CN116497042B - ANR gene clone of swallow flower and application thereof - Google Patents
ANR gene clone of swallow flower and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of plant genetic engineering breeding, and particularly relates to cloning and application of an ANR gene of swallow flower. The nucleotide sequence of the ANR gene of the swallow flower is shown as SEQ ID No.1, and the amino acid sequence encoded by the gene is shown as SEQ ID No. 2. It is found for the first time that over-expression of the ANR gene of the swallow flower in tobacco can lead to reduction of anthocyanin content in the corolla of the tobacco, and the color of the corolla becomes light. The ANR gene of the swallow flower not only can provide an important theoretical basis for the research on the color regulation and control of the swallow flower, but also can provide important gene resources for garden plant color breeding.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a swallow flower ANR gene clone and application thereof.
Background
In the plant breeding process, the flower color is one of the important characters of people, and especially in the field of ornamental plant breeding mainly based on ornamental flowers, a new flower color variety can generate great economic benefit. The genetic engineering breeding can directionally modify certain target characters of plants while retaining other original characters, greatly shortens the breeding period and provides a new idea for flower color breeding. The main component of plant flower color is anthocyanin, and the difference of anthocyanin content and components is a determining factor of flower color diversity of different varieties. The branch structure gene in the anthocyanin biosynthesis pathway can compete with the main structure gene for the reaction product of the last gene to block the accumulation of anthocyanin, lighten the plant flower color and enrich the plant flower color diversity.
The swallow flower (IRIS LAEVIGATA) is perennial herb of Iris of Iridaceae, has beautiful flower shape and strong cold resistance, is commonly applied to the sides of a pool, under a forest and beside a rock in gardens, and is an excellent ornamental flower in gardens. In nature, the swallow flower is mainly bluish purple, and the color is relatively single, so that the color breeding of the swallow flower is particularly necessary for meeting the requirement of color diversity in garden application. The action mechanism and biological functions of structural genes for positively regulating anthocyanin synthesis in swallow flowers are verified in model plants, but the influence of the branch gene ANR on plant flower colors has not been reported yet.
Disclosure of Invention
In view of the above, the invention provides a swallow flower ANR gene, a cloning method, a vector construction and an application method thereof in ornamental plant flower color breeding.
One of the purposes of the present invention is to provide a swallow flower ANR gene. The full length of the gene is 1287bp, the ORF region is 1020bp, and 339 amino acids are encoded.
The second purpose of the invention is to provide a protein encoded by the ANR gene of the swallow flower, and the protein can regulate and control the synthesis of anthocyanin in the tobacco corolla.
It is still another object of the present invention to provide a biological material related to the above-mentioned Yan flower ANR gene.
The fourth object of the present invention is to provide an application of the above-mentioned Yan flower ANR gene.
The aim of the invention is achieved by the following technical scheme:
An ANR gene of a swallow flower, which is a gene represented by A1) or A2) as follows:
A1 Nucleotide sequence is cDNA molecule or genome DNA of SEQ ID No.1 in the sequence table;
A2 90% or more similarity to the nucleotide sequence defined in A1), and a cDNA molecule or genomic DNA encoding the yan protein of claim 2.
The protein encoded by the swallow flower ANR gene is characterized by having a sequence represented by the following B1), B2), B3) or B4):
B1 Protein composed of an amino acid sequence shown as SEQ ID No.2 in a sequence table;
B2 Protein with the amino acid sequence with the homology of more than 90% with the amino acid sequence shown in SEQ ID No.2 and the same expression function;
B3 Protein derived from B1) with the same function and obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.2 in the sequence table;
b4 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of B1), B2) or B3).
A biological material related to the above-mentioned yan flower ANR gene, characterized by being any one of the following C1) to C5):
c1 A recombinant cloning vector comprising the ANR gene of swallow flower;
C2 A recombinant plant expression vector comprising a swallow flower ANR gene;
C3 A recombinant plant expression vector obtained by connecting a tag with the N end or/and the C end of the ANR gene of the swallow flower;
C4 A) a bioengineering bacterium comprising the recombinant vector of C2) or C3);
c5 A transgenic plant comprising the recombinant plant expression vector of C2) or C3).
