CN111088261B

CN111088261B - Petunia floral organ development gene PhDof28 and application thereof

Info

Publication number: CN111088261B
Application number: CN202010047408.XA
Authority: CN
Inventors: 岳远征; 杜菊花; 史俐莎; 刘家伟; 杨秀莲; 王良桂
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Jiade Ecological Environment Technology Co ltd
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2021-09-28
Anticipated expiration: 2040-01-15
Also published as: CN111088261A

Abstract

The invention discloses a petunia floral organ development gene PhDof28 and application thereof, belonging to the technical field of plant genetic engineering. The invention clones 1 gene PhDof28 which can play an important regulation role in the development of floral organs from petunia, the gene sequence of the gene PhDof28 is shown as SEQ ID NO.1, and the expression protein is shown as SEQ ID NO. 2. In the invention, a plant expression vector pCAMBIA2300-PhDof28 is constructed to transform petunia and tobacco, and the excess expression of the PhDof28 gene is found under the drive of a CaMV35S promoter, and the PhDof28 gene synthesized in large quantity promotes the increase of petunia corolla, the growth of receptacle, the increase of tobacco corolla diameter and the deepening of flower color, thus showing that the PhDof28 gene plays an important role in the aspect of regulating and controlling the development of flower organs. The invention provides a new gene resource for improving the growth of plant floral organs and improving the ornamental value.

Description

Petunia floral organ development gene PhDof28 and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a petunia floral organ development gene PhDof28 and application thereof.

Background

The Dof gene family protein is a specific transcription regulation factor of plants, interacts with other family protein members besides the family protein, and has important regulation and control functions in the aspects of plant tissue differentiation, seed development, stress, plant physiological metabolism and the like. Since the first ZmDof1(MNB1a) transcription factor was identified and reported in maize in 1993, the Dof gene has been found in a variety of monocots and dicots. Among them, 37 Dof genes are present in the arabidopsis genome (Yanagisawa, 2002), 30 in the rice genome (Lijavetzky et al, 2003), 26 in barley (Moreno-Risueno et al, 2007b), 28 in soybean (Wang et al, 2007), and Dof genes are also present in plants such as pumpkin (Kisuet al, 1998), tobacco (Baumann et al, 1999), wheat (Chen et al, 2005), and potato (Plesch et al, 2001). The Dof protein generally consists of 200-400 amino acids, and has a specific and highly conserved DNA binding domain, namely a Dof domain consisting of 52 amino acids at the N-terminal. The Dof protein interacts with the promoters of different plant-specific genes by its Dof domain, and AAAG sequences are present in the DNA binding sequence of each Dof protein. The AAAG sequence and its reverse complement CTTT are core sequences recognized by Dof proteins. In addition to the Dof domain, another major domain comprised by Dof proteins is the regulatory domain located at the C-terminus. Unlike the conserved Dof domain, the amino acid sequence of the transcriptional regulatory domain is more variable and not conserved, and it is likely to activate or inhibit gene transcription by reacting with different types of regulatory proteins or substances, and being regulated by different pathway signals. This diversity may be one of the bases for the diversity of Dof functions.

Petunia hybrida (Petunia hybrida) belongs to Solanaceae (Solanaceae) Petunia (Petunia), is one of the most important horticultural ornamental plants in the world, and can be propagated by seeds, cuttage, grafting and the like due to short growth cycle, clear genetic background and short growth cycle, so that the Petunia hybrida can be used as a model plant for researching the gene function and the flower development molecular mechanism. The related gene Dof gene is separated and cloned from petunia and the function of the gene Dof gene is disclosed, which is beneficial to researching the molecular regulation mechanism of the Dof gene family in the growth and development of plants so as to be further applied to the character improvement of the plants.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a petunia floral organ development gene PhDof 28. The invention also aims to provide application of the petunia floral organ development gene PhDof 28.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a petunia floral organ development gene PhDof28 has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the petunia organ development gene PhDof28 is shown in SEQ ID NO. 2.

The carrier, the recombinant bacteria or the cells containing the petunia organ development gene PhDof 28.

Further, the vector of the petunia floral organ development gene PhDof28 is a plant expression vector.

Furthermore, the plant expression vector is pCAMBIA2300-PhDof 28.

The petunia organ development gene PhDof28 is applied to improvement of plant flower ornamental characters.

