CN116535478B - Swallow flower MYB4 protein and application thereof in color regulation and control - Google Patents
Swallow flower MYB4 protein and application thereof in color regulation and control Download PDFInfo
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Abstract
The invention discloses a swallow flower MYB4 protein and application thereof in flower color regulation, belonging to the technical field of plant genetic engineering, and discloses swallow flower MYB4 gene cloning, plant over-expression vector construction and application methods in flower color regulation. The amino acid sequence of the MYB4 protein of the swallow flower is shown as SEQ ID No.1, and the nucleotide sequence of the encoded protein is shown as SEQ ID No. 2. It is found for the first time that over-expression of the MYB4 gene of the swallow flower in tobacco can reduce the anthocyanin content in the corolla of the tobacco and whiten the color. The MYB4 protein of the swallow flower provided by the invention provides theoretical reference for researching the color regulation and control of the swallow flower, and provides important gene resources for the color breeding work in the future.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a swallow flower MYB4 protein and application thereof in color regulation.
Background
Transcription factors are various and have complex functions, and are involved in the regulation and control of the aspects of plant morphogenesis, growth and development, biotic and abiotic stress and the like. The flower color is the most intuitive and attractive ornamental characteristic of the garden plants, and the improvement and creation of excellent, new and special flower color new varieties of the garden plants have been reported with a few success. MYB is involved in regulating plant anthocyanin, and is a transcription factor family which is studied more at present. For example, transcription factors PAP1, PAP2, MYB113 and MYB114 in the MYB family of Arabidopsis thaliana can positively regulate the transcription level of anthocyanin synthase genes, and promote the synthesis and accumulation of plant anthocyanin; atMYB4, atMYB3, atMYB7 and AtMYB32 are transcription factors which are found in Arabidopsis and inhibit anthocyanin synthesis.
The swallow flower (IRIS LAEVIGATA) is perennial herbal flowers of Iridaceae, has peculiar flower shape, strong cold resistance and low soil requirement, can be used for greening wetland such as lakeside, river bank, pond edge and the like, shallow water, flower bed and flower border, and can also be used for cutting flowers. At present, the flower color of the swallow flower is not rich enough, and the swallow flower is mainly blue in nature. The research on the color regulation of the swallow flowers mainly focuses on the cloning and expression analysis of structural genes, and the research on the color regulation of transcription factors is not reported yet, especially the regulation of MYB transcription factors on the color of the swallow flowers.
In order to meet the requirement of garden application on flower color diversity, the invention provides the swallow flower MYB4 protein, and the flower color regulating function of the swallow flower MYB4 protein is verified in mode plant tobacco, so that theoretical reference and important gene resources are provided for the swallow flower color breeding work.
Disclosure of Invention
One of the purposes of the invention is to provide the swallow flower MYB4 protein and the application thereof in color regulation and control, which lays a theoretical foundation for researching the swallow flower color regulation and control mechanism and provides gene resources for developing color breeding work in future.
In order to achieve the above purpose, the technical scheme of the invention is as follows: provides a swallow flower MYB4 protein, which is characterized by having an amino acid sequence shown as SEQ ID NO.1 in a sequence table.
The second object of the invention is to provide a gene for encoding the MYB4 protein of the swallow flower, which is characterized by having a nucleotide sequence shown as SEQ ID NO.2 in a sequence table.
It is a third object of the present invention to provide a biological material related to MYB4 protein of swallow flower, characterized by comprising any one of the following (A1) to (A5):
(A1) A recombinant cloning vector comprising a gene encoding a MYB4 protein of a swallow flower;
(A2) A recombinant plant overexpression vector comprising a gene encoding a MYB4 protein of swallow flower;
(A3) A recombinant plant overexpression vector obtained by connecting a label to the N end or/and the C end of the gene (A2);
(A4) A bioengineering bacterium comprising the recombinant plant overexpression vector of (A2) or (A3);
(A5) A transgenic plant comprising the recombinant plant overexpression vector of (A2) or (A3).
(A2) And/or (A3) the plant over-expression vector containing a gene encoding the above-mentioned YAB 4 protein of Yan flower means a DNA capable of over-expressing the above-mentioned MYB4 protein of Yan flower in a host cell, which DNA may contain not only a promoter for promoting transcription of the MYB4 gene but also a terminator for terminating transcription of the MYB4 gene.
