CN116814651A - Application of oat flower MYB4a transcription factor in regulating and controlling plant flower column elongation - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a swallow flower MYB4a transcription factor in regulating and controlling plant flower column elongation. The nucleotide sequence of the gene of the MYB4a transcription factor of the swallow flower is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the gene can enable a transgenic tobacco flower column to be elongated and higher than a stamen after ectopic overexpression in tobacco, so that the tobacco fertility is influenced. Through systematic research, the invention provides the biological function of the swallow flower MYB4a transcription factor in regulating and controlling plant flower column development, and provides a new way for garden plant fruit sterility cultivation as an excellent gene resource.

Description

Application of oat flower MYB4a transcription factor in regulating and controlling plant flower column elongation

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a swallow flower MYB4a transcription factor in regulating and controlling plant flower column elongation.

Background

In the process of long growth and evolution, plants form various complicated and fine regulation mechanisms so as to adapt to living environments. 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 morphogenesis of floral organs.

The columella is the connection part between the stigma and the ovary, and is also the passage of pollen tube into the ovary. The length of the flower column is different according to the species, and the internal structure of the flower column is simple and is generally composed of thin wall tissues wrapped by the epidermis. Generally, dicotyledonous plants have solid flower columns, and monocotyledonous plants have hollow flower columns. The main function of the flower column is to conduct the pollen tube, which is the passage for the pollen tube to enter the pearl hole of the ovary for fertilization. For example, the short flower column head surface of the sand pear can fall more pollen to improve the fruit setting rate of fruits, while the setting rate of the senecio in Europe depends on the falling amount of pollen on the column head, the column head of the long flower column is hardly or only little self-flower pollen is fallen, and the germination rate is low, so the setting rate of the long column is low.

The Iris laevigata Fisch is an aquatic flower of Iris, and is mainly distributed in the eastern three-province, inner Mongolia and Yunnan of China. The flower type flower is peculiar, graceful and luxuriant, the leaves are rich, the leaves are tall and straight and like a sword, and the flower type flower and leaf type plant is important in gardens. The stigma of the swallow flower is higher than that of the stamen, and the swallow flower is difficult to set without external intervention and pollination. Many foliar plants in garden applications have an impact on their ornamental value due to the presence of fruits after the flowering phase. Therefore, the gene resource for the development of the columella is obtained from the swallow flowers, and the influence of fruits on ornamental value of garden plants is avoided, so that the columella is an important target of breeding work. At present, research proves that MYB transcription factors are involved in the morphogenesis of different organs of flowers, but research on the aspects of the regulation of the elongation of flower columns of relevant MYB transcription factors in swallow flowers has not been reported yet.

Disclosure of Invention

The invention aims to provide an application of a swallow flower MYB4a transcription factor in regulating and controlling plant flower column elongation, provides a new way for garden plant fruit sterility cultivation, has important significance for exploring the function of the swallow flower MYB4a transcription factor, reveals a plant flower column formation mechanism, and provides an important reference for molecular breeding of tobacco plants.

The invention aims at providing a swallow flower MYB4a gene.

It is another object of the present invention to provide a biological material related to the MYB4a gene of the swallow flower.

The third object of the invention is to obtain a tobacco transformed plant containing the MYB4a gene of the swallow flower.

The invention aims at providing an application of a swallow flower MYB4a transcription factor in regulating and controlling tobacco flower column elongation.

The aim of the invention is realized by the following technical scheme:

a swallow flower MYB4a transcription factor is characterized in that the gene sequence is shown as SEQ ID NO. 1.

The protein sequence coded by the MYB4a gene of the swallow flower is shown as SEQ ID NO. 2.

The biological material related to the MYB4a gene of the swallow flower is characterized by comprising any one of the following (A1) to (A3):

(A1) A plant expression vector containing a swallow flower MYB4a gene;

(A2) A bioengineering bacterium comprising the plant expression vector of (A1);

(A3) A transgenic plant comprising the plant expression vector of (A1).

