CN110760524B - A specific DNA fragment com58276 and its application in regulating plant stress resistance - Google Patents

Abstract

The invention discloses a specific DNA fragment com58276 and application thereof in regulating and controlling plant stress resistance. The nucleotide sequence of the specific DNA fragment com58276 is shown as SEQ ID NO: 1 is shown. Experiments prove that the specific DNA fragment com58276 is introduced into the wild type Arabidopsis thaliana Columbia-0 subtype to obtain transgenic Arabidopsis thaliana; compared with the wild type Arabidopsis thaliana Columbia-0 subtype, the drought resistance of the transgenic Arabidopsis thaliana is improved, and the improvement of the drought resistance is reflected by the reduction of the malonaldehyde content, the increase of the soluble sugar content, the reduction of the water loss rate and the improvement of the survival rate. Therefore, the specific DNA fragment com58276 can regulate the plant stress resistance. The invention has important application value.

Description

A specific DNA fragment com58276 and its application in regulating plant stress resistance

technical field

The invention belongs to the field of biotechnology, and specifically relates to a specific DNA fragment com58276 and its application in regulating plant stress resistance.

Background technique

Caragana caragana is a mesquite that grows mainly in desert or semi-desert areas in sandy land. It has a highly developed root system, strong drought tolerance and cold tolerance. Caragana is not only an important forage and industrial raw material, but also can maintain water and soil, prevent soil desertification, and has important economic and ecological value (Fang X W, Li J H, Xiong Y C, Xu D H, Fan X W, Li F M. Responses of Caraganakorshinskii Kom. to shoot removal: mechanisms underlying regrowth [J]. Ecological Research, 2007, 23(5): 863-871.).

Up to now, the research on Caragana caragana mainly focuses on the biological characteristics, physiological changes and anatomical structure, etc., while the research on stress-related genes and stress-related fragments is very little. The CkWRKY1 gene is a gene isolated from Caragana caragana, and the encoded CkWRKY1 protein belongs to the WRKY transcription factor family. Studies have shown that CkWRKY1 protein is a stress resistance-related protein and plays an important role in the response of Caragana caragana to adversity stress (Yang Q, Yin J J, Li G, Qi L W, YangF Y, Wang R G, Li G J. Reference gene selection for qRT-PCR in Caraganakorshinskii Kom. under different stress conditions [J]. Molecular Biology Reports, 2014, 41(4): 2325-2334.).

SUMMARY OF THE INVENTION

The object of the present invention is to improve the stress resistance of plants, such as drought resistance.

The present invention firstly protects a specific DNA molecule whose nucleotide sequence can be shown as SEQ ID NO:1.

Expression cassettes containing the specific DNA molecules also belong to the protection scope of the present invention.

The expression cassette (from 5' to 3') may include a promoter region, a transcription initiation region, a region of the gene of interest (containing the specific DNA molecule), a transcription termination region and optionally a translation termination region. The promoter region and the target gene region can be natural/similar to the host, or the promoter region and the target gene region can be natural/similar to each other, or the promoter region and/or the gene region of interest is heterologous to the host or to each other. As used herein, "heterologous" refers to a sequence that is derived from a foreign species, or, if from the same species, has been substantially altered in composition and/or genomic locus from the native form by deliberate human intervention retouch. The optionally included transcription termination region may be homologous to the transcription initiation region, homologous to the operably linked region of the gene of interest, homologous to the plant host; or; the gene region of interest, the host, is foreign or heterologous. Transcription termination regions can be derived from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

The expression cassette may also include a 5' leader sequence. The 5' leader sequence enhances translation.

In preparing the expression cassette, adapters or linkers may be used to ligate specific DNA molecules, or, other manipulations may be involved to provide appropriate restriction sites, remove excess DNA, remove restriction sites, and the like. To this end, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, re-substitutions such as transitions and transversions can be performed.