The plant over-expression vector is GV1300.
The bioengineering bacterium is agrobacterium GV3101 (pSoup-p 19).
The invention aims at providing an application of the swallow flower MYB4 protein in regulating and controlling plant flower colors.
A method of regulating plant flower color comprising the steps of:
(B1) Introducing a recombinant plant over-expression vector containing a swallow flower MYB4 gene into a recipient plant;
(B2) Knocking out or silencing the MYB4 gene of the swallow flower to lose or reduce the function of the swallow flower.
The recombinant plant over-expression vector containing the swallow flower MYB4 gene is used for transforming plant tissues through an agrobacterium-mediated method, 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 swallowwort or common tobacco.
The invention has the following remarkable effects:
(1) The MYB4 gene is cloned from the perianth of the swallow flower, and after a plant over-expression vector is constructed and tobacco is genetically transformed, the over-expression of the MYB4 gene of the swallow flower can influence the color of the corolla of the tobacco.
(2) The anthocyanin content in the transgenic tobacco corolla is reduced, and the color becomes white, which shows that the swallow flower MYB4 gene has the function of regulating and controlling the plant color.
(3) The MYB4 gene of the swallow flower lays a foundation for researching the color regulation mechanism of the swallow flower, and provides gene resources for the color breeding of garden plants, especially Iris plants.
Drawings
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-MYB4-GFP recombinant plasmid, and 3-7 are 5 transgenic tobacco lines.
FIG. 2 shows a map of the crown coloration phenotype of wild-type and MYB 4-transgenic tobacco according to the invention, wherein WT is wild-type and OE1, OE2, OE3 are transgenes.
FIG. 3 shows a graph of anthocyanin relative to tobacco corolla with wild type and MYB4 transgenic according to the invention, where WT is wild type, OE1, OE2, OE3 are transgenic and lower case letters indicate significance.
FIG. 4 shows a pattern of expression of anthocyanin synthesis related genes in tobacco corolla of the transver MYB4 gene of the present invention, wherein WT is wild type and OE is transgene.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but it should be understood that the scope of the invention is not limited to the specific embodiments. The experimental methods in the following implementation methods are all conventional methods, and materials, reagents and the like used in the examples are all commercially available unless otherwise specified.
In the examples, the relevant medium formulations are as follows:
LB liquid medium: 5g/L yeast extract+10 g/L tryptone+10 g/L sodium chloride;
LB solid medium: 5g/L yeast extract+10 g/L tryptone+10 g/L sodium chloride+15 g/L agar;
YEP liquid medium: 10g/L yeast extract+10 g/L tryptone+5 g/L sodium chloride;
YEP solid medium: 10g/L yeast extract+10 g/L tryptone+5 g/L sodium chloride+15 g/L 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 medium: 1/2MS+6BA 1.0 mg/L+NAA0.05mg/L+Hyg25mg/L+Tintin 200mg/L;
MS4 medium: 1/2MS+IBA 0.2 mg/L+Hyg25mg/L+Tintin 200mg/L.
EXAMPLE 1 cloning of the MYB4 Gene of Yan flower
1. Total RNA extraction from plants
The total RNA of the plant is extracted by adopting OminiPlant RNAKit (Dnase I) (century, china) kit, and the operation steps are carried out according to the instruction book.
2. CDNA Synthesis
CDNA was synthesized using PRIMESCRIPT TM RT REAGENT KIT WITH GDNA ERASER (PERFECT REAL TIME) (Takara, japan) kit, and the procedure was followed according to the instructions, and cDNA was diluted 10-fold and used as a template for gene cloning.
3. Gene cloning
Primer 5 is used for designing a swallow flower MYB4 gene cloning specific Primer, and the Primer sequence is as follows:
MYB4-F1:5’-GAAGAAGAAATGGTGAGGACAAAGAATTCT-3’
MYB4-R1:5’-TCCAATATTATTAACCGGATGGGTGATGCT-3’
a50. Mu.L reaction system was prepared for PCR amplification, which included 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 PCR reaction procedure was: 94 ℃ for 2min; cycling for 35 times at 98 ℃ for 10s,58 ℃ for 30s and 68 ℃ for 90 s; and at 68℃for 10min.
