CN111088259A

CN111088259A - PhDof4 gene related to petunia anther development and application thereof

Info

Publication number: CN111088259A
Application number: CN202010044917.7A
Authority: CN
Inventors: 岳远征; 杜菊花; 宾婧; 李娅; 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: 2020-05-01
Anticipated expiration: 2040-01-15
Also published as: CN111088259B

Abstract

The invention discloses a PhDof4 gene related to petunia anther development and application thereof, belonging to the technical field of plant genetic engineering. The petunia Dof gene family member PhDof4 gene disclosed by the invention has a nucleotide sequence shown as SEQ ID NO.1, and an amino acid sequence of an expression protein shown as SEQ ID NO. 2. The RNAi expression vector for constructing the PhDof4 gene is used for transforming petunia, so that the transgenic plant can be dwarfed, the flowering phase is advanced, and the anther is abnormal in development, which indicates that PhDof4 is an important gene for regulating and controlling the growth and development of the anther of the petunia. The invention also discloses a gene engineering improvement method for regulating the anther fertility of the gene, which can provide reference for further researching the molecular mechanism of the anther development of the petunia and has practical application value for male sterility gene engineering, hybrid breeding of the petunia, heterosis utilization and variety production.

Description

PhDof4 gene related to petunia anther development and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a PhDof4 gene related to petunia anther development 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 Dof gene is separated and cloned from petunia and the function of the Dof gene is revealed, 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 invention aims to provide a petunia anther development related gene PhDof 4. The invention also aims to provide application of the petunia anther development related gene PhDof 4.

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

a petunia anther development related gene PhDof4 has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the petunia anther development related gene PhDof4 is shown in SEQ ID NO. 2.

The carrier, the recombinant bacteria or the cell containing the petunia anther development related gene PhDof 4.

Preferably, the vector of the petunia anther development related gene PhDof4 is pEASY-Blunt-PhDof4 or PhDof 4-RNAi.

The petunia anther development related gene PhDof4 is applied to regulation and control of plant anther fertility or plant morphology.

Preferably, the application of the anther development related gene PhDof4 in regulating and controlling plant anther fertility comprises the following steps:

1) constructing a vector containing a petunia anther development related gene PhDof 4;

2) transforming the constructed vector of the petunia anther development related gene PhDof4 into plants or plant cells;

3) and culturing and screening to obtain a transgenic plant strain with anther abortion.

Preferably, the application of the anther development related gene PhDof4 in regulating and controlling plant morphology comprises the following steps:

3) and culturing and screening to obtain transgenic plant lines with dark green leaf color, short and small plants or shortened internodes.

Preferably, the vector containing the petunia anther development related gene PhDof4 is PhDof 4-RNAi.

Preferably, the plant is petunia.

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

(1) the invention discloses a petunia PhDof4 gene as a member of a PhDof gene family. The nucleotide sequence is shown as SEQID NO.1, and the amino acid sequence of the expression protein is shown as SEQID NO. 2;

(2) the invention preliminarily predicts the gene function based on the early-stage research and bioinformatics analysis software, obtains the full length of a gene sequence through gene cloning, determines the expression mode of the gene sequence through Real-Time PCR, identifies and analyzes the gene function through the technologies of subcellular localization, constitutive vector, transgenosis and the like, finds that the transgenic plant has dark green leaf color, advanced flowering phase, shorter and smaller plant and shortened internode compared with CK, has the phenomena of reduced anther, malformation, abortion and the like in the aspect of anther development, carries out bagging seed collection on the transgenic plant, greatly reduces the seed setting rate of the plant, reduces the ovary and greatly reduces the seed quantity of each fruit. The results show that PhDof4 plays an important role in regulating and controlling anther development;

(3) the petunia PhDof4 gene can provide reference for further researching molecular mechanism of petunia anther development and provide an effective molecular tool for regulating and controlling plant male reproductive organ-anther development by utilizing genetic engineering. Can be helpful for constructing the male sterile line of petunia so as to promote the utilization of heterosis and has practical application value for the improvement of petunia varieties and the cultivation of new varieties.