The expression cassette may also include selectable marker genes for screening transformed cells or tissues. Selectable marker genes can be used to screen transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT), providing herbicidal compounds such as glufosinate, 2,4-D ) resistance genes. Other selectable markers include phenotypic markers such as fluorescent proteins. The selectable markers listed above are not limiting. Any selectable marker gene can be used in the present invention.

The recombinant plasmid containing the specific DNA molecule also belongs to the protection scope of the present invention. The recombinant plasmid may be a recombinant plasmid obtained by inserting the specific DNA molecule into the starting plasmid. Specifically, the recombinant plasmid can be a recombinant plasmid obtained by inserting the specific DNA molecule into the multiple cloning site of the starting plasmid.

A selectable marker gene for screening transformed cells may be included on the starting plasmid. Selectable marker genes can be used to screen transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT), providing herbicidal compounds such as glufosinate, 2,4-D ) resistance genes. Other selectable markers include phenotypic markers such as fluorescent proteins. The selectable markers listed above are not limiting. Any selectable marker gene can be used in the present invention.

The recombinant plasmid may include any of the above-mentioned expression cassettes containing the specific DNA molecule.

The recombinant plasmid can specifically be the recombinant plasmid pBinGlyRed3-com58276. The recombinant plasmid pBinGlyRed3-com58276 is to replace the small DNA fragment between the restriction endonuclease EcoRI and XmaI recognition sequences of the pBinGlyRed3 vector with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1 (shown in SEQ ID NO: 1). The DNA molecule is the specific DNA fragment com58276), the obtained recombinant plasmid.

Recombinant microorganisms containing the specific DNA molecules also belong to the protection scope of the present invention.

The recombinant microorganism can be obtained by introducing the recombinant plasmid into the starting microorganism.

The starting microorganism may be bacteria, yeast, algae or fungi. The bacteria can be Gram-positive bacteria or Gram-negative bacteria. The gram-negative bacteria may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens can specifically be Agrobacterium tumefaciens EHA105.

The recombinant microorganism can specifically be EHA105/pBinGlyRed3-com58276. EHA105/pBinGlyRed3-com5827 is to introduce the recombinant plasmid pBinGlyRed3-com58276 into Agrobacterium tumefaciens EHA105 to obtain a recombinant Agrobacterium.

Transgenic cell lines containing the specific DNA molecules also belong to the protection scope of the present invention.

None of the transgenic cell lines containing the specific DNA molecule included propagation material. The transgenic plant is understood to include not only the first generation of transgenic plants obtained by transforming the recipient plant with the specific DNA molecule, but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

The present invention also protects the application of the specific DNA molecule, which can be A1) or A2):

A1) Regulate plant stress resistance;

A2) Breeding of transgenic plants with altered stress resistance.

In the above application, the regulation of plant stress resistance may be to improve plant stress resistance.

In the above application, the cultivating transgenic plants with altered stress resistance may be cultivating transgenic plants with increased stress resistance.

The present invention also protects a method for cultivating a transgenic plant, which may include the following steps: introducing the specific DNA molecule into the starting plant to obtain a transgenic plant; compared with the starting plant, the stress resistance of the transgenic plant is improved.

In the above method, the "introducing a specific DNA molecule into the starting plant" may be the introduction of an expression cassette containing the specific DNA molecule or a recombinant plasmid containing the specific DNA molecule into the starting plant.

Any of the above-mentioned plants may be any one of the following c1) to c5): c1) dicotyledonous plants; c2) monocotyledonous plants; c3) cruciferous plants; c4) Arabidopsis thaliana; c5) wild-type thaliana Arabidopsis Columbia-0 subtype.

Any of the above-mentioned stress resistance may be drought resistance.

In the above, the increase in drought resistance may be embodied in at least one of a decrease in malondialdehyde content, an increase in soluble sugar content, a decrease in water loss and an increase in survival rate.