Further, the application comprises the following steps:

1) constructing a vector of the petunia floral organ development gene PhDof 28;

2) transforming the constructed petunia organ development gene PhDof28 vector into plants or plant cells;

3) and culturing and screening to obtain transgenic plants with enlarged crown diameter, lengthened flowers or deepened flower colors.

In the application, the plant is petunia or tobacco.

In the application, the carrier of the petunia organ development gene PhDof28 is pCAMBIA2300-PhDof 28.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the petunia floral organ development gene PhDof28 provided by the invention is a new floral development gene, can regulate and control the development of plant floral organs, and can change the size of the plant floral organs, especially the size of petals, so that the petunia floral organ development gene PhDof28 can be applied to transgenic improvement of the ornamental characters of plants;

(2) the invention lays a molecular foundation for researching the regulation and control relation of the Dof transcription factor in the petal extension process, deepens the cognition on the petal size regulation and control network, and provides a new gene resource.

Drawings

FIG. 1 is a full-length amplification diagram of PhDof28 gene;

FIG. 2 is a diagram showing the result of positive detection of the overexpression vector of pCAMBIA2300-PhDof 28;

FIG. 3 is a subcellular localization map of the PhDof28 gene;

FIG. 4 is a diagram showing the result of PCR positive detection of PhDof28 transgenic petunia plants;

FIG. 5 is a phenotypic picture of transgenic petunia 96 line overexpressing the PhDof28 gene; note: a is corolla, B is flower, C is peduncle, D is anther; e is stigma and F is calyx;

FIG. 6 is a diagram of the prominent elongated portions of petunia petals;

FIG. 7 is a diagram showing the result of PCR positive detection of PhDof28 transgenic tobacco plants;

FIG. 8 is a graph showing the results of semi-quantitative analysis of positive lines of PhDof28 gene transferred from tobacco;

FIG. 9 is a diagram showing the result of PCR expression analysis of PhDof28 gene Real-Time at different flowering stages of transgenic tobacco;

FIG. 10 is a phenotypic plot of transgenic tobacco 31 lines overexpressing PhDof 28;

FIG. 11 is a scanning electron microscope analysis diagram of PhDof28 transgenic tobacco 28 strain and 31 strain; note: displaying a petal picture on a sampling part of a scanning electron microscope; up refers to the cell morphology of the upper half part of the petal; mid refers to the partial cell morphology in the petals; down refers to the cell morphology of the lower half of the petals.

Detailed Description

The invention is further described with reference to specific examples.

Example 1: cloning of petunia floral organ development Gene PhDof28

Experimental materials: petunia hybrida variety 'fantasy' is provided by a key laboratory of the scenic garden school of Nanjing forestry university, sown in a growth room of the scenic garden school of Nanjing forestry university and grown under natural conditions.

The gene source is as follows: based on the existing petunia anther transcriptome database (unpublished) of the previous subject group, the selected petunia anther transcriptome database was named as PhDof 28.

The specific steps of the PhDof28 amplification in the petunia total RNA are as follows:

1) the total RNA of the petunia tissue sample is extracted according to an RNA extraction kit of Beijing Edley company, and then the quality and the concentration of the extracted RNA are detected by 1.0 percent agarose gel electrophoresis and a spectrophotometer.

2) Carrying out reverse transcription on petunia total RNA which is ten times diluted into cDNA according to a Beijing all-type gold reverse transcription kit, and storing the cDNA at-20 ℃ for later use;

reverse transcription system: reverse transcription system (20 μ L): mu.L of gDNA Remover, 1. mu.L of absorbed Oligo (dt)18Primer (0.5. mu.g/pL), 1. mu.L

RT/RI Enzyme Mix，10μL 2×TX Reaction Mix，7μL Total RNA。

Reverse transcription reaction conditions: 30min at 42 ℃ and 5s at 85 ℃.

3) Amplification primers were designed using PrimerPremier 5 as follows:

the upstream primer PhDof 28-F: 5'-GCTAAGGTGAAGTTTTCATAATTGT-3', respectively;

the downstream primer PhDof 28-R: 5 'AAAAAGGAGAGATATAGATCAAGGTAG-3'.

The full-length amplification of the target gene was carried out according to Prime STAR high fidelity enzyme of TaKara Biotechnology Ltd, and the target band was detected by 1.5% agarose gel electrophoresis and the product was recovered to obtain a full-length DNA fragment (FIG. 1).

