CN108676805A - Petunia ASRs genomes and its application - Google Patents

Abstract

The application belongs to petunia technical field of biological breeding, and in particular to the patent application of petunia ASRs genomes and its application.The genome includes tri- genes of ASR1, ASR2 and ASR3, and the transcription factor of this 3 gene code R2R3 MYB types is related to anthocyanidin synthesis regulation respectively；That is, related to regulation and control delphinidin synthesis respectively.The transcription factor that relevant R2R3 MYB types are synthesized with other anthocyanidin being had been found that in plant establishes chadogram and shows that these three genes and AN2, DPL, AN4, PHZ etc. are located on same evolutionary branching.Transgenosis overexpress the result shows that, the relevant access of the regulation and control delphinidin of ASR1/2/3 specificity synthesis.The molecular basis that the application regulates and controls blue/purple petal in petunia has carried out preliminary combing, lays a good foundation to formulate the relevant Flower New Variety kinds such as blue/purple using these three genes in the future.

Description

Petunia ASRs genomes and its application

Technical field

The application belongs to petunia technical field of biological breeding, and in particular to petunia ASRs genomes and its application it is special Profit application.

Background technology

Petal color is the important fancy points of flowers, decides the economic value of flowers to a certain extent.Studies have shown that The pattern of plant is mainly to be determined by anthocyanidin, and ingredient and content in petal cell decide the colour generation of petal.

Anthocyanidin is a kind of flavonoids, and synthesis is controlled by a series of anthocyanidin composite structure genes, and by some turns Record the regulation and control of the factor.To petunia pattern the study found that the transcription factor of regulation and control anthocyanidin synthesis has coding bHLH types Gene A N1 encodes the Gene A N11 of WD40 types and encodes Gene A N2, AN4, DPL and PHZ etc. of R2R3-MYB types.Its In：The transcription factor of R2R3-MYB types plays main function in the synthesis of regulation and control anthocyanidin, they decide that anthocyanidin closes At time and synthesising part；AN2 is mainly expressed in the petal of petunia, decides the colour generation of petal；AN4 is mainly in stamen Middle expression, and play an important role during the colour generation of stamen；DPL and PHZ mainly flower arteries and veins and corolla in specifically expressing, and It plays an important role during nutrition organs is by the colour generation that intense light conditions induce.Based on these existing researchs so that people couple There is preliminary understanding in the molecular mechanism of petunia petal color change.But complexity is various due to petal color change, Being expressed by several genes is influenced with regulation and control, thus reinforces studying for forming related gene with pigment, for point of flower color formation The cultivation of sub- mechanism study and further Flower New Variety kind all has highly important scientific research theory significance and practical value.

Invention content

The application is designed to provide a petunia（Petunia）In genome coding R2R3-MYB types transcription because The ASRs genomes of son, which includes tri- genes of ASR1, ASR2 and ASR3, to what is formed for petunia petal color Molecular basis is studied and rearing new variety based theoretical.

Details are as follows for the technical solution that the application is taken.

One petunia ASRs genome, the genome include tri- genes of ASR1, ASR2 and ASR3, base sequence difference As shown in NO.1 ~ 3 SEQ ID, this 3 genes are separately encoded the transcription factor of a R2R3-MYB type, respectively with anthocyanidin Synthesis regulation is related；

The ASR1（Namely：ASR1^inf）Base sequence, including 1511bp, referring specifically to embodiment partial content；

The ASR2（Namely：ASR2^inf）Base sequence, including 1535 bp, referring specifically to embodiment partial content；

The ASR3（Namely：ASR3^inf）Base sequence, including 1503bp, referring specifically to embodiment partial content.

Application of the petunia ASRs genomes in pattern regulation and control, ASR1, ASR2 and ASR3 of the genome tri- Gene is related to anthocyanidin synthesis regulation respectively, specifically, related to regulation and control delphinidin synthesis.

Using the overexpression recombinant vector constructed by the petunia ASRs genomes, specifically, utilizing gateway skills Art respectively enters tri- genetic transformation of ASR1, ASR2 and ASR3 of ASRs genomes in overexpression vector pK2GW2.0/rfa.

To the PCR amplification method of the petunia ASRs genomes, with the petunia kind containing ASRs genomic genes DNA as PCR amplification template, PCR amplifications are carried out to three genes respectively, when PCR amplification, specific primer sequence was designed as：

Primer is when expanding ASR1 genes：

ASR1-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTGTTATCTGTAC,

ASR1-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGAAGACTCCAGAGGTCA；

Primer is when expanding ASR2 genes：

ASR2-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTCCTATCTGTAC,

ASR2-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGTAGACTCCAGAGG；

Primer is when expanding ASR3 genes：

ASR3-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACAATTCCTATCTGTAC,

ASR3-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGTAGACTCCAGAGG.

The petunia kind containing ASRs genomic genes is, for example,：The kinds such as petunia M1, V30.

A method of adjustment petunia pattern, using the method for hybridization recombination, to contain the short of ASRs genomic genes Kind of leading a cow is maternal or male parent（Such as the kinds such as petunia M1, V30）, the petunia of ASRs genomic genes is free of with other Kind is male parent or female parent（Such as the kinds such as petunia R27, W115）, hybridized, hybridize petunia product so as to adjust filial generation Kind pattern；

Or technique for gene engineering is utilized, ASRs genomes are overexpressed to accumulate anthocyanidin, or carry out gene mistake Living, gene silencing or gene knockout to reduce anthocyanin accumulation, or the conversion of ASRs genomes are entered and is free of ASRs genes In the plant of group, so as to adjust pattern.