In one embodiment of the present invention, a recombinant plasmid pBinGlyRed3-com58276 (containing a specific DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1) is introduced into the wild-type Arabidopsis thaliana Columbia-0 subtype to obtain a transgenic Arabidopsis; Transgenic Arabidopsis has improved drought resistance compared to wild-type Arabidopsis Columbia-0 subtype, which is reflected in reduced malondialdehyde content, increased soluble sugar content, reduced water loss, and improved survival . It can be seen that the specific DNA molecule shown in SEQ ID NO: 1 can regulate plant stress resistance. The invention has important application value.

Description of drawings

Figure 1 shows the growth state of Arabidopsis seedlings to be tested before natural drought, after natural drought and after 3 days of rehydration.

Figure 2 shows the statistical results of the survival rate of Arabidopsis seedlings.

Figure 3 shows the statistical results of the water loss rate of in vitro leaves of Arabidopsis thaliana.

Figure 4 shows the statistical results of soluble sugar content in Arabidopsis leaves.

Figure 5 shows the statistical results of malondialdehyde content in Arabidopsis leaves.

Figure 6 shows the growth state of Arabidopsis thaliana on MS solid medium containing 180 mM mannitol.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

Quantitative experiments in the following examples are all set up to repeat the experiments three times, and the results are averaged.

Caragana chinensis seeds were collected by Pei Xinwu (010-82106120), the inventor of the present invention, from the Shasheng Botanical Garden in Minqin, Gansu Province in 2014.

Wild type Arabidopsis thaliana (Columbia-0 subtype) is described in: Kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J. A genetic link between cold responses and flowering time through FVE in Arabidopsisthaliana. Nature Genetics. 2004, 36: 167-171. Hereinafter, wild-type Arabidopsis thaliana (Columbia-0 subtype) is referred to as wild-type Arabidopsis.

** indicates extremely significant difference, P<0.01. * indicates significant difference, P<0.05.

Example 1. Acquisition of Arabidopsis thaliana transgenic com58276 and identification of drought resistance

1. Obtaining the specific DNA fragment com58276

1. Sow the Caragana caragana seeds in sandy soil and manage the normal growth to obtain Caragana caragana plants that have grown for 1 month; then stop watering for 20 days to obtain the Caragana caragana plants of drought treatment.

The drought-treated Caragana caragana plants had developed roots, white leaves and thick epidermal hairs, which had strong drought resistance.

2. Take the leaves of the drought-treated Caragana caragana plants obtained in step 1, first extract total RNA, and then reverse-transcribe to obtain the cDNA of Caragana caragana.

3. After the completion of step 2, using the cDNA of Caragana as a template, a primer pair consisting of primer F: 5'-TCGCTTTCTCCTTAACCGTA-3' and primer R: 5'-AAACCTTAAATTGAGATACT-3' was used for PCR amplification, and then recovered. PCR amplification product of about 322bp.

4. Sequence the PCR amplification product recovered in step 3.

The sequencing results show that the nucleotide sequence of the PCR amplification product recovered in step 3 is shown in SEQ ID NO: 1.

The nucleotide sequence shown in SEQ ID NO: 1 was named the specific DNA fragment com58276.

2. Obtaining the recombinant plasmid pBinGlyRed3-com58276

1. The pBinGlyRed3 vector was cut with restriction enzymes EcoRI and XmaI (Zhang C, Iskandarov U, Klotz E T, et al.Athraustochytrid diacylglycerol acyltransferase 2 with broadsubstrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds. Journal of Experimental Botany, 2013, 64(11):3189-3200.), recovering a vector backbone of about 9 kb.

2. Using the cDNA of Caragana caragana obtained in step 1 as a template, primer FF: 5'-ACGGGGGACT GA ATTC GCTTTCTCCTTAACCGTA-3' (underlined is the recognition site of restriction endonuclease EcoRI) and primer FR: A primer pair consisting of 5'-CCGCCTCGAG CCCGGG AAACCTTAAATTGAGATACT-3' (underlined is the recognition site of restriction endonuclease XmaI) was subjected to PCR amplification, and a PCR amplification product of about 354 bp was recovered.

