1989, Plant Cell 1: 839-853; Kridl et al., 1991, Seed Sci Res, 1: 209-219; Washida et al., 1999, Welham and Domoney, 2000, Plant Sci. 159: 289-299; Plant Mol. Biol. 40: 1-12; Furtado et al., 2008, Plant Biotechnology Journal 6: 679-693; Chung et al., 2008, Plant Cell Reports, 27: 29-37) have been developed.

Leaves are plant organs that transpire and respire, and in particular, they photosynthesize and provide nutrients to the entire plant, so it is very important to keep these leaves healthy. Not only can they improve crop traits, but they can also be used to effectively control the expression of genes that are beneficial to the growth and development of plants or to enhance resistance to various environmental stresses. Therefore, there is a continuous demand for research on promoters that are specifically expressed in leaves and on inducing the expression of target genes using these promoters.

The present invention is intended to solve the above-mentioned needs, and an object of the present invention is to provide a leaf-specific promoter having a base sequence represented by SEQ ID NO: 1.

In addition, another object of the present invention is to provide an expression vector comprising the promoter, a transformant comprising the vector, and a composition for transformation.

In addition, another object of the present invention is to provide a primer set capable of amplifying the promoter.

In addition, another object of the present invention is to provide a composition for specifically expressing a gene in a leaf of a plant, comprising a promoter consisting of a base sequence represented by SEQ ID NO: 1, and a vector comprising the same.

In addition, another object of the present invention is a step of inserting a foreign gene into an expression vector including a promoter consisting of a base sequence represented by sequence number 1; and

The present invention provides a method for specifically expressing a foreign gene in a leaf of a transformed plant, comprising the step of transforming a plant with an expression vector into which the foreign gene has been inserted.

In order to achieve the above object, the present inventors used Arabidopsis TAIR/ATTED DB to clone the promoter region of the At5G13590 gene showing leaf-specific expression, thereby producing a plant leaf-specific expression promoter, and confirmed the activity of the promoter in a transformant using alfalfa ( Medicago sativa L. ), a perennial crop belonging to the legume family and cultivated as an important livestock feed crop in many countries around the world, thereby completing the present invention, and confirmed that it was also expressed in another crop, tobacco ( Nicotiana benthamiana ).

Hereinafter, the present invention will be described in more detail.

It consists of a base sequence represented by sequence number 1 and relates to a leaf-specific expression promoter.

The above promoter is a promoter capable of initiating transcription, and is expressed in response to abiotic and/or mechanical stress. In addition, a variant of the promoter sequence may be included within the scope of the present invention. The variant includes all base sequences having functional characteristics similar to the base sequence of SEQ ID NO: 1, although the base sequence is changed. Specifically, the promoter may include a base sequence having 70% or more, 80% or more, 90% or more, or 95% or more homology with the base sequence of SEQ ID NO: 1, respectively.

The percent sequence homology for the above polynucleotides is determined by comparing the two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region may contain additions or deletions (gaps) unlike the reference sequence for the optimal alignment of the two sequences (which does not contain additions or deletions).

The expression site of the above promoter can appear throughout the plant, and is expressed, for example, in flowers and stems.

The above promoter has a base sequence of SEQ ID NO: 1, and is characterized in that the base sequence is derived from 1784 positions before the translation initiation site of the coding sequence of the Arabidopsis At5G13590 gene.

[Sequence number 1]

ctttgttctttcacttcaactctttggtttttccgcaaatctaaaagtgttgcaaaatggttgatcatcaaaaccccataaacatatgggttgataatgggaataaattaattttgggttttggttgtatcggagaagtaggggtgagaccgagtctgaagggagagatagatacagacgaagcttttgttttgtt tgcaggagttagctgttactaaaattt tgcaactttcgtttttccctttatttctgtaacgcctgactctctctctctctttcttcttttcttttgtaactttgtgtttacggtaaaatatctggctcattttggtaaatgactaaattcttgatattatttttcaatattccacagtaatactaataaatgtaggagatatttgactggtcaatatagtagttgctctctttaaat gtggagctagctct tttcaccaaaaaatattttattagtcaaaacccattacggaaagaattcataaggcccattatcaaaaccatggaccagagcagaaaaagtaagtgagttaattccggattagtgtgaaattaaacctaggcatactaatcggattaagccggcatgaccacacccatatatttagtccggcgacggcgcaataaagtacagtatatactcaatctttatttattttttcactttagaaataaatata atttcttaattttcaataatcataagatttaatatttattttgtttgagtcctaactaattttggatgacaaaaaaaaaaaaaaacatcataattataaaaaaaatgaatggattttgttttgagacaaggaccacaactgtttagtcagcaaaacttaaatgagaacactaaatttaaacatatagacatataaccat gtaataatgattttcaaaaattctgaaaaactacagtcttatacaaatctttattataattcgctgaatcgattttataaatccgaatatttataaataattcttgaaaaagccgtgtaaaaaaaaaaaagggtggatgagtcaaactgtgttataaagaggcaaagttgctgctgcagagttgtgtggcccttgtcaaagtttagcatctctctctct ttctctgtgtagccttctcttcatatatttcatatctaaactgtactttttatttaattttcctggaaactgcatcatcatcctcatcatccattatcactcaaacctctccatcactcttccttctttttggtagattcttctctctcccctaacacctttctctttcttccctaaatctggatttcttattctctttcacacttattttgttttttgt cggtagatttg cgaacctctaattctttgattactcttctctctgctctgtgagtcttacgtaactagcttcgatcttgtttttacgtattggattcaatcctttttttggatacttttttcagatttttctccactaaccatattactttctgatttactttttttgtaatcttatcgttgacttcattttttttcttcttgattgt tcttcctttctataggggccttgttgatc tctggattcactgtttacttgcgtctattagggtctagttagatagataccttgtgggatttatgtttgtttcatctgaaatccatttgaatttgaattcttctttttgcctttttctctgctttataatgatattgatcttgtaggtgtgttagttggagttagcctgtaaaaaggtacaagagtgaagttttgatttttattagg accctcgaagttcaact