The biological material related to the MYB4a gene of the swallow flower is characterized in that:

the plant expression vector is GV1300-GFP.

The biological material related to the MYB4a gene of the swallow flower is characterized in that:

the bioengineering bacteria is Agrobacterium tumefaciens EHA105.

The use of any one of the biological materials related to the MYB4a gene of swallow flower in regulating plant stem elongation;

the method for regulating and controlling the elongation of the plant flower column is characterized by comprising the following steps of:

(B1) Introducing the swallow flower MYB4a gene into a receptor plant to obtain a transgenic plant;

(B2) Over-expressing the swallow flower MYB4a gene in a receptor plant;

(B3) Silencing or inhibiting expression of MYB4a gene in the bird's nest.

The method for introducing a plant expression vector into a recipient plant is characterized by comprising the following steps:

plant expression vectors containing the Yan flower MYB4a gene are transformed into 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 acceptor plant is swallow flower or common tobacco.

The invention has the following beneficial effects:

(1) According to the invention, a MYB4a gene is cloned from a columella of a swallow flower, a GV1300-MYB4a-GFP plant expression vector is constructed, agrobacterium tumefaciens EHA105 is transformed by an electric shock method, the GV1300-MYB4a-GFP plant expression vector is transformed into tobacco by an agrobacterium transformation method, and the result shows that the MYB4a gene is over-expressed in the tobacco to elongate the columella of the tobacco.

(2) The elongation of the flower column of the transgenic tobacco plant shows that the MYB4a gene of the swallow flower has the function of regulating the morphological formation of the flower organ of the plant.

(3) The swallow flower MYB4a 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 ornamental plant 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 subcellular localization of the MYB4a protein of the invention, wherein GFP is a green fluorescent protein; autofluorescence is chloroplast Autofluorescence; bright is Bright field; merge is green fluorescence, chloroplast autofluorescence, and bright field fusion.

FIG. 2 shows a PCR identification electrophoresis diagram of tobacco transformed with MYB4a gene in the invention, wherein M is DL2000 Marker, lane 1 is wild tobacco, lane 2 is recombinant plant expression vector GV1300-MYB4a-GFP plasmid, and lanes 3-7 are 5 transgenic tobacco lines.

FIG. 3 shows a phenotype diagram of wild-type and transgenic tobacco in the present invention, where WT is wild-type tobacco and OE is transgenic tobacco.

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 swallow flower MYB4a transcription factor in regulating and controlling tobacco column elongation.

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 medium: 1/2MS+6BA 1.0mg/L+NAA 0.05mg/L+Hyg 25 mg/L+Tintin 200mg/L

MS4 medium: 1/2MS+NAA 0.1mg/L+Hyg 25 mg/L+Tintin 200mg/L

The specific test scheme of this example is as follows:

EXAMPLE 1 Gene cloning

The specific primer sequences for cloning the MYB4a gene of the swallow flower are as follows:

MYB4a-F1：5’-GAAGAAGAAATGGTGAGGACAAAGAATTCT-3’

MYB4a-R1：5’-TCCAATATTATTAACCGGATGGGTGATGCT-3’

a50. Mu.L PCR reaction system was prepared using the E.Yanhua column cDNA as a template, which included 2. Mu.L template cDNA, 25. Mu.L 2X PCR buffer for KOD FX, 10. Mu.L 2mM dNTPs, 1. Mu.L each of the upstream and downstream primers, 1. Mu.L KOD FX and 10. Mu.L ddH2O, and the sample addition was performed on ice, and after the addition, the mixture was homogenized and centrifuged.

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.

Amplifying by PCR to obtain an amplified product; the amplified product was ligated to cloning vector pEASY-Blunt-Zero (full gold, china), E.coli DH 5. Alpha. Was transformed and spread on LB solid containing 100mg/L Amp resistance, cultured upside down in an incubator at 37℃for 10-12 hours, the monoclonal strain was picked up for PCR identification of bacterial liquid, positive clones were expanded for 10mL in LB liquid medium containing 100mg/L Amp resistance, and then sent to Bio-company for sequencing.