C2 The plant expression vector containing the above-mentioned Yan flower ANR gene as described in C3) and/or above means a DNA capable of expressing the above-mentioned Yan flower ANR gene in a host cell, and the DNA may contain not only a promoter for initiating the transcription of the ANR gene but also a terminator for terminating the transcription of the ANR gene.
The cloning vector in the embodiment of the invention is PMD18-T vector.
The plant expression vector in the embodiment of the invention is GV1300.
The bioengineering bacterium in the embodiment of the invention is agrobacterium GV3101.
The application of the ANR gene of the swallow flower in regulating and controlling plant flower color.
Further, the method for cultivating the new plant flower color variety by utilizing the Yan flower ANR gene comprises the following steps:
d1 Introducing the ANR gene of the swallowwort into a recipient plant to obtain a transgenic plant;
D2 Introducing a recombinant plant expression vector of the swallow flower ANR gene into a recipient plant to obtain a transgenic plant;
D3 Knocking out or silencing the ANR gene of the swallow flower, and losing or reducing the function of the ANR gene.
The recombinant plant expression vector containing the Yan flower ANR gene is used for transforming plant cells or tissues by using agrobacterium mediation or gene gun, and the transformed plant tissues are cultivated into plants.
According to the technical scheme of the invention, the receptor plant is a monocotyledonous plant or a dicotyledonous plant, preferably, a swallowwort or a Nicotiana tabacum.
The technical scheme provided by the invention has the beneficial effects that: according to the invention, the ANR gene is cloned from the swallow flower, a recombinant plant expression vector is successfully constructed, and the agrobacterium transformation method is adopted to transform the recombinant plant expression vector containing the swallow flower ANR gene into model plant tobacco, so that compared with wild tobacco, the color of the tobacco corolla becomes lighter by introducing the swallow flower ANR gene, and the relative content of anthocyanin is reduced by more than 2 times compared with wild tobacco, which means that the swallow flower ANR gene has the functions of inhibiting anthocyanin synthesis and reducing anthocyanin content.
The Yan flower ANR gene provided by the invention can be used as an excellent gene resource, can be widely applied to the field of genetic breeding of iris or other flower plants, and has important significance for flower color breeding.
Drawings
FIG. 1 is a clone agarose gel electrophoresis of the ANR gene of Yan flower; wherein lane 1 is DL2000 marker, and lanes 2 and 3 are ANR gene clone PCR results.
FIG. 2 is an electrophoresis chart of recombinant plant expression vector identification, wherein lane 1 is DL15000 marker, lane 2 is GV1300-IlANR-GFP recombinant plasmid, and lane 3 is Sal I and BamHI double enzyme digestion verification.
FIG. 3 is a screening chart of transgenic ANR gene tobacco of the Yan flowers, and 1, 2 and 3 are positive seedlings of the transgenic tobacco.
FIG. 4 phenotype maps of wild type and transgenic E.Yan flower ANR gene tobacco corolla and anthocyanin relative content map, wherein WT is wild type, OE1, OE2, OE3 are three lines of transgenic tobacco.
Detailed Description
A further understanding of the nature and advantages of the present invention may be realized by the following detailed description. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: ANR gene clone of swallow flower
The total RNA of the swallowwort was extracted using OminiPlant RNA Kit (Dnase I) (century, china) kit and reverse transcribed into cDNA by PRIMESCRIPT TM RT REAGENT KIT WITH GDNA ERASER (PERFECT REAL TIME) (Takara, japan) kit.
The Primer 5 is used for designing a cloning specific Primer of the ANR gene of the swallow flower, the obtained swallow flower cDNA is diluted 10 times as a template, the ORF region of the ANR gene is amplified according to the following reaction, and the sequence of the specific Primer is as follows:
ANR-F1:5’-TTGGAAACTAAGCATGGCTCAGATATTGAC-3’
ANR-R1:5’-GCATAATACGTTTTCTCTCTTCAAGTTGCA-3’
50. Mu.L of reaction system: comprises 2. Mu.L of template cDNA, 25. Mu.L of 2X PCR buffer for KOD FX, 10. Mu.L of 2mM dNTPs, 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of KOD FX and 10. Mu.L of ddH 2 O.