The full-length gene amplification reaction system (20 mu L) specifically comprises the following steps: 1 μ L of cDNA, 1 μ L of antisense Primer (PhDof28-F) (10mM), 1 μ L of Sense Primer (PhDof28-R) (10mM), 10 μ L of PrimeSTAR Hi-Fi enzyme, 7 μ L of 1st Strand cDNA. Reaction conditions are as follows: 10s at 98 ℃; 25s at 58 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃ and infinity at 16 ℃.

4) And recovering a target fragment, connecting the target fragment with a pEASY-Blunt vector, transforming escherichia coli, detecting positive recombinant clone, and sequencing to finally obtain the cDNA nucleotide sequence of petunia PhDof28 with a complete coding region, wherein the cDNA nucleotide sequence is shown as SEQ ID NO.1 and has the length of 966bp, and the amino acid sequence of the coding protein is shown as SEQ ID NO.2 and has the length of 319 aa. The specific reaction system and reaction conditions are as follows:

PCR recovery product and pEASY-Blunt ligation reaction system:

cloning system (5 μ L): 4 μ L CRproduct, 1 μ L pEASY-Blunt. Reaction conditions are as follows: reacting at room temperature for 5min, and cloning at constant temperature of 25 ℃ for 15 min.

Example 2: construction of plant expression vector pCAMBIA2300-PhDof28

And (3) plasmid extraction:

the extraction of pCAMBIA2300 plasmid, V097 plasmid, V197 plasmid and T vector plasmid carrying target gene all adopt high-purity plasmid extraction kit of Beijing Tiangen Biotechnology Co.

Extracting T vector containing target fragment and expression vector pCAMBIA2300 plasmid DNA, carrying out double enzyme digestion, and respectively recovering the fragment containing PhDof28 complete open reading frame and pCAMBIA2300 plasmid fragment. Then, the connection is carried out, the connected recombinant plasmid pCAMBIA2300-PhDof28 is transformed into an escherichia coli competent cell, a positive plasmid is extracted, enzyme digestion electrophoresis detection is carried out, and sequencing verification is carried out.

Double digestion reaction system (50 μ L): 1 μ L plasmid, 1 μ L QuickCut enzyme I, 1 μ L QuickCut enzyme II, 5 μ L10 XQuickCut Buffer, ddH₂And O is supplemented to 50 mu L.

After the system is prepared, vortex mixing is carried out, instantaneous centrifugation is carried out, the mixture is placed in a constant-temperature water bath kettle at 37 ℃ for reaction for 1 hour, finally, 1.5% agar gel is used for detecting double enzyme digestion conditions, gel cutting and recovery are carried out, finally, a nucleic acid determinator is used for detecting the concentration of a recovered product, and 1% agar gel is used for detecting whether recovery is correct or not (figure 2).

The method mainly comprises the following steps:

1) the vector plasmid DNA and the inserted target gene DNA were mixed at a molar ratio of 0.03 pmol: 0.03 to 0.3pmol was mixed to prepare a DNA solution having a volume of 5 to 10. mu.L.

2) Adding Solution I into the DNA Solution, mixing uniformly by vortex, centrifuging instantaneously, and reacting for 1h in a constant-temperature water bath kettle at 16 ℃. mu.L of the ligation product was removed from the solution and used for general PCR template to determine whether ligation was successful.

3) To the successfully ligated solution, 1. mu.L of solution III was added to the remaining solution, gently mixed, then 5. mu.L of the solution was pipetted into 50. mu.L of competent cells Trans-T1, plated on a Kana-resistant medium, and cultured overnight by inversion at 37 ℃. And (3) selecting full monoclonals on each plate by using a sterile toothpick, firstly backing up on a new plate, and then dipping the plate into a prepared 20-mu-L PCR reaction system for bacteria detection, wherein the bacteria detection result is better transmitted to detection, bacteria preservation and plasmid extraction.

4) PCR detection reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 2min, and 35 cycles; extension at 72 ℃ for 10 min.