Drawings

FIG. 1 is a map of the subcellular localization of the PhDof4 gene in tobacco leaves; the upper half is shown carrying 35 s: : GFP: expression of Agrobacterium GV3101 of the empty vector pCAMBlAl300 in epidermal cells under tobacco; the lower half of the graph is carrying 35 s: : expression of Agrobacterium GV3101 of GFP-target gene PhDof4 in epidermal cells under tobacco;

FIG. 2 is a diagram showing the result of PCR positive detection of petunia plants transformed with PhDof4-RNAi vector; CK 1: a positive control; CK 2: negative control;

FIG. 3 is a phenotype diagram of petunia transformed with PhDof4-RNAi vector;

FIG. 4 is a scanning electron micrograph of PhDof4-RNAi vector transformed petunia anthers, CK: a wild type;

FIG. 5 is a half-thin slice of PhDof4-RNAi vector transformed petunia anthers, CK: a wild type;

FIG. 6 is a diagram of the expression analysis of PhDof4 of different tissue sites of petunia transformed by PhDof4-RNAi vector, wherein, Bud: flower bud, Stem: stem, CK: a wild type;

FIG. 7 is a graph showing the result of measuring the chlorophyll content of petunia transformed by PhDof4-RNAi vector, CK: and (4) a wild type.

Detailed Description

The invention is further described with reference to specific examples.

The methods used in the following examples are, unless otherwise specified, conventional methods, and may be performed by reference to conventional experimental methods in molecular biology, or according to kits or instructions.

Example 1: cDNA cloning and identification of petunia PhDof4 gene

Plant material: petunia hybrida fantasy 'W115' variety is provided by a landscape garden key laboratory of Nanjing forestry university, is sowed in a growth room of the landscape garden college of Nanjing forestry university, and is sowed and grown under natural conditions.

The gene source is as follows: based on the existing petunia anther transcriptome database (not published) of the previous subject group, the primers were selected and named, and then full-length specific primers were designed in combination with Primer Premier 5.0.

PhDof4-F：5′-AGGAGACGAGGATGATGATAAGG-3′；

PhDof4-R：5′-CACTGCCATCCTGTCTGATACTT-3′。

The method for amplifying the PhDof4 gene from the petunia total RNA specifically comprises the following steps:

1) taking 50-100mg of petunia root, stem, leaf and flower jelly samples stored at-80 ℃ in the early stage, and extracting total RNA by adopting a liquid nitrogen grinding instruction in an RNA extraction kit of Beijing Edley company.

2) Then, the RNA quality is detected by TBE gel electrophoresis and detected by a spectrophotometer. The obtained total RNA is used as a template, reverse transcription is carried out according to the instruction of a reverse transcription kit provided by Beijing Quanyujin company, and a cDNA first chain is synthesized and used as a target gene full-length amplification template.

Reverse transcription system (20 μ L): mu.L of gDNA Remover, 1. mu.L of adsorbed Oligo (dt)18Primer (0.5. mu.g/. mu.L), 1. mu.L of TransScript

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

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

3) Diluting the cDNA by 10 times, carrying out full-length amplification on a target gene by Prime STAR high-fidelity enzyme of Beijing TaKara biotechnology limited, detecting a target band by using 1% agar gel electrophoresis after the reaction is finished, and if the band is bright and the segment length is correct, carrying out product recovery by referring to the specification of an easy pure Qcick Gelextraction Kit provided by Beijing all-purpose gold company, and if the band is weak and the segment length is correct, carrying out product enrichment and then recovering.

The reaction system (20 μ L) for full-length amplification of the target gene is specifically as follows: 1 μ L of cDNA, 1 μ L of antisense Primer (PhDof4-F) (10mM), 1 μ L of Sense Primer (PhDof4-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) Finally obtaining a DNA full-length fragment, cloning the DNA full-length fragment to a pEASY-Blunt vector, transforming escherichia coli, detecting positive recombinant clone, sequencing, and sequencing to obtain the petunia PhDof4 cDNA sequence with a complete coding region, wherein the cDNA sequence is shown as SEQ ID NO.1, the length is 1282bp, the amino acid sequence of the coded protein is shown as SEQ ID NO.2, and the length is 424 aa. The specific reaction system and reaction conditions are as follows:

cloning system (5 μ L): 4 μ L of PCR Product, 1 μ L of pEASY-Blunt.