Furthermore, the present invention relates to an expression vector comprising the promoter, and an expression vector comprising the promoter and a gene operably linked thereto.

The above vector may preferably be a plant expression vector, and may include all types of vectors commonly used as plant expression vectors.

In the above expression vector, the promoter may be located in front of the operably linked gene. This may include a recombinant DNA containing the appropriate nucleic acid sequence essential for the gene to express the coding sequence, and may be, for example, an expression cassette component that functions as a unit for expressing a heterologous protein.

In an embodiment of the present invention, At5G13590 promoter:GUS was prepared by inserting the promoter of the present invention into a binary vector (pBI101) containing a GUS reporter gene, and the GUS reporter gene can be replaced with a foreign gene for another useful purpose.

In addition, the present invention relates to a transformant comprising the promoter or vector and a composition for transformation of a plant comprising the promoter or vector.

The above transformant may be a transformed plant transformed using the expression vector of the present invention. The plant is not limited in type as long as it is a plant capable of transformation by inserting a gene expression cassette containing a leaf-specific expression promoter or an expression vector containing a leaf-specific expression promoter. The plant may be any one selected from the group consisting of the plant itself, tissues, cells, and seeds.

The above plant cell can be any plant cell, and any selected from the group consisting of cultured cells, cultured tissues, cultured organs, and combinations thereof can be used.

The above plant tissue may be an undifferentiated or differentiated plant tissue, and includes, for example, roots, stems, leaves, pollen, seeds, cancer tissues, and various forms of cells used in culture, including single cells, protoplasts, shoots, and calli. In addition, the plant tissue includes those in planta, organ culture, tissue culture, or cell culture.

The above plant may be, for example, any one selected from the group consisting of food crops, vegetable crops, specialty crops, fruit trees, flowers, forage crops, and combinations thereof.

In one embodiment of the present invention, it was confirmed that the At5G13590 promoter of the present invention was expressed in alfalfa and tobacco (Figs. 2 and 4).

The composition for transformation may preferably include a culture of a microorganism containing the expression vector of the present invention. The culture may include any one selected from the group consisting of a culture product obtained by culturing a microorganism containing the vector of the present invention in a culture medium or culture solution, a supernatant, a concentrate, a dried product, and a combination thereof. In addition, the formulation is not limited and may be a liquid or a solid.

The composition for transformation may further include additional components used for transformation of a plant in addition to the promoter or expression vector of the present invention, and for example, may include adjuvants, excipients, carriers, etc. to improve transformation efficiency.

The method for inserting the promoter of the present invention or the expression vector containing it into a plant can use a method commonly used in the technical field of the present invention, and for example, a method selected from the group consisting of the Agrobacterium method, the osmotic pressure method, the electroporation method, the gene gun method, and a combination thereof can be used, but is not limited thereto. Preferably, the Agrobacterium method can be used.

A transformant transformed by the transformation method of the present invention can specifically express a gene in leaf tissue by the promoter of the present invention that specifically expresses a gene in a leaf, and by using this, it is possible to harvest a plant, produce useful proteins through expression of a target gene, etc.

In addition, the present invention provides a composition for selecting a plant transformed with an oligonucleotide of 2 to 50 nt capable of specifically hybridizing to a part of the base sequence represented by SEQ ID NO: 1 for amplifying a DNA fragment comprising the base sequence represented by SEQ ID NO: 1, and a promoter of the present invention comprising the same or a recombinant vector comprising the same; or a plant selection kit. The primer set relates to, for example, a primer set comprising a forward primer consisting of SEQ ID NO: 2 and a reverse primer consisting of SEQ ID NO: 3, and a plant selection kit comprising the primer set.