Example 3: subcellular localization study of PhDof28 Gene

Primers for amplifying a complete coding reading frame are designed according to the full-length cDNA sequence of PhDof28 obtained in example 1, and Sal I and Kpn I enzyme cutting sites are introduced into the primers. The primers were designed as follows:

F：5′-ctgcaggggcccggggtcgacATGCAAGATATACATGCGATTGGT-3′；

R：5′-gcccttgctcaccatggtaccAGGTAGAAAATTAAGTGACTGATCATTTT-3′。

PhDof28 containing the complete ORF was isolated from petunia cDNA by RT-PCR using the primers described above. The ORF of the fragment was fused to the 5' end of the GFP reporter gene in pCAMBIA1300 to form a GFP: : pCAMBIA1300-PhDof28 chimeric gene. After enzyme digestion verification, agrobacterium is transformed by an electrotransformation method with the empty vector pCAMBIA1300, and then the Benzenia is transformed by an instant transformation method, and the experimental result shows that petunia PhDof28 is mainly positioned in a cell nucleus (figure 3).

The method comprises the following specific steps:

1) preparing a bacterial liquid buffer solution: 10 mM. L^-1MgCl₂，10mM·L^-1Biological buffer MES, 150. mu.M.L^-1Acetosyringone (insoluble in water, solubilized with dimethyl sulfoxide).

2) GV3101, pCAMBIA1300-PhDof28 fusion expression vector, pCAMBIA1300 no-load, and P19 auxiliary expression vector of successfully transformed Agrobacterium were thawed on ice. Then, 50. mu.L of each of the solutions was added to 30ml of LB liquid culture medium (containing 100 mg. multidot.L)^-1Kanamycin) was cultured at 28 ℃ with shaking at 200rpm in the dark to OD₆₀₀Between 0.6 and 1.0. 5000 r. mjn at 4 DEG C^-1Centrifuging for 5min by centrifuge, collecting thallus, resuspending thallus with buffer solution, and mixing with the resuspended bacteria solution at a certain ratio (OD)₆₀₀Ratio of 7: 5) and standing at room temperatureStanding for 2-3h for later use.

3) Injecting mixed bacteria-containing liquid into the back of the ben tobacco leaf by using a 1mL medical injector, culturing in an incubator for 2-3d, observing the expression condition of pCAMBIA1300-PhDof28-GV3101 in the epidermal cells under the tobacco under a laser confocal microscope, respectively observing under a GFP green fluorescence Field (GFP), a Chloroplast pink fluorescence Field (Chloroplast), a white light Field (Bright Field) and a mixed Field of the three (Merged), and simultaneously, taking the expression condition of agrobacterium GV3101 carrying an empty vector pCAMBlA1300 in the epidermal cells under the tobacco as a control. As shown in fig. 3, the results indicated that petunia PhDof28 was mainly localized to the nucleus.

Example 4: phenotypic observation of petunia transformed with PhDof28 plant expression vector

(1) EHA105 competent cell transformed by expression vector electrotransformation

Adding 30-35 mu LEHA105 competent cells into 1 mu L of expression vector plasmid (a product obtained in the embodiment 2), carrying out ice bath for 30min together with a cleaned and dried electric rotating cup, carrying out electric rotation by using an electric rotating instrument, adding 400 mu L of LB culture solution without antibiotics into the electric rotating cup for activation, culturing for 1h by using a shaking table at 200rpm at 28 ℃, coating a bacterial solution on a solid culture medium containing kanamycin resistance, carrying out dark culture in an incubator at 28 ℃ for 2 days, picking out a monoclonal for detection, and selecting a full positive colony shake bacteria for tobacco and petunia transformation.

(2) Genetic transformation of petunia

Adding 30ml of fresh LB culture solution into 20. mu.L of the above bacterial solution, shaking at 200rpm and 28 ℃ overnight, and performing suspension culture until OD is reached₆₀₀The value is 0.5, the strain is collected by centrifugation at 5000rpm and 4 ℃ for 10min, and 30ml of LB culture solution is added for bacteria shaking recovery. Taking two or three fresh and tender leaves of petunia and tobacco, disinfecting the leaves with 75% alcohol for 10s, cleaning the leaves with ultrapure water for 3 times, disinfecting and sterilizing the leaves with 0.1% mercuric chloride for 30min, rinsing the leaves with ultrapure water for three times under an aseptic condition, finally cutting the leaves into 5mm by 5mm, soaking the cut leaves in the suspension for 10min, then culturing the cut leaves on a co-culture medium for 3d, inducing the sprouts to root and transplanting the cut leaves.