After reacting for 5min at room temperature, carrying out constant temperature cloning reaction for 15min at 25 ℃ in PCR to complete connection. Finally, the strain is transformed into Escherichia coli Trans-T1 for bacterium preservation.

Example 2: subcellular localization of petunia PhDof4 gene

The Nicotiana benthamiana with the growth cycle of 40d is used as an experimental material.

Primers for amplifying the complete coding reading frame were designed according to the full-length cDNA sequence of PhDof4 obtained in example 1, and the primers were designed as follows:

F：5′-ctgcaggggcccggggtcgacATGGAACCTACGACCGGAAAT-3′；

R：5′-gcccttgctcaccatggtaccTAAGCTCTCATTGAAATTTGATGACC-3′。

PhDof4 containing intact OFRs was isolated from petunia cDNA using the primers described above for semi-quantitation. The ORF of the fragment was fused to the 5' end of the GFP reporter gene in pCAMBIA1300 to form a GFP: : pCAMBIA1300-PhDof4 chimeric gene, plasmid GFP was constructed: : pCAMBIA1300-PhDof 4. After enzyme digestion verification, agrobacterium is transformed by an electrotransformation method through the empty vector pCAMBIA1300 and then is transiently transformed into agrobacterium and injected into tobacco leaves, and the result shows that petunia PhDof4 is a transcription factor positioned in cell nucleus and cell membrane (figure 1).

The method comprises the following specific steps of transient agrobacterium transformation:

1) preparing a bacterial liquid buffer solution: 10mM. L^-1MgCl₂，10mM·L^-1MES，150μM·L^-1Acetosyringone.

2) Successfully transforming AgrobacteriumGV3101, pCAMBIA1300-PhDof4 fusion expression vector, pCAMBIA1300 no-load, P19 auxiliary vector placed on ice to melt. 50. mu.L of each of the solutions was added to 30mL of LB liquid medium (containing 100 mg. multidot.L)^-1Kana) was shake-cultured at 28 ℃ and 200rpm/min in the dark to OD₆₀₀Between 0.6 and 1.0. At 4 ℃, 5000 r.min^-1Centrifuging for 5min, collecting thallus, resuspending thallus with buffer solution, and mixing with the resuspended bacteria solution at a certain ratio (OD)₆₀₀The ratio is 7: 5), standing for 2-3h at room temperature.

3) Injecting mixed bacteria-containing liquid into the back of the tobacco leaf by using a 1mL medical injector, culturing in an incubator for 2-3d, observing the expression condition of epidermal cells under the condition that agrobacterium GV3101 carrying pCAMBIA1300-PhDof4 injects into 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 (BrightField) and a mixed field (Merged) of the two, and simultaneously determining the subcellular localization condition of the petunia PhDof4 protein by taking the expression condition of the epidermal cells under the condition that the agrobacterium GV3101 carrying an empty vector pCAMBlA1300 injects into the tobacco as a reference. As shown in fig. 1, the results show that: petunia PhDof4 was shown to be a transcription factor localized to the nucleus and cell membrane.

Example 3: gene function identification of petunia PhDof4 gene

1) Constructing RNAi vector, and taking V097 without variation sites by sequencing and without errors by double enzyme digestion verification: : performing LR reaction on the PhDof4 recombinant plasmid and an interference expression vector V139, performing double-PCR bacterial examination, performing single enzyme digestion verification on the recombinant plasmid for detecting correct Dof4-RNAi-V139 by using Xba I and Hind III restriction enzymes, generating three bands of 250bp, 500bp and 750bp after the Xba I enzyme digestion, generating a band of which the length is more than 2000bp and less than the length of the vector when the Hind III enzyme digestion is performed, and detecting by using gel electrophoresis. And (3) converting the successful Dof4-RNAi-V139 recombinant plasmid into EHA105 by adopting an electrotransformation method, and converting the target gene-linked recombinant plasmid and the V139 vector recombinant plasmid into Agrobacterium EHA105 to prepare for subsequent transgenic transformation. The relevant reaction system and reaction conditions are as follows:

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 bufferr，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.