The sample adding operation is carried out on ice, after the sample adding is finished, the mixture is uniformly mixed and centrifuged, and the mixture is placed in a PCR instrument for amplification reaction according to the following procedures:
94 ℃ for 2min; cycling for 35 times at 98 ℃ for 10s,58 ℃ for 30s and 68 ℃ for 90 s; and at 68℃for 10min.
Amplified by PCR to give amplified products (FIG. 1), detected by 1% agarose gel electrophoresis (120V, 20 min), and then observed for banding using a gel imaging system.
Agarose gel containing the target strips was cut with a blade under an ultraviolet transmissive cutting station and placed in a pre-prepared centrifuge tube, and the gel recovery procedure was referred to TIANGEN kit instructions.
Mu.L of the gel recovery product was mixed with 1. Mu.L of 6X Loading Buffer, and the recovery was detected by agarose gel electrophoresis at 1%. If the brightness of the strip is proper, preparing a connecting reaction liquid in a PCR tube according to the following reaction system, uniformly mixing the reaction liquid after the sample is added, and connecting the reaction liquid at 16 ℃ overnight.
10. Mu.L of reaction system: comprises 4.5. Mu.L of the gel recovery product, 5. Mu.L of Solution I, 0.5. Mu. LPMD 18-T.
The ligation product is transformed into escherichia coli (DH 5 alpha), and the specific operation steps are as follows:
adding 10 mu L of the reaction solution into 50 mu L of escherichia coli competent cells, gently mixing, and then placing in ice for 30min;
Placing in a water bath kettle at a temperature of 42 ℃ for 30s, and then rapidly transferring into ice and placing for 2min;
600. Mu.L of LB liquid medium (5 g/L Yeast extract+10g/L tryptone+10g/L NaCl) was added, and the mixture was placed on a constant temperature culture shaking table at 37℃and 200rpm, followed by shaking culture for 1 hour;
Centrifuging the transformation solution, removing a little LB culture solution on the upper layer, uniformly mixing the rest transformation solution by using a gun head, then taking 100 mu L of transformation solution to be coated on LB solid medium (5 g/L Yeast extract+10g/L tryptone+10g/L NaCl+15g/L Agar) containing Amp, sealing by using a sealing film, and placing in a constant temperature incubator at 37 ℃ for overnight culture (inversion).
The next day, 10-20 monoclonal strains of positive clones are selected on an LB solid plate and cultured in a centrifuge tube of 30 mu L LB liquid medium at 37 ℃ at 200rpm for 1h in a shaking way; and then, using the bacterial liquid of shake culture as a template, using the universal primers M13F and M13R of the T carrier as detection primers, preparing a PCR reaction liquid according to the following reaction system, and verifying the size of the inserted target gene through PCR reaction.
20. Mu.L of reaction system: comprises 2. Mu.L of bacterial liquid template, 6. Mu.L of ddH 2 O, 1. Mu.L of upstream and downstream M13 primer respectively, and 10. Mu.L of Es-TAQ MASTER Mix.
Placing the PCR tube on ice, sampling on the ice, uniformly mixing and centrifuging after the sampling is finished, and then placing the PCR tube in a PCR instrument for PCR amplification, wherein the reaction procedure is as follows:
95 ℃ for 10min; cycling for 30 times at 94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 90 s; and at 72℃for 10min.
After the completion of the PCR reaction, the reaction solution was spotted on 1% agarose gel, followed by electrophoresis (120V, 15 min) to examine whether the inserted target band was correct or not, and if so, 10mL of LB liquid medium, 10. Mu.L of Amp and all the remaining bacterial solution were added to 15mL of shaking tube, and the shaking tube was placed on a shaking table at 37℃and shaken at 200rpm overnight. The next day, 1mL of the bacterial liquid was sent to the biological company for sequencing on an ultra clean bench, 200. Mu.L of each of the remaining bacterial liquids was stored in 60% glycerol of the same volume (200. Mu.L), and the obtained solution was stored in a refrigerator at-80 ℃.