(3) PCR identification and plant morphology observation

1) DNA detection

Using Beijing TiangenExtracting leaf DNA by using a DNA extraction kit of a biological company, diluting the extracted DNA by 50 times, and performing PCR detection, wherein the reaction system in the PCR reaction is as follows: upstream and downstream primers 1. mu.L, ddH₂O7. mu.L, Green enzyme 10. mu.L, diluted DNA template 1. mu.L. The PCR reaction program comprises pre-deformation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 90s, extension at 72 ℃ for 10min, 35 cycles and reaction termination at 16 ℃. The PCR products were detected by electrophoresis on 1% agar gel and the results were observed on an ultraviolet gel imager, which indicated that PhDof28 successfully transformed petunia (FIG. 4).

2) Plant morphology observation (FIGS. 5 and 6)

And (3) carrying out phenotype observation on the petunia plants with positive PCR detection, and observing the change of the flower organs of the transgenic plants from the development of flower buds. The flower organs, including calyx and petals, of the transgenic positive plants are observed to be larger and longer than the wild type. The parts mainly increasing the elongation are the upper part of the petals (corolla), the lower part of the petals and the middle part of the petals (receptacle), particularly the petals are obviously much larger than the wild type in the development period.

Example 5: phenotypic observation of PhDof28 plant expression vector transformed tobacco

1. Cultivation of tobacco aseptic seedling

(1) Preparation work:

and (3) sterilization: tips of 1000. mu.L and 200. mu.L (a small head is cut off from the tip of 1000. mu.L in advance to avoid too large seeds to be sucked up), toothpick, 1.5mL centrifuge tube, ddH₂O, 200mL Erlenmeyer flask (for waste liquid).

(2) Preparing samples and reagents: gun and tip of 1000. mu.L and 200. mu.L, small amount of sealing film, MS culture medium, 8% sodium hypochlorite (stored in dark place), 75% alcohol (stored in dark place), toothpick, 1.5mL centrifuge tube, ddH₂O, conical flask (for waste liquid).

(3) The specific operation steps are as follows:

1) taking a proper amount of seeds, putting the seeds into a 1.5mL sterile centrifuge tube, adding 1mL 8% sodium hypochlorite, fully vortex and oscillate for 5min, standing for 1min-3min, completely sterilizing, sucking out the sodium hypochlorite (avoiding sucking out the seeds as much as possible) by using a sterilized gun head, and sterilizing once.

2) Adding 1mL of 75% alcohol, vortexing and shaking for 30s, standing for 30s, immediately sucking out the alcohol to avoid reducing the activity of the seeds, and sterilizing once.

3) Add 1mL sterile ddH immediately₂And O, vortex and shake for 1min, stand for 1min, and wash for 3-5 times.

4) Blowing the prepared MS culture medium, adding a small amount of ddH into the cleaned seeds₂And O, sucking out and beating the mixture on the surface of the MS culture medium by using a 1000-mu L gun head with a small section of head cut off, slightly and uniformly spreading the mixture by using sterilized toothpicks to avoid over-dense growth of seeds, sealing the culture medium by using a sealing film and marking.

5) The sealed culture medium is sealed by tinfoil paper, placed in a refrigerator at 4 ℃ for vernalization for two days, and then placed on a culture shelf for illumination culture.

6) After two true leaves grow out, the leaves can be transferred into sterilized nutrient soil for cultivation.

2. Plant expression vector transformation plant

Adding 30-35 mu LEHA105 competent cells into 1 mu L of expression vector plasmid (a product obtained in the embodiment 2), carrying out ice bath for 30min together with a cleaned and dried electric rotating cup, carrying out electric rotation by using an electric rotating instrument, adding 400 mu L of LB culture solution without antibiotics into the electric rotating cup for activation, carrying out culture for 1h by using a shaking table at 200rpm at 28 ℃, coating a bacterial solution on a solid culture medium containing kanamycin resistance, carrying out dark culture in an incubator at 28 ℃ for 2 days, picking out a monoclonal for detection, and selecting a full positive bacterial colony for shake culture.

(2) Tobacco transformation by leaf disc method

Adding 30ml of fresh LB culture solution into 20. mu.L of the above bacterial solution, shaking at 200rpm and 28 ℃ overnight, and performing suspension culture until OD is reached₆₀₀The value is 0.5, the strain is collected by centrifugation at 5000rpm and 4 ℃ for 10min, and 30ml of LB culture solution is added for bacteria shaking recovery. Taking two or three fresh and tender leaves of tobacco, sterilizing with 75% alcohol for 10s, cleaning with ultrapure water for 3 times, sterilizing with 0.1% mercuric chloride for 30min, rinsing with ultrapure water for three times under aseptic condition, cutting into 5mm × 5mm, soaking in the suspension for 10min, culturing on co-culture medium for 3d, inducingThe buds are transplanted after rooting, and can be cultured on a strong seedling culture medium and a rooting culture medium to ensure that the seedlings grow as strong as possible and the root systems are transplanted as thick as possible.