3. Homologously recombine the vector backbone recovered in step 1 and the PCR amplification product recovered in step 2 to obtain a recombinant plasmid pBinGlyRed3-com58276.

The recombinant plasmid pBinGlyRed3-com58276 was sequenced. According to the sequencing results, the structure of the recombinant plasmid pBinGlyRed3-com58276 is described as follows: Replace the small DNA fragment between the restriction endonuclease EcoRI and XmaI recognition sequences of the pBinGlyRed3 vector with the DNA whose nucleotide sequence is shown in SEQ ID NO: 1 molecule (the DNA molecule shown in SEQ ID NO: 1, that is, the specific DNA fragment com58276), the obtained recombinant plasmid.

Third, the acquisition of recombinant Agrobacterium

The recombinant plasmid pBinGlyRed3-com58276 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant Agrobacterium, which was named as EHA105/pBinGlyRed3-com58276.

4. Obtainment of Arabidopsis thaliana via com58276

1. Adopt the Arabidopsis thaliana inflorescence soaking transformation method (described in the following documents Clough, SJ, and Bent, AF. Floraldip: asmplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. (1998) 16, 735-743. ) to transfer EHA105/pBinGlyRed3-com58276 into wild-type Arabidopsis thaliana to obtain T ₁ generation transgenic com58276 Arabidopsis thaliana seeds.

2. Observe the luminescence of Arabidopsis thaliana seeds transformed from T ₁ generation to com58276 under green light, and then plant the seeds that can glow red to obtain positive seedlings of T ₁ generation transformed to com58276. The seeds received by the T ₁ generation transgenic com58276 positive seedlings are the T ₂ generation trans com58276 Arabidopsis seeds.

3. Under the green light, observe the luminescence of the T ₂ generation transgenic Arabidopsis thaliana seeds of different strains screened in step 2. If the ratio of the number of seeds capable of red light to the number of seeds not capable of red light in a line is 3:1, the line is a line in which one copy of the specific DNA fragment com58276 is inserted, and the received The seeds are the seeds of Arabidopsis thaliana transformed from T ₃ generation to com58276.

4. Observe the luminescence of the T ₃ generation transgenic Arabidopsis thaliana seeds screened in step 3 under green light, and the lines that can all emit red seeds are the T ₃ generation homozygous trans com58276 Arabidopsis thaliana. Two of the T ₃ generation homozygous transgenic com58276 Arabidopsis lines were named line3 and line7, respectively, and follow-up experiments were carried out.

5. Molecular identification

The Arabidopsis seeds to be tested are the T ₃ generation seeds of line3, the T ₃ generation seeds of line7 or wild-type Arabidopsis seeds.

(1) Take the seeds of Arabidopsis to be tested, soak them in 70% (v/v) ethanol aqueous solution for 30s, and wash with sterile water 3 times; then spread them on MS solid medium, and vernalize at 4°C for 2 days.

(2) After step (1) is completed, the Arabidopsis thaliana seeds to be tested are taken and cultured alternately between light and dark at 22° C. (16 h light cultivation/8 h dark cultivation) for 28 days to obtain the Arabidopsis thaliana seedlings to be tested.

(3) extracting the genomic DNA of the Arabidopsis seedling to be tested and using it as a template, using a primer pair composed of 5'-GGACTCTTGACCATGG-3' and 5'-ATTCGAGCTGGTCACC-3' to carry out PCR amplification to obtain a PCR amplification product; Then make the following judgment: if a PCR amplification product contains a DNA fragment of about 500 bp, the Arabidopsis thaliana seedling to be tested corresponding to the PCR amplification product is a positive seedling.

The results showed that the seedlings obtained from the T ₃ generation seeds of line3 and the T ₃ generation seeds of line7 were all positive seedlings.

6. Identification of drought resistance in Arabidopsis transgenic com58276

The Arabidopsis seeds to be tested are the T ₃ generation seeds of line3, the T ₃ generation seeds of line7 or wild-type Arabidopsis seeds.