[Sequence number 2]

CAT TCTAGA CTT TGT TCT TTC ACT TCA ACT CTT TGG TTT TTC

[Sequence number 3]

CAT GGA TCC AGT TGA ACT TCG AGG GTC CTA ATA AAA ATC

The above DNA fragment may be represented by sequence number 1, and the base sequence of sequence number 1 is derived from the promoter of the Arabidopsis At5G13590 gene.

The primer set comprising the above sequence numbers 2 and 3 can amplify the leaf-specific expression promoter of the present invention. In addition, through these characteristics, a transformant comprising the leaf-specific expression promoter of the present invention can be selected.

In addition, the present invention can provide a composition for specifically expressing a gene in a leaf of a plant, comprising a promoter consisting of a base sequence represented by SEQ ID NO: 1, and any one of vectors comprising the same.

In addition, the present invention comprises a step of inserting a foreign gene into an expression vector including a promoter consisting of a base sequence represented by sequence number 1; and

A method for specifically expressing a foreign gene in a leaf of a transformed plant can be provided, comprising the step of transforming a plant with an expression vector into which the foreign gene has been inserted.

The Arabidopsis thaliana-derived leaf-specific expression promoter of the present invention can be usefully used in breeding new high value-added varieties having excellent quality or yield-increasing effects by inducing gene expression in plants.

Figure 1 is a schematic diagram of the pBI101 vector, which is an expression vector used for promoter cloning according to the present invention.
Figure 2 is a drawing confirming the expression pattern of the leaf-specific expression At5G13590 promoter according to the present invention in Arabidopsis thaliana.
Figure 3 shows the results of analyzing the expression of the leaf-specific At5G13590 promoter according to the present invention in alfalfa. The left figure is a positive control showing the expression of 35S promoter:GUS, the middle figure is a figure confirming the expression of At5G13590 promoter:GUS, and the right figure is a negative control showing the expression pattern of an empty vector without the promoter portion.
Figure 4 is a drawing confirming the expression of the leaf-specific expression At5G13590 promoter according to the present invention in tobacco.

The following examples are only intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

Example 1: Promoter cloning of the Arabidopsis At5G13590 gene

The region presumed to be the promoter of the above gene was estimated to be 1784 base pairs. First, the genomic DNA (gDNA) of wild Arabidopsis thaliana was extracted, and a primer represented by sequence 2,3 containing XbaI at the 5 ' end and BamHI at the 3 ' end was synthesized.

Phusion High-Fidelity DNA Polymerase (Phusion), a DNA polymerase having a proofreading function, using the above gDNA as a template and the above primer set The promoter region was amplified using DNA Polymerase, Thermo Scientific. And at this time, the PCR reaction was performed using Gene Amp^®Using PCR System 2700 (Perkin Elmer, USA), PCR was performed at 98°C for 1 minute of initial denaturation, followed by 30 cycles of 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 40 seconds.

The 1784 base pairs obtained from the PCR product were confirmed on an agarose gel, and the PCR product was cut at the end with XbaI and BamHI restriction enzymes and cloned into the XbaI and BamHI restriction enzyme recognition sites of the pBI101 vector containing the GUS reporter gene. Afterwards, E. coli colony-PCR was performed to confirm the insertion of the PCR product, and one of the clones was sequenced to confirm that the correct base sequence was cloned (see SEQ ID NO: 1).

Example 2: Transformation of Arabidopsis thaliana and transformation into crops (alfalfa, tobacco) with the At5G13590 promoter

The pBI101 vector used for cloning the At5G13590 promoter has a GUS (β-glucuronidase) reporter gene between the left board and the right board and is a binary vector that can be used to transform plants (see Figure 1).

This vector was transformed into Agrobacterium (GV3101) and then transformed into Arabidopsis thaliana by the flower dipping method (Clough et al., Plant J., 16(6): 735-743, 1998). Transformed Arabidopsis thaliana were selected on a medium containing the antibiotic kanamycin.

In addition, this vector was used to transform alfalfa and tobacco, which are crops, by injecting the vector into Agrobacterium (LBA4404) (agro-infiltration, Walter et al., Plant J., 40, 428-438, 2004).

Example 3: Histochemical staining analysis of transgenic plants

GUS activity was examined from each tissue of the selected transgenic plants using a histochemical staining method. Each tissue of the plant was immersed in a solution containing 1 mM X-Gluc (5-bromo-4-chloro-3-indoly-β-glucuronidase), 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100, and reacted at 37°C for 24 h. After removing the solution, 70% ethanol was added to remove chlorophyll for 2 h. This process was repeated three times, and then stored in 100% ethanol while observing. As a result, GUS staining was observed in the leaves, as shown in Figs. 2, 3, and 4. In addition, GUS staining was observed at the wounding site using tweezers or scissors. As shown in Figs. 2, 3, and 4, it was clearly confirmed that the expression of the inserted gene was induced in each leaf. Therefore, it was confirmed that the promoter of the present invention is active as a leaf-specific expression promoter in Arabidopsis thaliana and crops such as alfalfa and tobacco.