The nucleotide sequence of the obtained Yan flower MYB4a gene is shown as follows:

ATGGTGAGGACAAAGAATTCTTCCTCCTCCTCCTCCTCGGCCTCGGCCTGTCCTAAGGAAGGGCTGAAGAGGGGAGCGTGGACAAGCCAAGAAGACGAGCTGTTATCCGACTACATCAGTGCTCATGGCCTTGGGAGGTGGCGAACCTTACCGGCCAATGCAGGTTTGAACAGGTGTGGCAAGAGTTGCAGGCTGCGATGGTTGAACTATCTGAGACCACATATTAAAAGAGGGAATATCACTGAGGAAGAGGAGGAGTTGATCGTTCGCCTCCATAAACTACTTGGCAACAGATGGTCACTAATAGCAGGAAGGCTGCCCGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACCCATATCAGAAGGAAGCTCTTAGGCTGCACCTCAAAGAACACTACCAAGACTGAAGAACCAAGCAGTACTCCTCCTCCTCCTCAAGAAGTAGTAGATCAAGAGGCGGCTGCTGCTGCTGCTGCAGAGAAAGCTGATCATGTAGTGAAGGTAATCCGGACAAAGGCGGTGAGATGCACGAGAACTCTAGTCTTTGCCGATCCATATTTACCTGCAAGCTCGGAGATCAAGAACAATATCTCTCCTCCACCTACAGATCATTCCCAGCTGGCTGCGGCGGACATGGATCATGAACCTTGGATGGCAGAACAACTTGTGCAGATTCCTCAGCTCTCCTACAACGATAGCCCTTACACTAGCAACAGCGTCGACAGCTTGCATAATAATAATAATGTCGGGGGGGCTACTAACGACGTTTCTATGATCCATGAAGACGATGATCAGTATCTGCAGGGAGAGTGGTTCGGCAGCAGTGGGATATTGAAGGAAGGGGAGGAGGGGCTCTACTCTCTTCTTGATCCTGATACATGGGACTCCCTCACATTCACTCAGAAGAATATCCACTCCGAGCATCACCCATCCGGTTAA

the ORF region of the MYB4a gene of the swallow flower contains 945 bases and encodes 314 amino acids, and the amino acid sequence is shown as follows:

MVRTKNSSSSSSSASACPKEGLKRGAWTSQEDELLSDYISAHGLGRWRTLPANAGLNRCGKSCRLRWLNYLRPHIKRGNITEEEEELIVRLHKLLGNRWSLIAGRLPGRTDNEIKNYWNTHIRRKLLGCTSKNTTKTEEPSSTPPPPQEVVDQEAAAAAAAEKADHVVKVIRTKAVRCTRTLVFADPYLPASSEIKNNISPPPTDHSQLAAADMDHEPWMAEQLVQIPQLSYNDSPYTSNSVDSLHNNNNVGGATNDVSMIHEDDDQYLQGEWFGSSGILKEGEEGLYSLLDPDTWDSLTFTQKNIHSEHHPSG

EXAMPLE 2 plant expression vector construction

(1) Designing carrier homology arm primers with Sal I and BamH I enzyme cutting sites respectively by using a homologous recombination method, using MYB4a plasmid connected with a cloning carrier as a template, amplifying the Yan flower MYB4a gene with GV1300 carrier homology arms, and recovering target fragments. The vector homology arm primers (underlined as SalI and BamHI cleavage sites) were as follows:

MYB4a-F2：5’-TTGATACATATGCCCGTCGACATGGTGAGGACAAAGAATTCTTC-3’

MYB4a-R2：5’-CCCTTGCTCACCATGGATCCACCGGATGGGTGATGCTCGGAGT-3’

(2) The expression vector GV1300-GFP plasmid is digested by Sal I and BamH I restriction enzymes, the vector fragment is recovered, the linearization vector is connected with MYB4a gene fragment added with a vector homology arm, the competent DH5 alpha of the escherichia coli is transformed and coated on LB solid containing 100mg/L Amp resistance, the mixture is inversely cultured for 10-12 hours in a 37 ℃ incubator, a monoclonal strain is selected for bacterial liquid PCR identification, positive clone is amplified and cultured for 10mL in LB liquid medium containing 100mg/L Amp resistance, and then the positive clone is sent to a biological company for sequencing. The vector was constructed using ClonExpress II One Step Cloning Kit (Nor praise, china) homologous recombination kit according to the instructions.