Various media formulations:

co-culture medium: MS +0.3mg/L NAA +2.0 mg/L6-BA

Strong seedling culture medium: MS +0.01mg/LNAA +0.1 mg/L6-BA +50mg/L Kana +300mg/L Cef

Rooting culture medium: 1/2MS +50mg/L Kana +300mg/L Cef

3. PCR identification and plant morphology observation

(1) DNA detection

Extracting leaf DNA by using a DNA extraction kit of Beijing Tiangen biological company, diluting the extracted DNA by 50 times, and then performing PCR detection, wherein the reaction system in the PCR reaction is as follows: upstream and downstream primers 1. mu.L, ddH₂O7. mu.L, Green enzyme 10. mu.L, diluted DNA template 1. mu.L. The PCR reaction program comprises pre-deformation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 90s, extension at 72 ℃ for 10min, 35 cycles and reaction termination at 16 ℃. The PCR product was detected by electrophoresis on 1% agar gel, and the result was observed on an ultraviolet gel imager, which showed that PhDof28 successfully transformed tobacco (FIG. 7).

The detection primers are as follows:

35SF：5′-ACGCACAATCCCACTATCCTTC-3′；

Dof28-R：5′-AAAAAGGAGAGATATAGATCAAGGTAG-3′。

the PCR positive detection system (20. mu.L) was as follows: mu.L of DNA (50-fold dilution of template), 1. mu.L of 35SF, 1. mu.L of LDof28-R, 10. mu.L of green enzyme, 7. mu.L of ddH₂O。

The reaction conditions for PCR positive detection were as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 59 ℃, 1min at 72 ℃ and 35 cycles; 10mjn at 72 ℃; 16 ∞.

(2) Semi-quantitative detection of expression level

For the transgenic plants with positive DNA detection and phenotype, total RNA is extracted and subjected to reverse transcription (refer to embodiment 1), and then the quality and the expression quantity of the template are semi-quantitatively detected.

The primer sequences were designed as follows:

qRT-PCR primers for the PhDof28 gene were:

QPhDof28-F：5′-GGGAACTTTACAAATCTGATGACG-3′；

QPhDof28-R：5′-GAAACCCGAACTATTACCACCC-3′。

the primers of the tobacco Actin gene are as follows:

Actin-R：5′-TGGTTGTGACTTTTGGTCCCA-3′；

Actin-F：5′-ACAAACCCACGCTTGAGATCC-3′。

the semi-quantitative system is as follows:

assay template quality (20 μ Ι _): mu.L of cDNA diluted 10-fold template, 1. mu.L of LActin-F, 1. mu.L of LActin-R, 10. mu.L of chlorophase, 7. mu.L of ddH₂O。

Detecting the specificity and expression quantity of the primers: mu.L of cDNA diluted 10-fold template, 1. mu.L of QPhDof28-F, 1. mu.L of QPhDof28-R, 10. mu.L of green enzyme, 7. mu.L of ddH₂O。

The PCR reaction program comprises pre-deformation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 90s, extension at 72 ℃ for 10min, 30 cycles and reaction termination at 16 ℃. And detecting the PCR product by using 1% agar gel electrophoresis, and observing the result in an ultraviolet gel imager. The results showed good quality of cDNA template and good primer specificity (FIG. 8).

(3) Detection of PhDof28 expression level of PhDof28 in different flowering phases by Real-Time PCR

cDNA of tobacco petals in three periods of large bud period, semi-open period and full-bloom period is used as a template, a specific primer of qRT-PCR is designed, and actin genes of tobacco are used as internal references to carry out qRT-PCR, wherein the sequence of the primer is shown in step (2).