According to sequencing results of biological company, bacterial liquid with correct sequence is used for extracting plasmid, and specific operation steps are referred to a specification of a plasmid extraction kit (TIANGEN, china).
The sequence of ORF region of the obtained Yanhua ANR gene is as follows:
ATGGCTCAGATATTGACCAAAACTGCGTGTGTCACCGGAGGAAACGGGTTCCTCGCGACCATCTTGATCAAGCAACTGTTGGAGAAGGGCTACGCCGTCAATGCCACCGTTCGCGACCCCGAGAACAAGGCGAGGGTCGGTCACCTCTTGGACCTGCAAAGCCTGGGCGATCTCAAGCTCTTCCGAGCGGAACTGACCGAAGAAGGAAGCTTCGACGAGGCGATAAGCGGCTGCGAATACGTCTTCCATCTCGCCACCCCGGTGAACATCTTCTCCCAAGACCCAGAGAATGAGTTGATCAAACCTGCAATCGAAGGAACCCTGAATGTCATGAAGTCGTGCCTCAAGGCGAAGGTCAAGCGGGTCGTCCTGACGTCGTCGGCAGCCGCCGTGTCGGTGAACAAGCTCAGAGGGACGGACCTCATGATGGACGAGGAGTGCTGGTCGGACGTCGAGTTCCTGACCGCCGAAAAGCCCCCGACATGGGGCTACCTGGTCTCGAAGACGCTCGCGGAGAAGGAAGCGTGGAAATTCGCACGAGAGAACGGCATCGACCTCGTGACCATAGTACCGGTCTTGATGGTCGGCCCTCCGTTGAGCGGCGACGTCCCATCGAGTGTAGGCATGGCTTTGTGCTTGCTCCTTGGAGATGATCCCCGCATTGGCGGTCTGAAGATCATGCAATCGCTCTCGGGCTCGGTCTCGCTCGTGCACGTGGAGGACGCGAGCAGGGCCCAGATATTCGTCGCGGAGACCGAGTCGGCCTCCGGTCGATACATCTGCTGCGCCGTCAACACGAGCCTACCGGAGCTCGCGGAGTTCCTCTCGAAGAGATACCCAGAGTACAAAGTCCCTACTAATATCACCGACGTACCGAAGAAAGCTAAACTGAAACTCTCCTCCGAGAAGCTCATCAAGGAAGGGTTCAACTTCGAGAAGAAAGAACTTGGAGAGATCTACGACGGAGCCATCAGCTATGCTAAGAAAGCAGGATTTTTTCCCAAAAATGGTGCAACTTGA
The ORFs region of the ANR gene of the swallow flower comprises 1020 bases encoding 339 amino acids:
MAQILTKTACVTGGNGFLATILIKQLLEKGYAVNATVRDPENKARVGHLLDLQSLGDLKLFRAELTEEGSFDEAISGCEYVFHLATPVNIFSQDPENELIKPAIEGTLNVMKSCLKAKVKRVVLTSSAAAVSVNKLRGTDLMMDEECWSDVEFLTAEKPPTWGYLVSKTLAEKEAWKFARENGIDLVTIVPVLMVGPPLSGDVPSSVGMALCLLLGDDPRIGGLKIMQSLSGSVSLVHVEDASRAQIFVAETESASGRYICCAVNTSLPELAEFLSKRYPEYKVPTNITDVPKKAKLKLSSEKLIKEGFNFEKKELGEIYDGAISYAKKAGFFPKNGAT
The ANR gene sequence of the swallow flower was registered to GenBank under the accession number OQ433948.