In short, the transcription factors of the application 3 coding R2R3-MYB types of new discovery in petunia genome, respectively It is named as ASR1, ASR2, ASR3.The transcription of relevant R2R3-MYB types is synthesized with other anthocyanidin being had been found that in plant The factor establishes chadogram and shows that these three genes and AN2, DPL, AN4, PHZ etc. are located on same evolutionary branching.The super table of transgenosis Up to the result shows that, the regulation and control delphinidin of ASR1/2/3 specificity synthesizes relevant access.In general, the application is for short The molecular basis of middle blue/purple petal regulation and control of leading a cow is combed, in the future using these three genes formulate blue/ The relevant Flower New Variety kind such as purple is laid a good foundation.

Description of the drawings

Fig. 1 is the evolutionary relationship of ASR genomes R2R3-MYB transcription factors related to the synthesis of other anthocyanidin；

Fig. 2 is distribution character of the ASR genomes in different petunia kinds（Illustrate kind ASR genes with the presence of PCR amplification Group）；

Fig. 3 is the amino acid sequence analysis of ASR genome encodings, and wherein asterisk represents the amino guarded in R2R3-MYB structural domains Sour site；Arrow represents the conserved positions [DLx2Rx3Lx6Lx3] with bHLH effects；

Fig. 4 is tissue expression (developmental stage of behalf petal) of the ASR genes in different petunias；

Fig. 5 is overexpression ASR1^M1Gene promotes petunia colour generation（ASR1^infImproper coding, ASR1^M1Normal encoding）；

Fig. 6 is to overexpress ASR1 in petunia M1 × R27^M1、ASR2^M1、ASR3^M1The phenotypic characteristic of gene；

Fig. 7 is to overexpress ASR1 in petunia W115^M1The phenotypic characteristic of gene；

Fig. 8 is application of the ASR genomes in petunia pattern breeding；

Fig. 9 is that PCR detects the isogenic genotype of petunia L0 self progeny HF1, ASR and AN2.

Specific implementation mode

Explanation is further explained to the application with reference to embodiment, before introducing specific embodiment, with regard to following realities The briefly introduction of the backgrounds such as part biological material, the experiment reagent involved in example is applied to be described as follows.

Biomaterial：

Involved petunia material in following embodiments（Including Petunia inflata, Petunia axillaris, M1, W115, M1 × R27, M1 × V30 etc.）, carrier（pDONR207、pK2GW2.0/rfa）And agrobacterium strains Agl1, by University of Amsterdam Ronald Koes professors laboratory give；

E. coli competent JM109 is company long purchased from Zhengzhou；

Experiment reagent：

Trizol kits, DNase, reverse transcription reagent box, PCR kit, DNA marker, plastic recovery kit, plasmid are small Extraction reagent kit etc. is purchased from Dalian treasured biotech firm（Takara）.

Gateway enzyme preparation Gateway BP Clonase II Enzyme mix and Gateway LR Clonase Enzyme Mix are purchased from silent winged scientific and technological (China) Co., Ltd of generation that of match；

Ampicillin（Amp）, kanamycins（Kan）, rifampin（Rif）, gentamicin（Gen）, cephalosporin（Cef）, it is strong Miromycin（Spe）Equal antibiotic, MS culture mediums and other chemicals are Bioisystech Co., Ltd long purchased from Zhengzhou；

Petunia genetic transformation co-cultures used medium：+ 1 mg/ of+0.1 mg/L methyl α-naphthyl acetates of MS+20g sucrose+10g glucose The 6-benzyladenine of+2 mg/L of L zeatin and the agar powder of 8g, pH value 5.7；

Petunia genetic transformation screening culture medium：+ 1 mg/L corns of+0.1 mg/L methyl α-naphthyl acetates of MS+20g sucrose+10g glucose The 6-benzyladenine and 8g agar powders of+2 mg/L of element, pH value 5.7；Antibiotic is the kanamycins and 250 of 100mg/L The cephalosporin of mg/L；

Petunia genetic transformation root media：MS+20g sucrose+10g glucose, 8g agar powders, pH value 5.7；Antibiotic is The cephalosporin of the kanamycins of 50mg/L and 250 mg/L；

Primer and sequencing：Completion is provided by Beijing six directions Hua Da Gene Tech. Company Limited.

Embodiment 1

The present embodiment mainly introduces petunia（Petunia）Coding R2R3-MYB type transcription factors in genome ASR genomes（Including ASR1, ASR2 and ASR3 gene）Acquisition process.

Early period obtains process to the analysis of ASR genomes, is mainly closed with flavonoids/anthocyanidin according to known in arabidopsis At encoded amino acid sequences such as relevant R2R3-MYB genes TT2, PAP1, PAP2 and petunia AN2, AN4, DPL, PHZ It is compared, is then based on known petunia Petunia axilaris and Petunia two kinds of inflata Genome sequence, with petunia AN2, AN4, DPL, PHZ, the R2R3-MYB genes such as arabidopsis PAP1, PAP2, TT2 are with reference to sequence Row, three R2R3-MYB genes have been obtained by offline Blast technologies in petunia Petunia inflata genomes （ASR1, ASR2 and ASR3 gene）Correlated series, these three genes are not present in Petunia axilaris genomes.By It is to be compared to obtain by the relevant R2R3-MYB genes of anthocyanidin synthesis regulation, therefore be named as ASR in these three genes （ANTHOCYANIN SYNTHESIS REGULATOR）.