The reaction system was as follows (10. mu.L): mu.L of cDNA (10-fold diluted template), 0.4. mu.L of Actin-F/QPhDof28-F, 0.4. mu.L of LActin-R/QPhDof28-R, 5. mu.L of SYBR GreenMaster Mix, 0.2. mu.L of calibrator Dye, 3. mu.L of ddH₂O。

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, plate reading at 79 ℃ for 1s, and 40cycles in total, after the reaction is completed, dissolution curve analysis is performed from 57 ℃ to 95 ℃, and the test results are repeated 3 times for each sample. The amplification efficiency of each PCR reaction was taken as 100%. The PhDof28 gene was expressed in each petal stage, but the expression level was different, relatively high in bud stage and full-bloom stage, and highest in open stage (FIG. 9).

(4) Observation of plant morphology (FIG. 10)

And (3) performing phenotype observation on the tobacco plants and wild plants which are positive in PCR detection, and observing the change of flower organs of the transgenic plants from the development of flower buds. The observation shows that the flower organs of the transgenic positive plants, including calyx, corolla, petals and the like, are larger and longer than the wild type. In the period of the petals to be developed, the main extending parts are the lower half part (down), the upper half part (mid) and the petal part (up) of the corolla; in addition, the lower half part of the flower crown cylinder extends most obviously and tends to become narrow and slender; the upper half part and the petal part of the corolla become longer, the whole surface area becomes larger, especially the petals are obviously larger than the wild type in the developing period, and the color is darker. To further verify the results of phenotypic observations, the epidermal cells of these three sites were observed by scanning electron microscopy to further verify our results (fig. 11). Scanning electron microscope results show that the cell morphology of the lower half part of the corolla tube of the transgenic positive plant becomes slender, the length of a single cell becomes long, the width becomes narrow, and the cell arrangement is more compact and ordered. And compared with wild plants, the cells on the upper half part of the corolla sleeve have obviously larger single cell volume. The cells in the petal part show the constant cell number, but the cell gaps become bigger, and the cells are more loosely arranged. The structures all show that PhDof28 is related to the size development and control of petunia floral organs, and can regulate the morphogenesis of floral organs in a way of regulating the volume and the arrangement of cells. Table 1 below presents statistics of petal and stem size and flower length for tobacco PhDof28 transgenic positive plants, validating the above observations.

Table 1: statistical table of petal and stem size and flower length of PhDof28 transgenic positive plants