<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> A PROMOTER FOR TRANSGENIC EXPRESSION IN ALFALFA <130> P21U13C0947 <150> KR 10-2020-0135331 <151> 2020-10-19 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1784 <212> DNA <213> Arabidopsis thaliana <400> 1 ctttgttctt tcacttcaac tctttggttt ttccgcaaat ctaaaagtgt tgcaaaatgg 60 ttgatcatca aaaccccata aacatatggg ttgataatgg gaataaatta attttgggtt 120 ttggttgtat cggagaagta ggggtgagac cgagtctgaa gggagagata gatacagacg 180 aagcttttgt tttgtttgca ggagttagct gttactaaaa ttttgcaact ttcgtttttc 2 40 cctttatttc tgtaacgcct gactctctct ctctctttct tcttttcttt tgtaactttg 300 tgtttacggt aaaatatctg gctcatttg gtaaatgact aaattcttga tattattttt 360 caatattcca cagtaatact aataaatgta ggagatattt gactggtcaa tatagtagtt 420 gctctcttaa atgtggagct agctcttttc accaaaaaat attttattag tcaaaaccca 480 ttacggaaag aattcataag gcccattatc aaaaccatgg accagagcag aaaaagtaag 540 tgagttaatt ccggattagt gtgaaattaa acctaggcat actaat cgga ttaagccggc 600 atgaccacac ccatatattt agtccggcga cggcgcaata aagtacagta tatactcaat 660 ctttatttat ttttcacttt agaaataaat ataatttctt aattttcaat aatcataaga 720 tttaatattt attttgtttg agtcctaact aattttggat gacaaaaaaa aaaaaaaaca 780 tcataattat aaaaaaaatg aatggatttt gttttgagac aaggaccaca actgtttagt 840 cagcaaaact taaatgagaa cactaaattt aaacatatag acatataacc atgtaataat 900 gattttcaaa aattctgaaa aactacagtc ttatacaaat ctttattata attcgctgaa 960 tcgattttat aaatccgaat atttataaat aattcttgaa aaagccgtgt aaaaaaaaaaa 1020 aagggtggat gagtcaaact gtgttataaa gaggcaaagt tgctgctgca gagttgtgtg 1080 gcccttgtca aagtttagca tctctctctt tctctgtgta gccttctctt catatatttc 1140 atatctaaac tgtacttttt atttaatttt cctggaaact gcatcatcat cctcatcatc 1200 cattatcact caaacctctc catcactctt ccttcttttg gtagattctt ctctctcccc 1260 taacaccttt ctctttcttc cc taaatctg gatttcttat tctctttcac acttatttg 1320 tttttgtcgg tagatttgcg aacctctaat tctttgatta ctcttctctc tgctctgtga 1380 gtcttacgta actagcttcg atcttgtttt tacgtattgg attcaatcct tttttggata 1440 ctttttcaga tttttctcca ctaaccatat tactttctga tttacttttt ttgtaatctt 1500 atcgttgact tcattttttt cttcttgatt gttcttcctt tctatagggg ccttgttgat 1560 ctctggattc actgtttact tgcgtct att agggtctagt tagatagata ccttgtggga 1620 tttatgtttg tttcatctga aatccatttg aatttgaatt cttcttttgc ctttttctct 1680 gctttataat gatattgatc ttgtaggtgt gttagttgga gttagcctgt aaaaaggtac 1740 aagagtgaag ttttgatttt tattaggacc ctcgaagttc aact 1784 <210> 2 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> forward primer for At5G13590 promoter <400> 2 cattctagac tttgttcttt cacttcaact cttt ggtttt tc 42 <210> 3 < 211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for At5G13590 promoter <400> 3 catggatcca gttgaacttc gagggtccta ataaaaatc 39

Claims (6)

A leaf-specific expression promoter consisting of a base sequence represented by sequence number 1. An expression vector comprising the promoter of claim 1. An expression vector comprising the promoter of claim 1 and a gene operably linked thereto. A transformant comprising any one of the promoter of claim 1, the vector of claim 2, and the vector of claim 3. A composition for specifically expressing a gene in a leaf of a plant, comprising any one of the promoter of claim 1, the expression vector of claim 2, and the expression vector of claim 3. A step of inserting a foreign gene into the expression vector of the second clause; and
A method for specifically expressing a foreign gene in a leaf of a transformed plant, comprising the step of transforming a plant with an expression vector into which the foreign gene has been inserted.