CN116375835B - Application of Yan flower MYB4b protein in regulation and control of plant leaf morphology - Google Patents
Application of Yan flower MYB4b protein in regulation and control of plant leaf morphology Download PDFInfo
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Abstract
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a swallow flower MYB4b protein in regulation and control of plant leaf morphology. The nucleotide sequence of the YAB 4b protein of the Yan flower is shown as SEQ ID No.1, the amino acid sequence is shown as SEQ ID No.2, and the gene can narrow the leaf of common tobacco and deepen the leaf color, which shows that the MYB4b gene of the Yan flower has the function of regulating the morphogenesis of the leaf of plants. The swallow flower MYB4b protein and the coding gene thereof provided by the invention not only provide an important theoretical basis for the regulation and control of the leaf shape of the swallow flower, but also provide important gene resources for the breeding of other foliage plants.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a swallow flower MYB4b protein in regulation and control of plant leaf morphology.
Background
In the process of long growth and evolution, plants form various complicated and fine regulation mechanisms so as to adapt to living environments. Differentiation occurs between cells during the growth and development of plants, because of temporal and spatial differences in intracellular gene expression, one of the main causes of such differences being the regulation of transcription factors at the transcriptional level. MYB transcription factor is taken as one of the largest transcription factor families in plants, is widely involved in the whole growth and development process of plants, and has important regulation and control effects on the growth and development of plants. In recent years, MYB transcription factor genes have also been shown to be involved in leaf morphogenesis.
Leaves are the main sites for photosynthesis and respiration of plants, and green plants convert light energy into chemical energy through photosynthesis of leaves, and are the most common and cheapest energy conversion process. Yields of green plants, including roots, stems, leaves, seeds, etc., where only 5% -10% of the material comes from nutrients absorbed by the roots and 90% -95% of the material comes from the product of photosynthesis of the plant leaves. In general, the more green the plant leaves, the stronger the photosynthesis, and the more organic matter is accumulated. The leaves have various shapes, and different plants have similar and similar shapes and different leaf shapes. The same plant has different leaf forms in different growth periods or under different growth conditions, and has great ornamental value.
The Yan flower (IRIS LAEVIGATA Fisch.) is a plant of Iridaceae, and is mainly distributed in Heilongjiang, jilin, liaoning, inner Mongolia and Yunnan. The flower type flower is peculiar, graceful and luxurious, the leaves are rich and green, and the flower type flower is tall and straight and sword-like, and is a blue aquatic flower with extremely high ornamental value. In addition, the bird's nest leaves have more fibers, can be used for papermaking or hemp making, and have extremely high economic value. At present, research proves that MYB transcription factors are involved in leaf morphogenesis, but research on leaf shape regulation of the MYB4b transcription factors of the swallow flower has not been reported yet.
Disclosure of Invention
The invention aims to provide an application of the swallow flower MYB4b protein in regulating and controlling plant leaf morphology, provides a new way for cultivating leaf morphology, has important significance for exploring the function of the swallow flower MYB4b and revealing the plant leaf formation mechanism, and provides an important reference basis for molecular breeding of tobacco plants.
The invention aims at providing a swallow flower MYB4b protein.
The second object of the present invention is to provide a gene encoding a MYB4b protein of swallow.
It is a further object of the present invention to provide biological materials related to the MYB4b protein of the swallow flower.
The fourth object of the invention is to obtain a tobacco transformed plant containing the MYB4b gene of the swallow flower.
The invention aims at providing an application of a swallow flower MYB4b gene in regulating morphological development of tobacco leaves.
The aim of the invention is achieved by the following technical scheme:
a yan flower MYB4 protein, characterized in that the protein is the protein of (A1) or (A2) as follows:
(A1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2;
(A2) A protein which is derived from (A1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2;
A gene encoding a MYB4B protein of a swallow flower, wherein the gene is a gene of (B1) or (B2) as follows:
(B1) The nucleotide sequence is cDNA or genome DNA of SEQ ID No.1 in the sequence table;
(B2) A cDNA or genomic DNA having 80% or more similarity to the nucleotide sequence defined in (B1) and encoding the YA B4B protein of Yan flower of claim (A1).