Gained ASR1（Namely：ASR1^inf）Base sequence as shown in SEQ ID NO.1（It is calculated with open reading frame, from 150th base positions of initiation codon mutate namely the 257th bit base of DNA sequence dna）Specially（Including 1511bp）：

ATGAATACTATTGTTATCTGTACGCCGACGTTGGGAGTGAGAAAAGGTTCATGGACTGAAGAAGAAGATTTTC TTTTGAGAAAATGCATTGAAAAGTATGGTGAAGGAAAGTGGCATCTTGTTCCCGTTAGAGCTGGTAAACAAACTAAA ACTATAAGCCATACTCTTTTCCTCTTTATATTCCTTTAAATAGAAGTATATGCATATGTCTAATACTCTCCTTCTCT TTTACATATGAAACAGGTCTGAATAGATGAAGAAAGAGTTGCAGACTAAGGTGGTTGAATTATCTAAGGCCACATAT TAAGAGAGGTGACTTCTCTGAGGATGAAGTGGATCTCATTTTGAGGCTTCATAAGCTTCTAGGCAACAGGCAAGCTT TTTTCTTTTTAATTTTTTTGAAAGCTGATATTCGTAACACTGCTTCGGTTAATCTGATATCATGCCAGGTTGGCGAC ATGCAAGTGACAACTATTGGCGGAGTAAGGAATTAAAATAAAGTGGATTTTCAAAAACACTAGAATGTCACGGTTAA CTAGTTAAGATTTGAAATTAGATCCTAAAATAATATTTTGAATGAGGATTCAATAATCTATATGTAACATAAAAAAA TTTATTTTTGCCCTACTTTTACAGTTTAATATTTCGAAAAATTATTGAGTTTGTAGTTGAACAGATTAAGAAGGAGA TTATAGAGAGGATAGAGATGTTACAAAGCTCAAAGAGAGCTCATAGAAGTAGGTTGTTTATTCAGCATTTATTGTAA GTAGTTGCGACTGTGGGAGGCCTAAAGTTCAGTTTGGAACTTGTAGATGCTCTTTAGGTTTAAGGTTTTGGATGTAA TGGTAATCATTTACAGTTTATCAAAAAAAATAGTATATATATATTTCGACAAAATTCATGAATTGAAAGATCTCCCA AAACAGAAACAGAGGGAGTAATATTAAGTACACTTCACCCACAATATTGATTTCCATGCATAACCAAGAACATATAT ATTGAGCTTGTCTCCTCTTATCATATGCATTCCATGTAGGTGGTCACTTATTGCGGGTAGACTTCCTGGAAGAACAG CAAATGATGTGAAAAACTATTGGAACACTAACTTTCGAAGGAAATTATCTCCTCAAAGACAAGGGAAAAGGTGCAGA TCTGCCATTAAGATCACTGAGAATACCATAATAAGGCCTCAACCTCGGATCTTTTCAAGCGCAAAGAATGTTTCTTT GGGCAGCAACAAGAGTATCACAAACAGTACTATCCATAAGGATGAAAGCAGCAAAAATATAATAAGGACTAACGAGA AGCAAATGACTGAAGCATCGACAGAAAATGGAGTTCAATGGTGGGCAAGTTTATTGGATGATCCTAATGAATTTGAT AAGGAAGCAGCAACTGGAAGCATACCTTCGAATTCATGTAATATCAACTCCAAGGAAGAATTACCAACATTCTTGCA AGAAGGAGAAACTGGCTGGGGTGATATTGACCTCTGGAGTCTTCTTAATTAG。

Gained ASR2（Namely：ASR2^inf）Base sequence as shown in SEQ ID NO.2（It is calculated with open reading frame, from 148th base positions of initiation codon mutate namely the 255th bit base of DNA sequence dna）Specially（Including 1535bp）：

ATGAATACTATTCCTATCTGTACGCCGACGTTGGGAGTGAGGAAAGGTTCATGGACTGAAGAAGAAGATTTTC TTTTGAGAAAATGCATTGAAAAGTATGGTGAAGGAAAGTGGCATCTTGTTCCCGTTAGTGCTGGTAAACAAACTAAA ACTATAAGCCGTACTCTTTTCCTCTTTATATTCCTTTAAATAGAAGTATATGCATAGGTCTAATACTCTCCTTGTCT TTTATATATGAAACAGGTCTGAATAGAATGTAGAAAGAGTTGCAGACTAAGGTGGTTGAATTATCTAAGACCACATA TTAAGAGAGGTGATTTCTCTGAGGATGAAGTGGATCTCATTTTGAGGCTTCATAAGCTTCTAGGCAACAGGCAAGTT TTTTTCTTTTTTATTTTTTTGAAAGCTGATATTCGTAACACTGCTTCGGTTAATCTGATATCATGCCAGGTAGGCGA CATGCAAGTGACCACTATTGGCGGAGTAAGGAATTAAAATAAAGAGGATTTTCAAAAACACACGAAATGTCACGGTT AACTAGTTAAGATTTGAAATTAGACCTAAATAATATTTTGAATCCCCTTTGTTACAACTGAATTTTTGTTTACGTAA AGAAGATTCAATAATCTATATTATAACATAAAATTTTTTATTTTTGCCTTACTTGTACAGAATAATTTTTCGGCAAA GGTTAATTAATGAACCCCCTTCTGTGAATCTATCTATGCCCCTAATTACCACCACCAACACATTTTATATGAAGGTA TTTCACTGAATACATAATATTTAAATTTTCGGTCTTAAACATATCATTATAGTTGTATAAAATTATGTCATCGAGGA TATAATTAGATTAAAGTATTCTAAACCAGTAGAGGTGTCAATATTTTTGGAAACGGATTAAAAAAGAAAGATGTCCG AAAATGAAACCAGAAAGTAATATTTACTACACTTTCACCCACAATCCCCAGTTGATGTTGATCTCCAACTAAGAACA TATGCTGCCTTATGCTGCATACATATATTGAGTCTTGTCTGATTTTATCATGTACACTTCCTTTAGGTGGTCACTTA TTGCGGGTAGACTTCCCGGAAGAACAGCAAACGATGTGAAAAACTATTGGAACACTCACTTTCAAAGGAAGTTATTT TCTCCACAAATACAAGGGAAAAAGTGCAGATCTGCCGTTAAGATCACTGAGAATACCATAATAAGACCTCAACCTCG GAACTTCTCAAGCACGAAGAATGTTTCTTTGGGCAGAAACAAGAGTATCACAAACGGTACTATACATAAGGATGAAA CCAGCAAAACAATTATAAGGACTAACGAGAAGCAAACGACTGAAGCATCAACAGATAATGGAGACCAGTGGTGGGCA AGTTTATTGGATGATCGTAATGAATTTGATGAGGAAGCAGCAGCTGGAAAAACACCTTCATGTAAGATCAACTCTGA GCTAGAAACACCAACATTGCTGCAAGAAGGAGAAACTGGCTGGGGTGATATTGACCTCTGGAGTCTACTTAATTAG。