The culture conditions were: 22°C; 12h light/12h dark; light intensity was 12000Lx.

1. Identification of natural drought resistance

The experiment was repeated three times to obtain the average value, and the steps for each repetition were as follows:

1. Take 3 culture pots, plant the Arabidopsis thaliana seeds to be tested in each pot, and cultivate for 3 weeks; after that, keep 2 Arabidopsis thaliana seedlings to be tested with basically the same growth in each pot.

2. After completing step 1, let it dry naturally for two weeks.

3. After completing step 2, rehydrate, and count the survival rate of the Arabidopsis seedlings to be tested after 3 days.

Before the natural drought, after the natural drought and after 3 days of rehydration, the growth state of the Arabidopsis seedlings to be tested is shown in Figure 1 (WT is wild-type Arabidopsis). The results showed that there was no significant difference in the phenotype between wild-type Arabidopsis and two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) before natural drought; after 2 weeks of natural drought, wild-type Arabidopsis thaliana All the leaves of the mustard turned yellow, curled, and even the plants died directly, while the leaves of the two T ₃ -generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) turned yellow, partially curled, and bolted; after 3 days of rehydration, The leaves of wild-type Arabidopsis thaliana were dead and the plants died, while the two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) all survived.

The statistical results of the survival rate of the Arabidopsis seedlings to be tested are shown in Figure 2 (WT is wild-type Arabidopsis). The results showed that the survival of the two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) was significantly improved compared to wild-type Arabidopsis.

2. Determination of water loss rate of isolated leaves

The experiment was repeated three times to obtain the average value, and the steps for each repetition were as follows:

1. Plant the Arabidopsis thaliana seeds to be tested and cultivate for 3 weeks to obtain the Arabidopsis thaliana seedlings to be tested.

2. After completing step 1, take the Arabidopsis thaliana seedlings to be tested with basically the same growth, pick the leaves, and weigh them with a 1/10,000 scale, which is the fresh weight.

3. After the completion of step 2, the leaves are placed in a petri dish, placed at room temperature for 2h, 4h, 6h, 8h, 10h or 12h, and weighed with a scale of 1/10,000, which is the weight after dehydration.

4. After completing step 3, calculate the water loss rate of the isolated leaves.

Water loss rate (%) = (fresh weight (g) - weight after water loss (g))/(fresh weight (g)) × 100%

Taking the leaf placement time as the abscissa and the corresponding water loss rate as the ordinate, draw the water loss curve. The water loss curve is shown in Figure 3 (WT is wild-type Arabidopsis). The results showed that the water loss rates of the two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) were significantly reduced compared to wild-type Arabidopsis.

3. Determination of soluble sugar content

The experiment was repeated three times to obtain an average value, and the steps of each repetition were as follows: when performing step 1 and 2, the leaves of the Arabidopsis thaliana seedlings to be tested were collected, and then the soluble sugar content was detected by a plant soluble sugar content detection kit (product of Solarbio). .

The detection results are shown in Figure 4 (WT is wild-type Arabidopsis). The results showed that the soluble sugar content of the two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) was significantly increased compared to wild-type Arabidopsis.

Fourth, the determination of malondialdehyde (MDA) content

The experiment was repeated three times to obtain the average value, and the steps of each repetition were as follows: when performing step 1 and 2, the leaves of the Arabidopsis thaliana seedlings to be tested were collected, and then the malondialdehyde (MDA) content detection kit (product of Solarbio) was used to detect Malondialdehyde content.

The detection results are shown in Figure 5 (WT is wild-type Arabidopsis). The results showed that the malondialdehyde content of two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) was significantly reduced compared to wild-type Arabidopsis.

5. Identification of drought resistance under mannitol stress (i.e., by adding mannitol to simulate drought)

The experiment was repeated three times to obtain the average value, and the steps for each repetition were as follows:

1. Take 20 seeds of Arabidopsis thaliana to be tested, treat with 70% (v/v) ethanol aqueous solution for 5 min, sterilize 2.6% (v/v) sodium hypochlorite aqueous solution for 10 min, and then wash with sterilized Tween water for 5 times.