The biological material related to the MYB4b gene of the swallow flower is characterized by comprising any one of the following (C1) to (C5):
(C1) A recombinant cloning vector containing a gene encoding a MYB4b of a swallow flower;
(C2) A recombinant plant expression vector comprising a gene encoding a MYB4b of a swallow flower;
(C3) A recombinant plant expression vector obtained by connecting a tag to the N-terminal or/and the C-terminal of the gene (C1);
(C4) 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).
The plant expression vector in the embodiment of the invention is GV1300;
The bioengineering bacterium in the embodiment of the invention is agrobacterium GV3101.
A method of regulating plant leaf morphology comprising the steps of:
(D1) Introducing a gene encoding the swallow flower MYB4b protein into a recipient plant to obtain a transgenic plant;
(D2) The MYB4b gene of the swallow flower is overexpressed in a receptor plant;
according to the technical scheme of the invention, the receptor plant is dicotyledonous plant, preferably tobacco.
The invention has the following beneficial effects:
(1) According to the invention, the MYB4b 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 MYB4b gene into model plant tobacco, so that the leaf morphology of the tobacco is influenced by the over-expression of the MYB4b gene.
(2) The leaf of the tobacco transgenic plant is narrowed and the leaf color is deepened, which shows that the MYB4b gene of the swallow flower has the function of regulating and controlling the morphogenesis of the plant leaf.
(3) The Yan flower MYB4b 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 leaf breeding.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PCR identification electrophoresis patterns of transgenic tobacco of the present invention, wherein M is DL2000 marker,1 is wild type, 2 is GV1300-MYB4b-GFP recombinant plasmid, and 3-7 are 5 transgenic tobacco lines.
FIG. 2 shows the protein sequence alignment and homologous protein clustering of the Yan flower MYB4B in the invention, wherein the A diagram is homologous protein clustering and the B diagram is protein sequence alignment.
FIG. 3 shows a phenotype diagram of wild type and transgenic tobacco in accordance with the present invention, where WT is wild type and OE is transgenic.
FIG. 4 shows the expression of anthocyanin synthesis-related genes in tobacco leaves of the Piyana MYB4b gene of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The experimental methods used in the examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples are commercially available unless otherwise specified.
The invention aims to overcome the defects of the prior art and provides an application of a MYB4b protein of a transhihlin in regulating and controlling the morphosis of tobacco leaves.
The relevant culture medium formulation method in the examples is as follows:
LB liquid Medium (100 ml): 0.5g yeast extract+1 g tryptone+1 g sodium chloride
LB solid medium (100 ml): 0.5g yeast extract+1 g tryptone+1 g sodium chloride+1.5 g agar
YEP liquid medium (100 ml): 1g yeast extract+1 g tryptone+0.5 g sodium chloride
YEP solid medium (100 ml): 1g yeast extract+1 g tryptone+0.5 g sodium chloride+1.5 g agar
MS1 medium: MS+6BA 1.0mg/L+NAA 0.05mg/L
MS2 medium: 1/2MS+6BA 1.0mg/L+NAA 0.05mg/L+Hyg 20 mg/L+Tintin 200mg/L
MS3 culture medium 1/2MS+6BA 1.0mg/L+NAA 0.05 mg/L+Hyg25mg/L+Tintin 200mg/L
MS4 culture medium 1/2MS+NAA 0.1mg/L+Hyg 25 mg/L+Tintin 200mg/L
The specific test scheme of this example is as follows:
1. Extraction of total RNA from plants
The extraction of total RNA of plants is carried out by adopting OminiPlant RNA Kit (Dnase I) (century, china) kit according to the instruction.
2. CDNA Synthesis
CDNA synthesis was performed using PRIMESCRIPT TM RT REAGENT KIT WITH GDNA ERASER (PERFECT REAL TIME) (Takara, japan) kit according to the instructions.
3. Gene cloning
The ORF region encoded by the MYB4b gene of the swallow flower is cloned by designing a primer as follows:
MYB4b-F1:5’-GAAGAAGAAATGGTGAGGACAAAGAATTCT-3’
MYB4b-R1:5’-TCCAATATTATTAACCGGATGGGTGATGCT-3’
the reaction system was 50. Mu.L, including 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 ddH2O.