Gained ASR3（Namely：ASR3^inf）Base sequence as shown in SEQ ID NO.3, specially（Including 1503bp）：

ATGAATACAATTCCTATCTGTACGTCGACGTTGGGAGTGAGGAAAGGTGCATGGACTGAAGAAGAAGATTTTC TTTTGAGAAAATGCATTGAAAAGTATGGTGAGGGAAAGTGGCATCTTGTTCCCGTTAGAGCTGGTAAACAAACTTGT ACTATTAAGTACTCCTCTTTATATTCGTTTAAATATATAGAAGTATATGCATATGTCTAATACTCTCCTTGTGTTTT ATATGTGAAAATATACAGGTCTGAATAGATGTAGGAAGAGTTGCAGACTGAGGTGGTTGAATTATCTAAGGCCACAT ATTAAGAGAGGTGACTTCTCTGAGGATGAAGTGGATCTCATTTTGAGGCTTCATAAGCTTCTAGGCAACAGGCGAGT TTGTTAATTTTGTTTTTTTCGCTAGCTGATATTCGTAACACTGATTCGATTATTCTGGTATCTTGCCAGGTAGGCGA CATGCAAGTGACCACTAGTGGCGGGTTAGGAGTTCAGATATAGAGGGTTATCAGAAACACTAGAATGTCACATCTAA GATATGCTAATGAATTTTCGTTTATGTTCAAAGAATTCAATATATAGTCTATACCTAATGTAACAAAGTTTCCTTTT GCCCTACATGTACAATGTAGGGCAAAGAAAGACTACGAACCTCGTTAATTCTACGAACCTCGCTATACCCATAGTTA CCACTACCCTCAACACTATGTGGAGATGTTTTACTGAGCAAGTTGTTTAAAAAAAGAAAGACTTTTGGTACTTGTGG TCTTGACATATTAATTTGTGTAAAATCATGTCATTGAGGATAAAAGAATTATCTAAATAAATTGTTCAAATATTATA TAGAGGTGTCAATCTTTCTGGAAACAGATTTAAAAGAAAGAGTCCCAAAAATGAAACAGAGGGATTAATATTTACTA CCCTTTCACCCACCATCCCCAGTTGATCTCCATGCATAACCAAGAACATATGCTGCATAAATATATATGGAGTCTTG TCTGACTTTATCATATAAAGTACATTTCCTTTAGGTGGTCACTTATTGCGGGTAGAATTCCAGGAAGAACAGCAAAC GACGTGAAAAACTATTGGAACACTCACTATCGAAGGAAGTTAATTTCTCTTCAAAGACAAGGGAAAAAGTGCAGATC TGCCATTAAGATCACTGAAAATACCATTATAAGACCTCAACCTCGGAGCTTCTCAAGCACAAAGAATGTTTCTTTGG GCAGCAACAAGAGTATCACAAACAGTACAATACATAAGGATGAAAGCAGCAAAAGAGTTGTAAGGACTAACGAGAAA CAAACGACTGAAGCATTGACAGACAATGGATTTCAATGGTGGGCTAGTCTATTGGATGATCCTAATGAATTTGATGA GGAAGCAGCAGCTGGAAGCACAGCTTTCTGTAAGATCAACTCCGAGGAAGAAACACCAACATCGTTGCAAGAAGGAG AAACTGGCTGGGGTGATATTGACCTCTGGAGTCTACTTAATTAG。

The structural analysis of these three gene orders is shown：Three genes all contain there are three exon, wherein ASR1 and ASR2 genes are unable to normal encoding R2R3-MYB nucleic acid sequences due to being mutated in second exon region（ASR1 is From the 150th base positions of initiation codon, ASR2 is the 148th base positions）, and the CDS of ASR3 can be with normal encoding Normal R2R3-MYB amino acid.

The chadogram that ASRs genomes are built by software MEGA6.1, as shown in Figure 1, analysis result shows：ASRs tri- The genes such as gene and AN2, AN4, DPL, PHZ are located at the same evolutionary branching；Illustrate that three genes are related to anthocyanidin synthesis.

Embodiment 2

The present embodiment mainly introduces the acquisition process of Cloning of Genes Related, vector construction and transgenic petunia.