Example 2: construction of plant over-expression vector of ANR gene of swallow flower
The ORF region of the ANR gene of the swallow flower, from which the stop codon was removed, was constructed between Sal I and BamH I cleavage sites of the plant expression vector GV1300 by using the method of homologous recombination to obtain a recombinant plant expression vector GV1300-IlANR-GFP (FIG. 2, lane 2). The vector construction was performed using ClonExpress II One Step Cloning Kit (Northenan, china) homologous recombination kit according to the specification, and the vector homology arm primers (underlined at Sal I and BamH I cleavage sites) were as follows:
ANR-F2:5’-TTGATACATATGCCCGTCGACTGGAAACTAAGCATGGCTCAGATATTGAC-3’
ANR-R2:5’-CCCTTGCTCACCATGGATCCGCATAATACGTTTTCTCTCTAGTTGCA-3’
to further verify the ligation of the recombinant plasmid, double restriction enzyme digestion was performed on the recombinant plant expression vector GV1300-IlANR-GFP with restriction enzymes Sal I and BamHI, and the results of the double restriction enzyme digestion were shown to be two bands and the positions of the bands were correct (FIG. 2, lane 3), indicating that the ANR gene of the Yan flower was ligated with the GV1300-GFP vector, and that the construction of the plant expression vector was successful.
Double cleavage identified 20 μl reaction system: includes 6. Mu.L of recombinant plasmid GV1300-IlANR-GFP, 2. Mu.L of buffer, 10. Mu.L of ddH 2 O, restriction enzymes Sal I and BamHI, 1. Mu.L each.
Example 3: yanhua ANR gene genetically transformed tobacco
The recombinant plasmid GV1300-IlANR-GFP was transferred into competent cells of Agrobacterium GV3101 by freeze-transfer, the transformation procedure being as described in the specification.
The agrobacterium genetically transformed by the leaf disc method for transforming the recombinant plasmid GV1300-IlANR-GFP plant over-expression vector comprises the following steps:
(1) GV1300-IlANR-GFP agrobacterium liquid is taken out from a refrigerator at the temperature of minus 80 ℃ to be streaked on a YEP solid culture medium (10 g/L Yeast extract+10g/L tryptone+5g/L NaCl+15g/L Agar), placed in a 28 ℃ incubator to be inversely cultured for 36 hours, a monoclonal strain is selected to carry out PCR identification, and then the correctly identified agrobacterium is amplified and cultured in a 50mLYEP liquid culture medium (10 g/L Yeast extract+10g/L tryptone+5g/L NaCl).
(2) Agrobacteria were shaken to OD 600 to 0.7 and used to infect tobacco leaf discs.
(3) The well-grown tobacco aseptic seedlings were selected, tender leaves were cut into 1cm 2 squares, and then placed on a preculture medium (MS+20 g/L sucrose+7.8 g/L agar+1 mg/L6-BA+0.05 mg/L NAA) for dark culture for 2d.
(4) The bacterial cells were collected by centrifugation at 4000rpm for 5min, the supernatant was discarded, the bacterial cells were resuspended in a super clean bench with a heavy suspension (1/2MS+20g/L sucrose), then the pre-cultured tobacco leaves were immersed in the heavy suspension and gently shaken to infect for 5min, the leaves were placed on filter paper for air drying, and then were returned to the pre-culture medium (with the upper surface of the leaves facing upwards), co-cultured in a dark inversion at 28℃for 2d, while a leaf disk not infected with Agrobacterium was set as a negative control.
(5) The co-cultured leaves are placed on a screening culture medium (1/2MS+20g/L sucrose+1mg/L6BA+0.05 mg/L NAA+20mg/L Hyg+500mg/L Cef) for illumination culture, and the culture medium is changed every 15 days, so that the concentration of Cef can be properly reduced by the culture medium, and the purpose of promoting transgenic tobacco leaves to grow resistant buds and inhibiting agrobacterium is achieved. The non-transgenic tobacco is also subjected to resistance culture, and under normal conditions, the non-transgenic tobacco leaves die gradually.
(6) When the adventitious buds are differentiated from the leaves, the adventitious buds are cut off and transferred into a rooting medium (1/2MS+20g/L sucrose+0.1 mg/L NAA+25mg/L Hyg+200mg/L Cef) for rooting culture, and adventitious roots can grow out about 10 days.