The reaction procedure is: 94 ℃ for 2min; cycling for 35 times at 98 ℃ for 10s,58 ℃ for 30s and 68 ℃ for 90 s; and at 68℃for 10min.
Amplifying by PCR to obtain an amplified product; the amplified product was ligated to cloning vector PEASY-Blun-Zero (full gold, china), E.coli transformed, and positive clones were selected for sequencing.
The sequence of the coding ORF region of the MYB4b gene of the obtained Yan flower is as follows:
ATGGTGAGGACAAAGAATTCTTCCTCCTCCTCCTCCTCGGCCTCGGCCTGTCCTAAGGAAGGG CTGAAGAGGGGAGCGTGGACAAGCCAAGAAGACGAGCTGTTATCCGACTACATCAGTGCTCATGGCCTTGGGAGGTGGCGAACCTTACCGGCCAATGCAGGTTTGAACAGGTGTGGCAAGAGTTGCAGGCTGCGATGGTTGAACTATCTGAGACCACATATTAAAAGAGGGAATATCACTGAGGAAGAGGAGGAGTTGATCGTTCGCCTCCATAAACTACTTGGCAACAGATGGTCACTAATAGCAGGAAGGCTGCCCGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACCCATATCAGAAGGAAGCTCTTAGGCTGCACCTCAAAGAACACTACCAAGACTGAAGAACCAAGCAGTACTCCTCCTCCTCCTCAAGAAGTAGTAGATCAAGAGGCGGCTGCTGCTGCTGCTGCAGAGAAAGCTGATCATGTAGTGAAGGTAATCCGGACAAAGGCGGTGAGATGCACGAGAACTCTAGTCTTTGCCGATCCATATTTACCTGCAAGCTCGGAGATCAAGAACAATATCTCTCCTCCACCTACAGATCATTCCCAGCTGGCTGCGGCGGACATGGATCATGAACCTTGGATGGCAGAACAACTTGTGCAGATTCCTCAGCTCTCCTACAACGATAGCCCTTACACTAGCAACAGCGTCGACAGCTTGCATAATAATAATAATGTCGGGGGGGCTACTAACGACGTTTCTATGATCCATGAAGACGATGATCAGTATCTGCAGGGAGAGTGGTTCGGCAGCAGTGGGATATTGAAGGAAGGGGAGGAGGGGCTCTACTCTCTTCTTGATCCTGATACATGGGACTCCCTCACATTCACTCAGAAGAATATCCACTCCGAGCATCACCCATCCGGTTAA
the coding ORF region of the MYB4b gene of the swallow flower comprises 945 bases and codes 314 amino acids:
MVRTKNSSSSSSSASACPKEGLKRGAWTSQEDELLSDYISAHGLGRWRTLPANAGLNRCGKSCRL RWLNYLRPHIKRGNITEEEEELIVRLHKLLGNRWSLIAGRLPGRTDNEIKNYWNTHIRRKLLGCTSKNTTKTEEPSSTPPPPQEVVDQEAAAAAAAEKADHVVKVIRTKAVRCTRTLVFADPYLPASSEIKNNISPPPTDHSQLAAADMDHEPWMAEQLVQIPQLSYNDSPYTSNSVDSLHNNNNVGGATNDVSMIHEDDDQYLQGEWFGSSGILKEGEEGLYSLLDPDTWDSLTFTQKNIHSEHHPSG
EXAMPLE 2 plant expression vector construction
(1) By utilizing a homologous recombination method, designing carrier homologous arm primers with Sal I enzyme cutting sites and BamH I enzyme cutting sites respectively to amplify the MYB4b genes of the swallow flowers, and recovering target fragments. The underlined parts of the vector homology arm primers are Sal I and BamH I cleavage sites, as follows:
MYB4b-F2:5’-TTGATACATATGCCCGTCGACATGGTGAGGACAAAGAATTCTTC-3’
MYB4b-R2:5’-CCCTTGCTCACCATGGATCCACCGGATGGGTGATGCTCGGAGT-3’
(2) The GV1300-GFP plasmid of the expression vector is digested by Sal I and BamH I restriction enzymes, the vector fragment is recovered, the linearization vector is connected with the MYB4b gene fragment, the competent E.coli is transformed and the plate is coated, the monoclonal strain is selected, and the recombinant plant expression vector GV1300-MYB4b-GFP is obtained through bacterial liquid PCR detection and sequencing verification. The vector construction was carried out using ClonExpress II One Step Cloning Kit (Nor praise, china) homologous recombination kit according to the instructions.