（One）Extracting genome DNA, RNA extractions, reverse transcription prepare cDNA

The extraction of petunia RNA is carried out using Trizol methods, and concrete operations are with reference to as follows：

It is added in Trizol to the 2mL centrifuge tubes of 1.5mL, takes 1g~2g samples, be added in centrifuge tube after liquid nitrogen grinding, whirlpool Shake 5min；

300 μ L chloroforms are added, concussion mixing 2min, 12000rpm centrifuge 15min；

It slowly draws in 850 μ L supernatants to new 2mL centrifuge tubes, 850 μ L isopropanols is added, reverse mixing, room temperature are quiet for several times Set 2h or so；

12000rpm centrifuges 10min；Supernatant is absorbed, back-off drains extraction raffinate 1min or so；

70% alcohol, 750 μ L are added, shake 30min；12000rpm centrifuges 1min, absorbs alcohol, and back-off drains extraction raffinate；It is added 494 The ddH of μ L₂O flicks centrifuge tube and is completely dissolved to RNA；

The DNase buffer of 5 μ L are added（100×）With the DNase mixings of 1 μ L, 37 DEG C of incubation 60min remove DNA；

The chloroform/water saturated phenol mixed liquor of 500 μ L is added, mixing whirlpool shakes 5min；

12000rpm centrifuges 10min；It takes 400 μ L supernatants to 1.5 new mL centrifuge tubes, 400 μ L isopropanols and 40 μ L is added NaAC solution（3M）, overturn mixing, -20 DEG C of placement 30min or so；

12000rpm centrifuges 10min, absorbs supernatant, and be stored at room temperature；It is completely cleared to liquid；

70% alcohol, 750 μ L are added, shake 30min；12000rpm centrifuges 1min, absorbs alcohol, and back-off drains extraction raffinate；

The ddH of 50 μ L is added₂O；It shakes to RNA and is completely dissolved；

Nucleic acid concentration detector measured concentration and purity are used to the RNA that is extracted, after further quantitative, the RNA- that will be extracted 20 DEG C save backup.

Takara PrimerScript TM II 1st Strand cDNA Synthesis are used to the RNA extracted Kit kits carry out reverse transcription and prepare cDNA, and specific operation process reference explanation book carries out；By the cDNA- after reverse transcription 20 DEG C save backup.

2 × CTAB methods can be used in petunia DNA extractions, and concrete operations can refer to as follows：

Take the fresh blade of 0.1g to be put into 1.5mL centrifuge tubes, grinding is slurried, then be added in centrifuge tube 650 μ L 2 × CTAB, after be put into water-bath 10min in 65 DEG C of thermostat water baths,

Centrifuge tube is taken out after water-bath, the mixed liquor of chloroform/water-saturated phenol of 450 μ L is added to it, gently mixing 5min；

12000 rpm centrifuge 10min, suct clear 400 μ L, are added in 1.5 new ml centrifuge tubes；

Isometric isopropanol is added, after overturning mixing, is placed at room temperature for 1h；

10min is centrifuged, supernatant is abandoned, dries 3min at room temperature；

75% ethyl alcohol 750uL is added, fully shaking 30min makes DNA fully wash；

12000 rpm centrifuge 2min；Supernatant is abandoned, and is stored at room temperature completely cleared to alcohol；

100uL ddH are added₂O shakes 30min, DNA is made fully to dissolve

DNA-20 DEG C after extraction is saved backup.

（Two）Gene diffusion

Gained is analyzed in reference implementation example 1Petunia inflataIn ASR genome sequences, design following serial primer sequence Row, to carry out further PCR clones；Specific primer sequence design is as follows（It is 5 ' -3 '）：

ASR1-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTGTTATCTGTAC,

ASR1-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGAAGACTCCAGAGGTCA；

ASR2-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTCCTATCTGTAC,

ASR2-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGTAGACTCCAGAGG；

ASR3-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACAATTCCTATCTGTAC,

ASR3-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGTAGACTCCAGAGG

PCR amplification：With step（One）Middle extracted petunia DNA is template, utilizes above-mentioned designed primer sequence, reference PrimeSTAR GXL DNA Polymerase high-fidelity kits（Takara companies）Specification carries out PCR amplification, 50 μ L Reaction system design is as follows：

5 × PrimeSTAR GXL Buffer, 10 μ L；

DNTP, 4 μ L；

Primer, each 0.5 μ L (10 μM)；

DNA profiling, 4 μ L；

GXL high fidelity enzymes, 1 μ L；

Moisturizing is to 50 μ L；

Response procedures are as follows：98℃、10s；55℃、15s；68℃、2 min；35 cycles；4 DEG C terminate reaction.

By pcr amplification product and DNA molecular Marker under 1 × TAE buffer conditions, with a little ethidium bromide of addition 1.0% Ago-Gel detaches（Fragment length meets expection）；Product is subjected to glue recycling（Glue recycling is with reference to plastic recovery kit Progress）, -20 DEG C save backup.

It should be noted that when PCR amplification, using the extracted DNA of the kinds such as petunia M1, V30, R27, W115 as template into When row amplification, ASR genomes（Including ASR1, ASR2 and ASR3 gene）Acquisition can be expanded in M1, V30, shows petunia Contain ASR genomes in M1, V30 genome；And fail amplification in the kinds such as R27, W115 and obtain ASR genomes, show ASR genomes are free of in the petunias kind such as R27, W115（Fig. 2）.