(7) The rooted tobacco seedlings are transferred to nutrient soil for continuous culture, partial leaves are taken to extract DNA, the PCR verification is carried out by adopting ANR homology arm primers, the positive tobacco seedlings are continuously cultured until seeds are harvested, and the next generation positive plants are continuously screened (figure 3).
Example 4: the anthocyanin content of the obtained T 3 generation transgenic tobacco is identified, and the specific process is as follows:
drying the harvested T 1 tobacco seeds, sterilizing and sowing the tobacco seeds on a 1/2MS screening culture medium containing 25mg/L Hyg, wherein the sterilization method comprises the following steps: sterilizing with 75% alcohol for 1min, cleaning with sterile water for 3 times, sterilizing with 2% sodium hypochlorite solution for 10min, and cleaning with sterile water for 5 times; after vernalization for 2d at 4 ℃, normal culture is carried out in a tissue culture room, when four leaves grow slightly larger, the tobacco is moved into nutrient soil (turfy soil: vermiculite=3:1) for culture, the illumination environment is illumination/darkness for 14h/10h, water is poured once a week, seeds of T 2 generations are harvested, and the transgenic tobacco of T 3 generations is obtained by using the same seed disinfection and screening method.
0.1G of full-open T 3 -generation transgenic tobacco corolla is taken, ground by adding liquid nitrogen, then 10mL of 0.1mol/L ethanol hydrochloride (8.3 mL of concentrated hydrochloric acid is diluted to 1L with 95% ethanol) is added, and the mixture is leached for 30min under the water bath condition of 60 ℃. After centrifugation at 12000rpm, the supernatant was examined for optical density values at wavelengths of 530nm, 620nm and 650 nm. Wherein wild-type tobacco plants were used as controls, 3 biological replicates were set. The anthocyanin content calculation method comprises the following steps:
Calculating the optical density value of anthocyanin: OD λ＝(OD530-OD620)-0.1(OD650-OD620)
Anthocyanin content (nmol/g) =od λ/epsilon×v/m×1000000
OD λ: optical density of anthocyanin at 530nm wavelength
Epsilon: anthocyanin molar extinction coefficient 4.62×10 6
V: volume of extractive solution (mL)
M: sampling quality (g)
1000000: The calculation result is converted into the fold of nmol
Experimental results showed that the anthocyanin content in the wild type tobacco corolla was 114.52nmol/g, while the anthocyanin content in the corolla of the 3 ANR gene-transferred tobacco lines was 63.39nmol/g, 49.60nmol/g and 53.38nmol/g, respectively (FIG. 4). The data fully demonstrate that the anthocyanin content in the ANR gene-transferred tobacco corolla is obviously reduced, and the color of the tobacco becomes shallow.
Claims (7)
1. The ANR gene of the swallow flower is characterized in that the nucleic acid sequence of the gene is shown as SEQ ID No. 1.
2. The protein encoded by the ANR gene of the swallow flower is characterized in that the amino acid sequence of the protein is shown as SEQ ID No. 2.
3. A biological material related to the ANR gene of swallowwort according to claim 1, which is any one of the following (A1) to (A3):
(A1) A recombinant cloning vector containing a swallow flower ANR gene;
(A2) A recombinant plant expression vector comprising a swallow flower ANR gene;
(A3) A bioengineering bacterium comprising the recombinant vector of (A2).
4. The biological material related to the ANR gene of swallow flower according to claim 3, wherein: the bioengineering bacteria is agrobacterium GV3101.
5. Use of the biological material related to the ANR gene of the swallow flower according to any one of claims 3 to 4 for regulating and controlling the color of the corolla of the common tobacco, wherein the regulating and controlling is to lighten the color of the corolla of the common tobacco.
6. The use according to claim 5, characterized in that: introducing the Yan flower ANR gene of claim 1 into a recipient plant to obtain a transgenic plant.
7. The use according to claim 6, characterized in that: a recombinant plant expression vector containing the swallow flower ANR gene is transformed into plant cells or tissues by using agrobacterium-mediated or gene gun and the transformed plant tissues are grown into plants.
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