EXAMPLE 3 genetic transformation of tobacco
1. Cultivation of aseptic seedlings of tobacco
Placing a plurality of wild tobacco seeds into a sterile 1.5ml centrifuge tube, sterilizing with 75% ethanol in a sterile workbench for 1min, washing with sterile water for 3 times, sterilizing with 1% NaClO for 10min, washing with sterile water for 5 times, inoculating onto MS culture medium, and culturing under illumination at 25deg.C to obtain aseptic tobacco seedling.
2. Preparation of bioengineering bacteria
(1) Agrobacterium was transformed with GV3101 (Veidi, china) competent for the transformation procedure as described in the specification.
(2) Picking single colony on a transformation plate to 10ml of YEP (or LB) liquid medium containing 50mg/L Kan and 25mg/L Rif, and carrying out shaking culture at 28 ℃ and 180rpm for 12-16h;
(3) Sucking 1ml of bacterial liquid, adding the bacterial liquid into 50ml of YEP (or LB) liquid culture medium containing 50mg/L Kan and 25mg/L Rif, and carrying out shaking culture at 28 ℃ and 180rpm until OD 600 reaches 0.6-0.8;
(4) Centrifuging the cultured bacterial liquid at 5000rpm for 5min, discarding supernatant, suspending bacterial precipitate with equal volume (50 ml) of 1/2MS osmotic culture medium (pH 5.8) under aseptic condition, and placing on ice until tobacco is infected.
3. Tobacco infection
(1) Pre-culturing. Cutting sterile tobacco seedling leaf into 1cm×1cm small blocks, placing in MS1 solid culture medium (leaf upper surface upward), and pre-culturing for 2 days (light culture and dark culture);
(2) And (5) infection. Pouring the resuspended bacterial liquid into a sterile small conical flask in an ultra-clean workbench, taking out tobacco leaves which are pre-cultured for 2 days, putting the tobacco leaves into the bacterial liquid, soaking for 5min, taking out the leaves, putting the leaves on sterile filter paper to suck the bacterial liquid attached to the surfaces of the leaves, and simultaneously setting a leaf disc which is not infected by agrobacterium as a negative control;
(3) Co-culturing. Inoculating the infected tobacco leaves on an MS1 culture medium (the upper surfaces of the leaves face upwards), sealing by using a sealing film, and performing dark co-culture at 28 ℃ for 2 days;
(4) And (5) selecting and culturing. Co-cultured leaves were inoculated on MS2 medium for light culture and resistance screening was performed (note that the edges of the leaves were gently pressed into the medium to increase selection pressure). The non-transgenic tobacco is subjected to resistance culture at the same time, and under normal conditions, the non-transgenic tobacco leaves die gradually;
(5) And (5) subculturing. About 1-2 weeks, inoculating tobacco leaves on MS3 culture medium for subculture, and properly reducing the concentration of Timesin, wherein the aim is to promote the transgenic tobacco leaves to grow resistant buds and inhibit agrobacterium;
(6) And (5) rooting. When the plant grows to form adventitious buds of about 1cm, cutting off the adventitious buds, transferring the adventitious buds to an MS4 culture medium to induce rooting, and growing adventitious roots about 10 days;
(7) Hardening off seedlings. After the regenerated seedlings grow developed root systems, uncovering and culturing for 2 days for hardening seedlings, taking out, washing agar carried by the plant root systems with sterile water, transplanting the agar into a small basin containing sterilized soil, and culturing at room temperature;
(8) Extracting DNA from the obtained transgenic tobacco plant, and carrying out PCR identification by adopting MYB4b homology arm primers to obtain a transgenic positive tobacco plant;
(9) Carrying out fluorescent quantitative PCR detection on transgenic positive tobacco leaves: taking transgenic tobacco leaves, adding liquid nitrogen for grinding, extracting RNA, carrying out reverse transcription to obtain cDNA, and detecting the change of the expression quantity of related structural genes on an anthocyanin synthesis path in a transgenic strain by real-time fluorescent quantitative PCR, wherein a reaction system is 20 μl and comprises 7 μl deionized water, 2 μl cDNA template and 0.5 μl and 10 μl2×SYBR dye of each specific upstream and downstream primer; the amplification procedure was: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, cycling 45 times, extension at 72 ℃ for 7min, the reaction was completed on a LIGHT CYCLER-96 fluorescence quantitative PCR instrument, the results were calculated with 2 -△△CT, the significance of the data was analyzed with SPSS, and the results were plotted with Origin Pro 8.0. Wherein wild-type tobacco plants were used as controls, 3 biological replicates were set.