EXAMPLE 3 subcellular localization

By a method for transiently transforming Nicotiana benthamiana, subcellular localization detection is carried out on MYB4a protein, and the subcellular localization is concretely carried out as follows:

(1) Transforming the plasmid GV1300-MYB4a-GFP into agrobacterium tumefaciens EHA105 (Veidi, china) by a freeze thawing method;

(2) After positive clones were obtained, single colonies were picked and inoculated into LB liquid medium containing 50mg/L Kana, 25mg/L Rif, and cultured overnight with shaking at 180rpm in a shaker at 28 ℃;

(3) Inoculating 50 μL of fresh bacterial liquid into 10mL of LB liquid medium containing 50mg/L Kana, 25mg/L Rif, 10mM MES and 40 μM AS, shake culturing at 28deg.C for 14h, and expanding culturing to strain growth log phase;

(4) The next day, 4000g was centrifuged for 10min to collect the cells, the supernatant was discarded, and 10mM MgCl was used ₂ Resuspension of the cells, and adjusting the OD of each cell by a spectrophotometer ₆₀₀ 1.5, adding a certain amount of AS (acetosyringone) into all the resuspension bacteria liquid to make the final concentration 200 mu M, and standing for 3 hours at room temperature;

(5) The method comprises the steps of (1) pouring water to Nicotiana benthamiana one day before injection, placing the Nicotiana benthamiana in a low light condition as much as possible before injection, selecting tobacco leaves with good growth state, pricking a hole on the back of the leaves by using a needle head of a 1mL syringe, removing the needle head, and sucking mixed bacterial liquid from the back of the tobacco to inject into the leaves;

(6) Culturing the injected tobacco in dark for 12h, taking out, culturing under normal illumination for 3d, tearing off the lower epidermis of the injection leaf, and placing on a glass slide to prepare the temporary water-filled sheet. GFP expression was observed using a laser confocal microscope (LSM 800 with Airyscan, ZEISS, germany).

EXAMPLE 4 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 seedlings.

2. Preparation of bioengineering bacteria

(1) Agrobacterium was transformed with EHA105 (Veidi, china) competent for the transformation procedure as described in the specification.

(2) Picking single colony on a transformation plate to 10mL of YEP liquid culture medium containing 50mg/L Kana and 25mg/L Rif, and shaking and culturing at 28 ℃ and 180rpm for 12-16h;

(3) 1mL of the bacterial liquid is absorbed and added into 50mL of YEP (or LB) liquid medium containing 50mg/L Kana and 25mg/L Rif, and the bacterial liquid is cultured at 28 ℃ and 180rpm in a shaking way until the bacterial liquid reaches OD ₆₀₀ Reaching 0.6 to 0.8;

(4) The cultured bacterial liquid is centrifuged at 4500rpm for 5min, the supernatant is discarded, the bacterial cells are resuspended in an equal volume (50 mL) of 1/2MS osmotic medium (pH 5.8) under aseptic conditions, and the resuspension is placed on ice for tobacco infection.