Further ASR1, ASR2 and ASR3 gene obtained in M1, V30 kind is sequenced and is analyzed, show ASR1, Tri- genes of ASR2, ASR3 all contain 2 intrones, ASR1, ASR2 gene that M1, V30 are cloned can normal encoding R2R3-MYB amino acid（Fig. 3）.

Based on above-mentioned sequencing result, inventor is further to tri- genes of ASR1, ASR2 and ASR3 in petunia kind The histoorgans such as petal, blade, stem, gynoecium, the stamen of ' M1 × R27 ', ' M1 × V30 ' P. inflata different times Expression characterization is analyzed, and method is carried out using RT-PCR, and using actin as reference gene.

When RT-PCR is analyzed, primer sequence（It is 5 ' -3 '）Design is as follows：

ASR1/2/3-F：CAGACGCCCATATCATGAATAC,

ASR3-R：GTTGATCTTACAGAAAGCTGTGC,

ASR1-R：ATTACATGAATTCGAAGGTATGC,

ASR2-R：CACTGGTCTCCATTATCTGTTG,

ASR1-R：CATTGAACTCCATTTTCTGTCG,

actin1-F：CTACGAGGGTTATGCTTTGCC,

actin1-R：GCTGGAATGTGCTAAGGGATG；

When RT-PCR, using above-mentioned designed primer, with reference to TaKaRa Ex Taq kits（Takara companies）Specification, The design of 25 μ L reaction systems is as follows：

Ex Taq, 0.125 μ L；

10 × Ex Taq Buffer, 2.5 μ L；

DNTP, 2 μ L；

CDNA products, 2.5 μ L；

Primer, each 0.25 μ L (10 μM)；

Moisturizing is to 25 μ L；

Response procedures are as follows：94℃、30s；55~60 DEG C（According to primer annealing temperature）, 30s, 72 DEG C, 1 min, 26~35 PCR cycle（The PCR cycle of actin genes is 26, and the PCR cycle of each genes of ASR is 35）；4 DEG C terminate reaction.

The result shows that express trend in different petunia kinds consistent for tri- genes of ASR1, ASR2 and ASR3.It is main It shows：No matter all apparent in corolla or floral tube ASR1 and ASR2 genes are early stage three petunia petals are developed, Expression, and be gradually reduced with the development expression quantity of petal；And two genes also have strong expression in anther and gynoecium, and It is expressed in stem, leaf weaker.ASR3 genes are mainly expressed in anther and gynoecium, and are hardly expressed in other organs（Figure 4）.

（Three）Carrier construction

Using gateway technologies by step（Two）In from petunia M1 institutes DNA amplification base sequence,（Respectively ASR1^M1、 ASR2^M1、ASR3^M1, can normal encoding）It is integrated into carrier, to build overexpression vector pK2GW2.0/rfa.Specific mistake Journey is as follows.

Reaction：

Using Gateway BP Clonase II Enzyme mix enzymes, by step（Two）In expanded from petunia M1 PCR product is integrated into carrier pDONR207；The design of 5 μ L reaction systems is as follows：

Step（Two）Middle PCR product, 4 μ L；

PDONR207 carriers, 0.5 μ L；

BP enzymes, 1 μ L；

25 DEG C are reacted 1 hour.

The Proteinase K Solution of 1 μ L are added, 10min is reacted at 37 DEG C；

Reaction product addition e. coli jm109 is converted, screening and culturing；The step is according to kit specification Can, or with reference to following steps：

2.5 μ L of reaction product are added to the e. coli jm109 competence of 50 μ L, cryostat 30min；

42 DEG C of conditions, heat shock 1min are then rapid to place cryostat 2min on ice；

The LB culture solutions of 800 μ L, 37 DEG C of shake culture 60min are added into centrifuge tube；

Bacterium solution 12000rpm is centrifuged into 10s；

Resuspended bacterium solution, and it was evenly coated on containing gentamicin concentration be 40mg/L LB solid mediums on, 37 DEG C of mistakes Night cultivates；Picking positive colony is incubated overnight on containing the fluid nutrient medium that gentamicin concentration is 40mg/L.

After culture, extracts plasmid and be sequenced whether detection sequence is purpose gene order, sequencing is done without mutation Afterwards i.e. it is believed that gene is successfully integrated into pDONR207, referred to as entry vector.

Reaction：

Relevant operation, the design of 2.5 μ L reaction systems are carried out using Gateway LR Clonase Enzyme Mix kits It is as follows：

Entry vector, 1 μ L；

Expression vector pK2GW2.0/rfa, 1 μ L；

LR enzymes, 0.5 uL；

25 DEG C are reacted 1 hour；

The Proteinase K Solution of 1 μ L are added, 10min is reacted at 37 DEG C；

Reaction product addition e. coli jm109 is converted, screening and culturing；The step is according to kit specification, or ginseng It is carried out according to the above Escherichia coli step of converting；

It should be noted that the LB solids and fluid nutrient medium of the step spectinomycin containing 80mg/L；

After culture, picked clones are cultivated in the LB liquid medium containing 80mg/L spectinomycins, and are carried out PCR is detected, and extracts plasmid to verifying correct positive colony, -20 DEG C save backup.