5. Analysis of results
(1) Sequence alignment and phylogenetic analysis of MYB4b of swallow flowers
By alignment of homologous protein sequences, it was found that the N-terminal of the sequence of MYB4B had a highly conserved R2R3 domain, indicating that MYB4B belongs to R2R3-MYB (FIG. 2-B). The cluster analysis evolutionary tree found that the gyb 4b of the swallowwort was closest in evolutionary relationship to AoMYB308,308 of asparagus officinalis of the genus asparagus of the family liliaceae, the protein homology was 46.69%, and secondly ZoMYB1 of ginger of the family zingiberaceae, the protein homology was 49.81% (fig. 2-a).
(2) Identification of transgenic positive tobacco plants
DNA was extracted from the 6 obtained transgenic tobacco plants, PCR identification was performed using vector homology arm primers for MYB genes, wild-type tobacco was used as a control, GV1300-MYB4b-GFP recombinant plasmid was used as a positive control, and 5 transgenic tobacco lines were each provided with a target band, which indicated that the MYB4b gene had been successfully inserted into the tobacco genome (FIG. 1).
(3) Leaf morphology observation of transgenic positive tobacco plants
By comparison of leaves of wild-type tobacco and transgenic tobacco plants, it was found (FIG. 3) that leaves of transgenic tobacco plants were significantly narrower than wild-type and also had a darker leaf color than wild-type.
(4) Quantitative analysis of tobacco anthocyanin synthesis pathway related structural genes
Overexpression of the MYB4b gene in the Yan flower significantly increased the expression levels of DFR and LAR in tobacco leaves, 345.9 and 712.5 fold compared to control (fig. 4), suggesting that darkening of leaf color in transgenic tobacco may be due to the massive accumulation of catechins.
Claims (3)
1. The application of over-expression of the MYB4b gene of the swallow flower in regulating and controlling leaf shape and leaf color of common tobacco is characterized in that a recombinant plant over-expression vector containing the MYB4b gene of the swallow flower is introduced into tobacco, the amino acid sequence of the MYB4 gene coding protein is shown as SEQ ID NO.2, and the regulation and control refers to narrowing the leaf shape and deepening the leaf color of the tobacco.
2. The use according to claim 1, wherein the nucleic acid sequence of the MYB4b gene is shown in SEQ ID No. 1.
3. Use according to claim 1 or 2, characterized in that plant cells or tissues are transformed by using agrobacterium-mediated or gene gun and the transformed plant tissues are cultivated into plants.
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| JP2010111602A (en) * | 2008-11-05 | 2010-05-20 | Atm Kk | Antioxidant |
| CN113527454A (en) * | 2021-07-16 | 2021-10-22 | 中国热带农业科学院热带作物品种资源研究所 | Anthurium AaMYB6 transcription factor and encoding protein and application thereof |
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| JP2010111602A (en) * | 2008-11-05 | 2010-05-20 | Atm Kk | Antioxidant |
| CN113527454A (en) * | 2021-07-16 | 2021-10-22 | 中国热带农业科学院热带作物品种资源研究所 | Anthurium AaMYB6 transcription factor and encoding protein and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Comparative analysis of R2R3-MYB transcription factors in the flower of Iris laevigata identifies a novel gene regulating tobacco cold tolerance;J Yang;Plant Biololy;20220702;第24卷(第6期);全文 * |
| Genbank database.transcription factor MYB1-like [Phoenix dactylifera],NCBI Reference Sequence: XP_038970680.1.NCBI Genbank database.2021,全文. * |
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