Constructing LR recombination reaction by RNAi vector:

taking V097 without mutation sites by sequencing and double enzyme digestion verification: : the PhDof4 recombinant plasmid and the interference expression vector V139 are subjected to LR reaction, the reaction system and the specific operation are as follows,

LR reaction system: 25-75ng of the intermediate vector V097, less than 75ng of the empty V139, 1. mu.L of LRclonase^TMII，ddH₂O is supplemented to 5 mu L

Vortex, mix well, centrifuge briefly, and react at 25 ℃ for 6 h. Transformation of E.coli is carried out by methods such as pre-transformation, culture on plates containing Spe resistance, double PCR bacterial detection, detection of primers: 35SF + geneF and T35SR + geneF

The RNAi single enzyme digestion verification system is as follows:

and (3) placing the enzyme digestion system at 37 ℃ for reaction for 30min-1h, and detecting by gel electrophoresis.

Note: X.mu.L 1000 ng/plasmid concentration (ng.mu.L)^-1)

2) Genetic transformation of petunia

And (3) transforming the constructed PhDof4-RNAi vector leaf disc method into petunia by adopting an electrotransformation method.

Firstly, melting the constructed agrobacterium carrying PhDof4-RNAi vector on ice, adding 20 mu L of bacterial liquid into 30mL of fresh liquid LB culture solution (containing 100 mg. L)^-1Kanamycin), shake to OD₆₀₀The value was 0.4. Transferring to a 30mL sterile enzyme-free centrifuge tube, collecting bacterial liquid at 5000rpm/min and 4 ℃ for 10 min. 30mL of LB liquid culture medium (containing no antibiotic) was added to the centrifuge tube, and shaking cultured at 28 ℃ and 200rpm/min for 2 hours.

Taking several young leaves from the top of petunia, sterilizing the surface with 75% alcohol for 10s, and adding ddH₂Cleaning with O for 3-5 times, sterilizing by soaking in 0.1% mercuric chloride for 30min, and sterilizing with ddH₂And O cleaning for 3-5 times. Cutting the leaves into 5mm small blocks, soaking in the prepared solution for 10min, and removing surface bacteria liquid by suction, and culturing in co-culture medium for 3 d. And after the co-culture is finished, sequentially transferring to a screening culture medium, a strong bud culture medium and a rooting culture medium for culture, and finally finishing the transformation and transplanting.

3) Positive validation of petunia

The obtained transgenic plants are verified by DNA and RNA (figure 2), which shows that the detected transgenic plants are positive. Further observing the positive transgenic plants, the flowering time of the transgenic plants is advanced, and in addition, as shown in figure 3, compared with CK, the leaf color is obviously dark green, the plants are shorter and smaller, and the internode shortening phenomenon is generated; in the aspect of anther development, transgenic plants have the phenomena of anther reduction, deformity and abortion, and pollen tube germination and pollen activity detection confirm the result. In addition, when the anther is subjected to electron microscope scanning and semi-thin slicing (fig. 4 and fig. 5), the number of pollen grains of a transgenic plant is greatly reduced, the pollen grains are mostly deformed and shriveled, germination holes are hardly seen, the surface lines of the pollen grains are closely and disorderly arranged, tapetum cells of the pollen grains are loosely arranged and have a tendency of early degradation, and the shape and the size of microsporocytes are irregular.

In order to verify the relationship between the anther abortion phenotype of a transgenic plant and the expression level of the PhDof4 gene, a wild type petunia plant is used as a control, the expression of the PhDof4 gene in the transgenic plant is verified through Real-Time PCR, and the fact that the positive petunia line interfering the expression of the PhDof4 gene has the strong and weak gene expression is shown, the difference of obvious expression levels exists among 7 transgenic lines L14, L6, L8, L10, L11, L19 and L20 (figure 6), the expression level is related to the phenotype of the plant, the expression level of the PhDof4 gene in the L14 line is the lowest, the anther abortion of the line is more obvious, and the above results show that the PhDof4 gene regulates the anther abortion, and the reduction of the expression level of the PhDof4 gene can cause the fertility of the petunia anther abortion.