Sequence listing

<110> Nanjing university of forestry

<120> petunia floral organ development gene PhDof28 and application thereof

<130> 100

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 966

<212> DNA

<213> Petunia hybrida

<400> 1

gctaaggtga agttttcata attgtattga gatatgcaag atatacatgc gattggtggt 60

ggaggtgcgc gatttttcgg aggtggtggt ggaggagata gaaggctaag gcctaacaat 120

caacataatc ttcaagcact aaagtgtccg cgttgcgact ctatcaatac aaaattttgt 180

tactacaaca actacaatct ctctcaacca cgtcacttct gcaaaagttg cagaaggtac 240

tggactaagg ggggcgttct tcggaacgtc cccgttggtg gtggatgccg aaaaagcaaa 300

cgttccaaac ccaaatcaaa ccccacaact acaactgatg ttgtagttga tggttcagaa 360

gaacctaaat cgaactctca ctcgagtagt gagagttcga gtctcaccgc taccacaact 420

gctgcagcag cgaatactgg aacaacttcc agttctgctg ctgctgcagc agaagttgtg 480

tcagcaaatt cttccaatgt ctcttcagca atgtttctca acttttcaga atccaacttc 540

tttgtaccta acagtacgaa cagtggcttt gatcatcatc atcatcatca acccgcgtta 600

attgatcata ctacagaagg tcagatgttc caagatattg ggaactttac aaatctgatg 660

acgccatcga acgacccagg gtcgtttaat atggtggaaa ttccggcctg gaatcatcaa 720

cattcgaaga tggatatgga aactgatcac gtgaagatgg aacagatggg tggtaatagt 780

tcgggtttca tgagtcaaac gagtcgtgtt ggacataaca gtgaagttgg tgcactggat 840

tggcaaattg gacatgggtt gtatgattta acagggaccg ttgatcaatc ttactggagt 900

caaacacagt ggggtgaaaa tgatcagtca cttaattttc taccttgatc tatatctctc 960

cttttt 966

<210> 2

<211> 319

<212> PRT

<213> Petunia hybrida

<400> 2

Ala Lys Val Lys Phe Ser Leu Tyr Asp Met Gln Asp Ile His Ala Ile

1 5 10 15

Gly Gly Gly Gly Ala Arg Phe Phe Gly Gly Gly Gly Gly Gly Asp Arg

20 25 30

Arg Leu Arg Pro Asn Asn Gln His Asn Leu Gln Ala Leu Lys Cys Pro

35 40 45

Arg Cys Asp Ser Ile Asn Thr Lys Phe Cys Tyr Tyr Asn Asn Tyr Asn

50 55 60

Leu Ser Gln Pro Arg His Phe Cys Lys Ser Cys Arg Arg Tyr Trp Thr

65 70 75 80

Lys Gly Gly Val Leu Arg Asn Val Pro Val Gly Gly Gly Cys Arg Lys

85 90 95

Ser Lys Arg Ser Lys Pro Lys Ser Asn Pro Thr Thr Thr Thr Asp Val

100 105 110

Val Val Asp Gly Ser Glu Glu Pro Lys Ser Asn Ser His Ser Ser Ser

115 120 125

Glu Ser Ser Ser Leu Thr Ala Thr Thr Thr Ala Ala Ala Ala Asn Thr

130 135 140

Gly Thr Thr Ser Ser Ser Ala Ala Ala Ala Ala Glu Val Val Ser Ala

145 150 155 160

Asn Ser Ser Asn Val Ser Ser Ala Met Phe Leu Asn Phe Ser Glu Ser

165 170 175

Asn Phe Phe Val Pro Asn Ser Thr Asn Ser Gly Phe Asp His His His

180 185 190

His His Gln Pro Ala Leu Ile Asp His Thr Thr Glu Gly Gln Met Phe

195 200 205

Gln Asp Ile Gly Asn Phe Thr Asn Leu Met Thr Pro Ser Asn Asp Pro

210 215 220

Gly Ser Phe Asn Met Val Glu Ile Pro Ala Trp Asn His Gln His Ser

225 230 235 240

Lys Met Asp Met Glu Thr Asp His Val Lys Met Glu Gln Met Gly Gly

245 250 255

Asn Ser Ser Gly Phe Met Ser Gln Thr Ser Arg Val Gly His Asn Ser

260 265 270

Glu Val Gly Ala Leu Asp Trp Gln Ile Gly His Gly Leu Tyr Asp Leu

275 280 285

Thr Gly Thr Val Asp Gln Ser Tyr Trp Ser Gln Thr Gln Trp Gly Glu

290 295 300

Asn Asp Gln Ser Leu Asn Phe Leu Pro Ser Ile Ser Leu Leu Phe

305 310 315

<210> 3

<211> 25

<212> DNA

<213> PhDof28-F primer sequence (Artificial)

<400> 3

gctaaggtga agttttcata attgt 25

<210> 4

<211> 27

<212> DNA

<213> PhDof28-R primer sequence (Artificial)

<400> 4

aaaaaggaga gatatagatc aaggtag 27

<210> 5

<211> 45

<212> DNA

<213> primer sequence PhDof28F (Artificial)

<400> 5

ctgcaggggc ccggggtcga catgcaagat atacatgcga ttggt 45

<210> 6

<211> 50

<212> DNA

<213> primer sequence PhDof28R (Artificial)

<400> 6

gcccttgctc accatggtac caggtagaaa attaagtgac tgatcatttt 50

Claims

1. Petunia organ development genePhDof28The nucleotide sequence is shown in SEQ ID NO. 1.

2. Petunia floral organ development gene of claim 1PhDof28The amino acid sequence of the expression protein is shown as SEQ ID NO. 2.

3. A petunia floral organ development gene comprising the petunia floral organ development gene of claim 1PhDof28The vector, the recombinant bacterium or the cell of (1).

4. Root of herbaceous plantThe gene containing organ development of petunia according to claim 3PhDof28The vector of (1), wherein the vector is a plant expression vector.

5. The genes for organ development containing petunia according to claim 4PhDof28The vector is characterized in that the plant expression vector is pCAMBIA2300-PhDof28。

6. Petunia floral organ development gene of claim 1PhDof28The application of the plant in improving the ornamental character of the plant flowers is that the plant is petunia or tobacco.

7. Use according to claim 6, characterized in that it comprises the following steps:

1) construction of petunia organ development GenePhDof28The overexpression vector of (1);

2) the constructed petunia organ development genePhDof28Transforming the overexpression vector into a plant or plant cell;

8. The use of claim 7, wherein the petunia organ developmental gene isPhDof28The vector is pCAMBIA2300-PhDof28。