It should be noted that during correlated response, the plastid of plastid extraction process foundation Takara companies is small to carry reagent Box specification, or carried out with reference to following steps：

The overnight E. coli broth of 1.5mL is added into the centrifuge tube of 2mL, 12000rpm centrifuges 30s and collects thalline；

250 μ L solution P1 are added into the centrifuge tube there are bacterial sediment, are precipitated using the thorough suspended bacterial of turbula shaker；

250 μ L solution P2 are added into centrifuge tube, leniently spinning upside down 6-8 times makes thalline fully crack；

350 μ L solution P3 are added into centrifuge tube, leniently spins upside down 6-8 times, mixes well, 12000rpm centrifuges 10min；

The supernatant that previous step is collected is transferred to pipettor in adsorption column CP3,12000rpm centrifuges 30s, outwells collecting pipe In waste liquid, adsorption column CP3 is put into collecting pipe；

500 μ L protein liquid removals PD, 12000rpm centrifugation 30s are added into adsorption column CP3, outwells the waste liquid in collecting pipe, will inhale Attached column CP3 is put into collecting pipe；

600 μ L rinsing liquids PW, 12000rpm centrifugation 30s are added into adsorption column CP3, outwells the waste liquid in collecting pipe, will adsorb Column CP3 is put into collecting pipe；

Above step is repeated, is relaundered 1 time；

Adsorption column CP3 is put into collecting pipe, 12000rpm centrifuges 2min, the alcohol of cleared remnants；

Adsorption column CP3 is placed in a clean centrifuge tube, 50 μ L elution buffer EB are added dropwise to the intermediate position of adsorbed film, It is placed at room temperature for 2 min；12000rpm centrifuges 2min, collects plasmid, -20 DEG C save backup.

（Four）Conversion Agrobacterium and the genetic transformation for carrying out petunia

By step（Three）In constructed carrier convert to Agrobacterium plant Agl1, prepare transfection bacterium solution.To Agrobacterium-mediated Transformation Transgene carrier is carried out with reference to following steps：

Agrobacterium competence plant Agl1 is placed on ice, until melting；

It takes 2.5 uL plasmids in the centrifuge tube of 1.5mL, and the Agrobacterium competence of 50uL, ice bath 30min is added to it；Liquid nitrogen speed Freeze 1min；

Centrifuge tube is placed into 37 DEG C of warm bath culture 5min, it is rapid later to place cryostat 2min on ice；

The LB liquid medium of 800 μ L is added into centrifuge tube, sets 28 DEG C of shake culture 3h；

2800rpm centrifuges 5min, collects thalline, is uniformly coated on the solid containing 20mg/L rifampins, 80mg/L spectinomycins On culture medium, 28 DEG C of culture 2d；

Picking monoclonal, on the fluid nutrient medium containing 20mg/L rifampins, 80mg/L spectinomycins, 28 DEG C are incubated overnight； Bacterium solution is subjected to PCR detections, it is spare to verifying -80 DEG C of guarantor bacterium of correct positive colony.

The genetic transformation of petunia, i.e. During Agrobacterium, using the leaf disc transformation method of During Agrobacterium, to petunia kind ' M1 × R27 ' and W115 have carried out genetic transformation operation.Dip dyeing operation refers to following steps（Or reference：Conner et al. 2009, Transformation and regeneration of petunia. - In: Greats, T., Strommer, J:Evolutionary, Developmental and Physiological Genetics. Pp. 395- 409. Springer, New York）：

The petunia blade of 15 or so health of clip, sterilizes 2s in 70% alcohol, rinses 1 time in sterile water, then by leaf Piece is placed in 0.5% liquor natrii hypochloritis and sterilizes 10min, and after aqua sterilisa rinses 3 times, 5cm × 5cm is cut into blade Left and right size, the leaf dish being square；

Take the fresh bacterium solution of 5mL being incubated overnight（I.e. above-mentioned prepared transfection bacterium solution）, a concentration of 20 μM of 10 μ L acetyl fourth The aqua sterilisa of ketone musk and 10mL uniformly mix composition transfection liquid；

The petunia leaf dish that cutting is completed is placed in transfection liquid and disseminates 15min or so, the blade after dip dyeing is gently placed on It co-cultures and is cultivated 2 days on culture medium；

Then blade is moved to from co-cultivation culture medium and is cultivated 40 ~ 50 days on screening and culturing medium, until petunia young shoot is grown；

Petunia young shoot is moved on root media again and is cultivated 20 ~ 30 days, until petunia grows healthy and strong root；

Petunia after taking root moves to progress hardening culture, condition of culture in nutritive cube：25 ~ 30 DEG C, illumination condition be 6000 ~ 10000lx；Attention thin film moisturizing during culture hardening；Between culture（Or greenhouse）Middle culture 70 ~ 90 days, until petunia petal It is open.

The PCR of transfer-gen plant is detected：With the DNA of extraction（The DNA extractions of petunia refer to aforementioned extracting method）For mould Plate carry out PCR detections, and with corresponding recombinant plasmid make positive control, using unconverted petunia DNA as negative control into Row PCR reactions.Design primer sequence is as follows：

p35s（F）：AGAAGACGTTCCAACCACGTCT,

T35s（R）：TGCTCAACACATGAGCGAAAC；

PCR reaction systems and response procedures refer to aforementioned operation.

By pcr amplification product and DNA molecular Marker under 1 × TAE buffer conditions, with a little ethidium bromide of addition 1.0% Ago-Gel detaches, and the observation detection conversion results in ultraviolet gel imager.

（Five）Petunia transgenic experiments result

The transgenic petunia plant for being converted into work(continues to cultivate, and carries out relevant Physiology and biochemistry detection and analysis.It is related real It tests and is briefly discussed below.

（1）M1 × R27 transfer-gen plant phenotypic analyses

Three genes have anthocyanidin in callus and plant breeding to be shown to transfer-gen plant offspring's phenotypic analysis The phenotype of accumulation（Fig. 5）, apparent anthocyanin accumulation, including blade, stem and flower is presented in the transfer-gen plant obtained after overexpression Valve（Fig. 6）, especially early stage petal is developed, anthocyanin accumulation is significantly raised.