The phenotypes of the petunia with dark green leaf color and pollen abortion were further verified, the chlorophyll content was determined by spectrophotometry (fig. 7), the pollen viability was determined by acetocarmine, the pollen germination rate was determined by in vitro culture solution, the pollen morphology scanning electron microscope and half thin section were performed, and the results all verify the observed phenotypic changes.

Sequence listing

<110> Nanjing university of forestry

<120> petunia anther development related PhDof4 gene and application thereof

<130>100

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>1282

<212>DNA

<213>Petunia hybrida

<400>1

aggagacgag gatgatgata aggatcagat ggaacctacg accggaaatg agttgcagga 60

tgagaattca gatctaacga aaagtgatat cataaaagag ccacctgttg ataatgactg 120

ttcagcaaca aaaacctcaa aaagtgaaga tgagcagggc gaaccaagta atccgcaaga 180

gaaaatcctc aaaaagccag acaagatact tccctgccct cgctgtaata gcatggaaac 240

caaattttgt tatttcaaca attacaatgt caaccagcct agacacttct gcaagaactg 300

ccagagatat tggacagctg gtgggaccat gaggaatgtg cctgtcggtg ctggccgtcg 360

gaagaacaag aactcagttc cacattaccg ccaaatatct gtctctgaag aacttcccaa 420

tgcgctaata gattatccaa atggaatcca gcaacctgtt cttgtatttg gctcccatgc 480

accactctgt gaatcgatgg cttccgtttt gaacattggt gacaaagcaa tgcagaattg 540

ttcaccgaac gggtcccaga aaccagagga gtcaggggtt caagttactt atgaagctgg 600

agacaatgga gatgaccatt ccagggtcac tgccgcaagc tcaaaggata aagtcgataa 660

aaccgtacct gacatgctcg ggcagaatta ccgcagtttt cctagcttgc cttgctatcc 720

tggggctcct tggccatatc catggagctc tgcagttcct cctcctggtt attgccctcc 780

tctctttcct atgcctttct acccagcagc tgcttattgg ggttgtggtg tagctggttc 840

gtggaatgtc ccttgggtgt ccccacctac tgtttcccta acccaaacac cttcaacttc 900

aggtcctaat tctccaactc tggggaaaca ctcgagggag gaaaacatac taaaaccgct 960

gagcagcgag gaagagcctc agaaagagag taatcctgag aagtgcctgt gggttccgaa 1020

aactctgcga attgatgatc caggagaagc tgcaaagagt tctatatggg cgacgttggg 1080

aataaaacat gatggtgttg attcagttgg tggaagtctt ttcagtgctt ttcggtcgaa 1140

gaatgatgaa aaaactagtg tttcggaaaa ctctactgta ttacaagcaa atccggcagc 1200

attttctagg tcatcaaatt tcaatgagag cttataagca ggtgtgaatc attaatgtta 1260

agtatcagac aggatggcag tg 1282

<210>2

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<212>PRT

<213>Petunia hybrida

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Gly Asp Glu Asp Asp Asp Lys Asp Gln Met Glu Pro Thr Thr Gly Asn

1 5 10 15

Glu Leu Gln Asp Glu Asn Ser Asp Leu Thr Lys Ser Asp Ile Ile Lys

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Glu Pro Pro Val Asp Asn Asp Cys Ser Ala Thr Lys Thr Ser Lys Ser