After the PCR reaction was completed, the amplification result was detected by 1% agarose gel electrophoresis, the target band was recovered, then the recovered product was connected to cloning vector PEASY-Blunt-Zero (full gold, china), E.coli was transformed and spread on LB plate containing 100mg/L Amp, and cultured overnight in 37℃incubator, after the next day single clone was picked up and verified by bacterial liquid PCR using PEASY-Blunt-Zero vector universal primers M13-F and M13-R, positive clones were amplified in LB liquid medium for 10ml and then sent to Bio-company for sequencing.
The obtained oat flower MYB4 gene ORF region comprises 945 bases:
the ORF region of the MYB4 gene of the swallow flower codes 314 amino acids:
Example 2 construction of a Swallow MYB4 Gene plant overexpression vector
(1) Designing a carrier homology arm primer with Sal I enzyme cutting site and BamHI enzyme cutting site respectively, carrying out PCR amplification by taking a cloning carrier plasmid connected with the MYB4 gene of the swallow flower as a template, wherein a PCR reaction system and a reaction program are the same as those of gene cloning, and recovering a target fragment by glue after the PCR is finished, wherein the carrier homology arm primer is as follows:
MYB4-F2:5’-TTGATACATATGCCCGTCGACATGGTGAGGACAAAGAATTCTTC-3’
MYB4-R2:5’-CCCTTGCTCACCATGGATCCACCGGATGGGTGATGCTCGGAGT-3’
(2) The expression vector GV1300-GFP is digested by Sal I and BamH I restriction enzymes, the expression vector fragment is recovered by glue, the linearization vector is connected with a target gene added with homology arms by ClonExpress II One Step Cloning Kit (Nuo-zan, china) homologous recombination kit, the competent E.coli is transformed and coated on LB solid medium containing 50mg/L Kana, the culture is inverted overnight at 37 ℃, the monoclonal bacterial liquid PCR is picked up the next day for detection, positive clones are amplified and cultured in LB liquid medium for 10ml and then sent to biological company for sequencing, and the recombinant plant overexpression vector GV1300-MYB4-GFP is obtained, and the homologous recombination step is carried out according to the kit instruction.
Example 3 application of Yan flower MYB4 Gene in color control
1. Culturing tobacco aseptic seedlings
Placing a plurality of wild common tobacco seeds into a sterile 1.5ml centrifuge tube, sterilizing with 75% alcohol in an ultra-clean workbench for 1min, washing with sterile water for 3 times, sterilizing with 1% NaClO for 10min, washing with sterile water for 5 times, inoculating the tobacco seeds onto MS culture medium, and culturing under normal illumination at 25 ℃ to obtain the tobacco aseptic seedling.
2. Preparation of an infectious microbe liquid
Agrobacterium competent GV3101 (Veidi, china) was transformed, the transformation procedure was as described above, and then spread on YEP solid medium containing 50mg/L Kana and 25mg/L Rif, and cultured upside down at 28℃for 36h; selecting a monoclonal to perform bacterial liquid PCR verification, and performing amplification culture on a positive bacterial colony containing a target gene in a YEP (or LB) liquid culture medium containing Kana for 10ml under the culture condition of 28 ℃ and 180rpm shaking culture for 12-16h; sucking 1mL of the cultured bacterial liquid, adding the bacterial liquid into 50mL of fresh YEP (or LB) liquid culture medium containing corresponding antibiotics, and continuously shaking and culturing until OD 600 is about 0.6-0.8; the cultured bacterial liquid was centrifuged at 5000rpm for 5min, the supernatant was discarded in an ultra clean bench to collect the bacterial cells, and the bacterial cells were resuspended in an equal volume (50 ml) of 1/2MS infiltration medium (pH 5.8) for tobacco infection.