3. Tobacco infection

(1) Pre-culturing. 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;

(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 4min, 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. The co-cultured leaf was inoculated on MS2 medium for light culture, and Hyg resistance selection was performed. Non-transgenic tobacco as a control was also inoculated on a medium containing Hyg resistance, and under normal conditions, non-transgenic tobacco leaves gradually died;

(5) And (5) subculturing. Inoculating tobacco leaves on MS3 culture medium for subculture about 1-2 weeks;

(6) And (5) rooting. When adventitious buds grow out of the edges of the leaves by about 1cm, cutting off the adventitious buds, transferring the adventitious buds to an MS4 culture medium, and inducing rooting;

(7) Transplanting. After the developed root system of the seedling grows out, uncovering and culturing for 2 days to exercise the seedling, adding a small amount of sterile water into a culture bottle to keep a culture medium moist, cleaning agar carried by the plant root system before transplanting, planting the plant root system into a small basin containing sterilized soil (garden soil: vermiculite=3:1), and culturing at room temperature;

(8) Extracting DNA of the transgenic tobacco plant, and carrying out PCR identification by adopting MYB4a homology arm primers to obtain the transgenic positive tobacco plant;

4. analysis of results

(1) Subcellular localization analysis of MYB4a protein of swallow flower

Through transient transformation of Nicotiana benthamiana leaves, GFP green fluorescent signal distribution is observed under a laser confocal microscope, the empty carrier green fluorescent signal is found to be uniformly distributed in the whole cell, and the swallow flower MYB4a is positioned in the cell nucleus (figure 1), which shows that the swallow flower MYB4a has the general characteristics of transcription factors, belongs to transcription factor proteins, plays a transcription regulation function in the cell nucleus and participates in a specific biological process.

(2) Identification of transgenic positive tobacco plants

The DNA extraction was performed on the 5 transgenic tobacco plants obtained, and PCR identification was performed using homology arm primers (MYB 4a-F2 and MYB4 a-R2), wherein wild type tobacco was the control and GV1300-MYB4a-GFP recombinant plasmid was the positive control, and as a result, the 5 transgenic tobacco lines all contained the desired band, indicating that MYB4a gene had been successfully inserted into the tobacco genome (fig. 2).

(3) Post phenotype observation of transgenic positive tobacco plants

By comparing the columns of wild-type tobacco and transgenic tobacco plants, it was found (FIG. 3) that the wild-type tobacco columns were flush with or slightly below the stamens in order to facilitate pollination and setting; the style of the transgenic tobacco plant is obviously 2-3mm higher than that of the stamen, and is not easy to pollinate and firm.

Claims (9)

1. A swallow flower MYB4a transcription factor is characterized in that the gene sequence is shown as SEQ ID NO. 1.

2. The protein sequence coded by the oat flower MYB4a gene of claim 1 is shown as SEQ ID NO. 2.

3. The biological material related to the oat flower MYB4a gene of claim 1, characterized by comprising any one of the following (A1) to (A3):

(A1) A plant expression vector containing a swallow flower MYB4a gene;

(A2) A bioengineering bacterium comprising the plant expression vector of (A1);

(A3) A transgenic plant comprising the plant expression vector of (A1).

4. The biological material related to the MYB4a gene of swallow flower according to claim 3, wherein:

the plant expression vector is GV1300-GFP.

5. The biological material related to the MYB4a gene of swallow flower according to claim 3, wherein:

the bioengineering bacteria is Agrobacterium tumefaciens EHA105.

6. Use of the biological material related to the MYB4a gene of swallow flower according to any one of claims 3-5 for regulating elongation of plant flower column.

7. The method of controlling plant pillar elongation according to claim 5, wherein:

(B1) Introducing the swallow flower MYB4a gene into a receptor plant to obtain a transgenic plant;

(B2) Over-expressing the swallow flower MYB4a gene in a receptor plant;

(B3) Silencing or inhibiting expression of MYB4a gene in the bird's nest.

8. The method of introducing a plant expression vector into a recipient plant according to claim 7, wherein:

plant expression vectors containing the Yan flower MYB4a gene are transformed into plant tissues through an agrobacterium-mediated method, and the transformed plant tissues are cultivated into plants.

9. The biomaterial of claim 3 or the use of claim 6 or the method of any one of claims 7, wherein the recipient plant is a bird's nest or a nicotiana tabacum.