35 40 45

Glu Asp Glu Gln Gly Glu Pro Ser Asn Pro Gln Glu Lys Ile Leu Lys

50 55 60

Lys Pro Asp Lys Ile Leu Pro Cys Pro Arg Cys Asn Ser Met Glu Thr

65 70 75 80

Lys Phe Cys Tyr Phe Asn Asn Tyr Asn Val Asn Gln Pro Arg His Phe

85 90 95

Cys Lys Asn Cys Gln Arg Tyr Trp Thr Ala Gly Gly Thr Met Arg Asn

100 105 110

Val Pro Val Gly Ala Gly Arg Arg Lys Asn Lys Asn Ser Val Pro His

115 120 125

Tyr Arg Gln Ile Ser Val Ser Glu Glu Leu Pro Asn Ala Leu Ile Asp

130 135 140

Tyr Pro Asn Gly Ile Gln Gln Pro Val Leu Val Phe Gly Ser His Ala

145 150 155 160

Pro Leu Cys Glu Ser Met Ala Ser Val Leu Asn Ile Gly Asp Lys Ala

165 170 175

Met Gln Asn Cys Ser Pro Asn Gly Ser Gln Lys Pro Glu Glu Ser Gly

180 185 190

Val Gln Val Thr Tyr Glu Ala Gly Asp Asn Gly Asp Asp His Ser Arg

195 200 205

Val Thr Ala Ala Ser Ser Lys Asp Lys Val Asp Lys Thr Val Pro Asp

210 215 220

Met Leu GlyGln Asn Tyr Arg Ser Phe Pro Ser Leu Pro Cys Tyr Pro

225 230 235 240

Gly Ala Pro Trp Pro Tyr Pro Trp Ser Ser Ala Val Pro Pro Pro Gly

245 250 255

Tyr Cys Pro Pro Leu Phe Pro Met Pro Phe Tyr Pro Ala Ala Ala Tyr

260 265 270

Trp Gly Cys Gly Val Ala Gly Ser Trp Asn Val Pro Trp Val Ser Pro

275 280 285

Pro Thr Val Ser Leu Thr Gln Thr Pro Ser Thr Ser Gly Pro Asn Ser

290 295 300

Pro Thr Leu Gly Lys His Ser Arg Glu Glu Asn Ile Leu Lys Pro Leu

305 310 315 320

Ser Ser Glu Glu Glu Pro Gln Lys Glu Ser Asn Pro Glu Lys Cys Leu

325 330 335

Trp Val Pro Lys Thr Leu Arg Ile Asp Asp Pro Gly Glu Ala Ala Lys

340 345 350

Ser Ser Ile Trp Ala Thr Leu Gly Ile Lys His Asp Gly Val Asp Ser

355 360 365

Val Gly Gly Ser Leu Phe Ser Ala Phe Arg Ser Lys Asn Asp Glu Lys

370 375 380

Thr Ser Val Ser GluAsn Ser Thr Val Leu Gln Ala Asn Pro Ala Ala

385 390 395 400

Phe Ser Arg Ser Ser Asn Phe Asn Glu Ser Leu Ala Gly Val Asn His

405 410 415

Cys Val Ser Asp Arg Met Ala Val

420

<210>3

<211>23

<212>DNA

<213> PhDof4-F primer sequence (Artificial)

<400>3

aggagacgag gatgatgata agg 23

<210>4

<211>23

<212>DNA

<213> PhDof4-R primer sequence (Artificial)

<400>4

cactgccatc ctgtctgata ctt 23

<210>5

<211>42

<212>DNA

<213> primer sequence PhDof4F (Artificial)

<400>5

ctgcaggggc ccggggtcga catggaacct acgaccggaa at 42

<210>6

<211>47

<212>DNA

<213>PhDof4R(Artificial)

<400>6

gcccttgctc accatggtac ctaagctctc attgaaattt gatgacc 47

Claims

1. A petunia anther development related gene PhDof4 has a nucleotide sequence shown in SEQ ID NO. 1.

2. The expressed protein of petunia anther development related gene PhDof4 of claim 1, wherein the amino acid sequence thereof is represented by SEQ ID No. 2.

3. A vector, recombinant bacterium or cell containing the petunia anther development related gene PhDof4 of claim 1.

4. The vector of the petunia anther development related gene PhDof4 as claimed in claim 3, wherein the vector is pEASY-Blunt-PhDof4 or PhDof 4-RNAi.

5. The use of the petunia anther development-related gene PhDof4 in claim 1 for regulating plant anther fertility or plant morphology.

6. The use of the gene PhDof4 related to anther development as claimed in claim 5, wherein the use of the gene PhDof4 related to anther development for regulating the fertility of plant anthers comprises the following steps:

7. The use of claim 5, wherein the use of the anther development associated gene PhDof4 for regulating plant morphology comprises the steps of:

8. The use of claim 6 or 7, wherein the vector containing petunia anther development related gene PhDof4 is PhDof 4-RNAi.

9. The use according to any one of claims 5 to 7, wherein the plant is petunia.