3. Infection of tobacco
Cutting sterile tobacco seedling leaf into 1cm×1cm small blocks, and pre-culturing in MS1 solid culture medium (leaf upper surface upward) for 2 days (light culture and dark culture); 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, slightly shaking the small conical flask during soaking, taking out the leaves, and placing the leaves on sterile filter paper to absorb bacterial liquid attached to the surfaces of the leaves; 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; co-cultured leaves were inoculated on MS2 medium for light cultivation, and resistance screening was performed (note that the edges of the leaves were gently pressed into the medium to increase selection pressure), while leaf discs that were not infected with Agrobacterium were set as negative controls. Under normal conditions, tobacco leaves infected by agrobacterium grow callus at the edges, and tobacco leaves not infected by agrobacterium die gradually; 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; when the plant grows about 1cm of resistant buds, cutting off the resistant buds, transferring the resistant buds to an MS4 culture medium to induce rooting, and growing adventitious roots about 10 days; after the regenerated seedlings grow developed root systems, transplanting the regenerated seedlings into a small basin containing sterilized soil, and placing the small basin in room temperature for culture.
4. Molecular characterization of transgenic tobacco
And extracting DNA from the obtained transgenic tobacco plants, and carrying out PCR detection by using MYB4 homology arm primers to identify the transgenic positive tobacco plants. Wild tobacco was used as control, GV1300-MYB4-GFP recombinant plasmid was used as positive control, and 5 transgenic tobacco lines were each provided with the desired band, indicating that the MYB4 gene had been successfully inserted into the tobacco genome (FIG. 1).
5. Detection of anthocyanin content in transgenic tobacco
0.1G of bloomed 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 by 95% ethanol) is added, and leaching is carried out 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 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 extract (ml)
M: sampling quality (g)
1000000: The calculation result is converted into the fold of nmol
By recording the growth and development process of transgenic positive tobacco plants, it was found that overexpression of the swallow flower MYB4 gene in tobacco significantly whitened the tobacco corolla color compared to the control (fig. 2).
The anthocyanin content detection result shows that: the anthocyanin content in the corolla of the wild tobacco is 108.15nmol/g, the anthocyanin content in the corolla of the 3 MYB 4-transgenic tobacco lines is 30.83nmol/g, 41.34nmol/g and 35.63nmol/g respectively, and the anthocyanin content in the corolla of the MYB 4-transgenic tobacco is remarkably reduced compared with the wild type (figure 3).
6. Expression pattern detection of anthocyanin synthesis related genes in transgenic tobacco corolla
Taking corolla tissue of transgenic tobacco, adding liquid nitrogen for grinding, extracting RNA, carrying out reverse transcription to obtain cDNA, detecting the expression quantity change of related structural genes on an anthocyanin synthesis path in a transgenic strain by real-time qPCR, wherein NtTUBA is used as an internal reference gene of the tobacco, and a primer for quantification is shown in a sequence table. The reaction system and the reaction procedure are described in the UltraSYBR Mixture specification. The reaction was performed on a Roche LIGHT CYCLER 96 fluorescent quantitative PCR apparatus, and the obtained data was used for calculating the relative expression level by using 2 -△△CT, and the result was plotted by using Origin Pro 8.0. Wherein wild-type tobacco plants were used as controls, 3 biological replicates were set.
The detection result of the expression pattern of anthocyanin synthesis related genes in transgenic tobacco corolla shows that: over-expression of the MYB4 gene in the swallow flower significantly increases the expression level of ANS and ANR1 in the tobacco corolla by 3.3 and 7.5 times as compared to wild tobacco plants, thus, it was hypothesized that the color whitening of transgenic tobacco might be that a large amount of colored anthocyanins in the corolla are catalyzed by ANR to form epicatechin (fig. 4).
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent variations of the claims herein will fall within the scope of the invention.
Claims (3)
1. The application of over-expression of the MYB4 gene of the swallow flower in the color fading of the corolla of common tobacco is characterized in that a recombinant plant over-expression vector containing the MYB4 gene of the swallow flower is introduced into tobacco, and the amino acid sequence of the MYB4 gene coding protein is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the nucleic acid sequence of the MYB4 gene is shown in SEQ ID No. 2.
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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| Comparative analysis of R2R3-MYB transcription factors in the flower of Iris laevigata identifies a novel gene regulating tobacco cold tolerance;J Yang;Plant Biology;20220702;第24卷(第6期);全文 * |
| transcription factor MYB1-like [Phoenix dactylifera]，NCBI Reference Sequence: XP_038970680.1;Genbank database;NCBI;20210127;全文 * |
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