2. After completing step 1, sow the Arabidopsis seeds to be tested in the medium (MS solid medium, MS solid medium containing 150 mM mannitol, MS solid medium containing 180 mM mannitol, or MS solid medium containing 200 mM mannitol). solid medium), vernalized at 4°C for 3 days.

3. The medium obtained in step 2 was vertically cultured for 6 days, and the growth state of Arabidopsis was observed.

The growth state of Arabidopsis on MS solid medium containing 180 mM mannitol is shown in Figure 6 (WT is wild-type Arabidopsis). The results showed that there was no significant difference in phenotype between wild-type Arabidopsis thaliana and two T ₃ generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) on MS solid medium; On the medium, compared with wild-type Arabidopsis, the survival rates of the two T ₃ -generation homozygous transgenic com58276 Arabidopsis lines (line3 and line7) were increased to a certain extent.

The above results indicate that the introduction of specific DNA fragment com58276 into wild-type Arabidopsis can improve the drought resistance of Arabidopsis. The improved drought resistance was reflected in a decrease in malondialdehyde content, an increase in soluble sugar content, a decrease in water loss and an increase in survival rate.

<110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> A specific DNA fragment com58276 and its application in regulating plant stress resistance

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 322

<212> DNA

<213> Caragana korshinskii Kom.

<400> 1

tcgctttctc cttaaccgta gctagtcgtc ttggccggaa tcacagatgg ccgcgcgcca 60

tctctcccct ctcgcgctcc ttcccgttcg cgcatcaccc ctctccctct cgatctctcc 120

ttttcgcgcc tctccccttt tttcctctcg cgacttcact gcagtcattc gtaatagcca 180

cgcacggcca aggatggcgc gccggtcacc ttctccccac cttcttctct atcacgaccg 240

ccctcccttt tctctcctcc tgatgcactt tcctatcttt ctagctgctg ctgctactta 300

gcagtatctc aatttaaggt tt 322

Claims (17)

1. A specific DNA molecule, the nucleotide sequence of which is shown in SEQ ID NO:1. 2. An expression cassette comprising the specific DNA molecule of claim 1. 3. A recombinant plasmid containing the specific DNA molecule of claim 1. 4. The recombinant microorganism containing the specific DNA molecule of claim 1. 5. the application of the described specific DNA molecule of claim 1, is A1) or A2): A1) Regulate the drought resistance of plants; A2) Breeding of transgenic plants with altered drought resistance. 6. The application according to claim 5, wherein the plant is a dicotyledonous plant. 7. The application of claim 5, wherein the plant is a monocotyledonous plant. 8. The application of claim 6, wherein the dicotyledonous plant is a cruciferous plant. 9. The application of claim 8, wherein the cruciferous plant is Arabidopsis thaliana. 10. The application of claim 9, wherein the Arabidopsis is a wild-type Arabidopsis Columbia-0 subtype. 11. A method for cultivating a transgenic plant, comprising the steps of: introducing the specific DNA molecule of claim 1 into the starting plant to obtain a transgenic plant; compared with the starting plant, the drought resistance of the transgenic plant is improved. 12. The method of claim 11, wherein: the introduction of the specific DNA molecule of claim 1 into the starting plant is to introduce into the starting plant an expression cassette containing the specific DNA molecule or containing the specific DNA molecule. Recombinant plasmids of DNA molecules. 13. The method of claim 11 or 12, wherein the plant is a dicotyledonous plant. 14. The method of claim 11 or 12, wherein the plant is a monocotyledonous plant. 15. The method of claim 13, wherein the dicotyledonous plant is a cruciferous plant. 16. The method of claim 15, wherein the cruciferous plant is Arabidopsis thaliana. 17. The method of claim 16, wherein the Arabidopsis is a wild-type Arabidopsis Columbia-0 subtype.

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