In addition analysis shows, ASR1^M1To anthocyanidin synthesis regulating and controlling effect it is most strong, gynoecium and stamen in genetically modified plants All present apparent anthocyanin accumulation, followed by ASR3^M1, regulating and controlling effect it is most weak be ASR2^M1。

（2）Petunia petal pH value measures

With reference to Quattrocchio et al. (2006, PH4 of Petunia is an R2R3-MYB protein that activates vacuolar acidification through interaction with basic-helix-loop- Helix transcription factors of the anthocyanin pathway), the pH of petal is measured.It surveys Timing should be noted：Since petal pH is influenced by external environment condition, all test samples must trained on an equal basis The condition of supporting carries out；In addition the carbon dioxide meeting interference measurement in air is as a result, therefore the continuous mode of pH is complete within 1 min At.

2 wide-open petals of sample to be tested are cut off when measurement respectively, is quickly placed after liquid nitrogen grinding and contains 6 mL Pure water（ddH₂O）Test tube, after mixing well, quickly measured with pH meter, numerical value recorded after numerical stability.

PH value is all about 5.6 between measurement result shows transgenic petunia and compares, without apparent difference spy Property, illustrate that the pH value of tri- gene pairs petunia petal cells of ASR does not have regulating and controlling effect.

（3）Liquid chromatogram（HPLC）Analysis

With reference to ' the measurement high performance liquid chromatography of anthocyanidin in People's Republic of China's agricultural industry criteria-plant-derived food ' （NYT 2640-2014）, using liquid-phase chromatography method, transgenic petunia petal anthocyanidin content is measured, correlative measurement Surely commission Ke Biao Technology R ＆ D Centers（Qingdao）Co., Ltd completes, and measures the quantitative standard specimen of anthocyanidin（Delphinidin, arrow vehicle Asterin, petunia pigment, pelargonidin, peonidin, malvidin）It is Sigma Products.

The component analysis of anthocyanidin in petunia petal is shown（ASR1 is analyzed using HPLC technologies^M1、ASR2^M1、 ASR3^M1It overexpresses transgenic petunia and compares the ingredient and content of anthocyanin in petal）, overexpress ASR genomes（Including ASR1^M1、 ASR2^M1And ASR3^M1Gene）It can promote the accumulation of anthocyanidin total content, wherein ASR1^M1Genetically modified plants petal In anthocyanidin content reach 493.62 mg/kg, ASR2^M1Reach 486.75 mg/kg, ASR3^M1Reach 488.63 mg/kg, And the anthocyanidin content in petunia control petal only has 297.34 mg/kg.

Further to 6 kinds of Anthocyanins analysis shows, in addition to delphinidin is not detected among in transgenic plants It arrives, different degrees of induction occur in other 5 kinds of pigments, and wherein paeonidin and malvidin increases apparent.

It can be seen that ASR genomes（Including ASR1, ASR2 and ASR3 gene）Specificity regulation and control be with delphinidin The anthocyanidin at center synthesizes access, i.e. ASR genomes are directly related with the synthesis of delphinidin.

（4）W115 transgenic petunia phenotypic analyses

It is directly related with the synthesis of delphinidin further to verify ASR genomes, by ASR^M1Three genes are transformed into respectively W115 petunia plant.Due in W115 there is complete delphinidin to synthesize passageway related genes.But due to the product ASRs genome and AN2 gene of the kind without containing origin, petal coloration are white, and ASR pairs can be directly verified by transgenosis The effect of petal coloration.

The result shows that apparent anthocyanin accumulation, including blade, stem, flower is presented in the transfer-gen plant obtained after overexpression Valve and stamen and gynoecium（Fig. 7）, early stage petal is developed, anthocyanin accumulation it is apparent more obvious.

Also, ASR1^M1To anthocyanidin synthesis regulating and controlling effect it is most strong, gynoecium and stamen all present in genetically modified plants Apparent anthocyanin accumulation, followed by ASR3^M1, regulating and controlling effect it is most weak be ASR2^M1。

It can further demonstrate that, ASR genomes（Including ASR1, ASR2 and ASR3 gene）Regulation and control that can be specific are to fly Anthocyanidin centered on swallow grass pigment synthesizes access.

Embodiment 3

The present embodiment is mainly introduced further to be tested by what hybridization means applied ASR genomes in petunia color adjustment Card.

For to ASR genomes（Including ASR1, ASR2 and ASR3 gene）Function in petunia pattern carries out further Verification, inventor further carried out genetic cross experiment（Hybrid process is as shown in Figure 8）, related experiment briefly introduction is such as Under.

Turn first to controlling anthocyanidin synthesis in petunia kind 1 × R27 of M self progenies, Petunia axlaris The record factor is identified, the results showed that：

The one of kind of M 1 × R27 self progenies ' R30 ' contains normal ASR genomes（Including ASR1, ASR2 and ASR3 genes ,+/+）With AN2 genes, 5 ' H genes of F3 ' have a mutation, delphinidin content and few, and phenotype is brick-red；

In Petunia axlaris, AN2 gene mutations, no ASR genomes（Including ASR1, ASR2 and ASR3 gene, -/-）, It is white that 5 ' H genes of F3 ', which have partial function, pattern phenotype,.

R30 is hybridized with Petunia axlaris, the phenotype spy of aubergine is presented in the filial generation ' L0 ' of acquisition Sign has genotype ASRs（+/-）

Petunia ' L0 ' is further selfed, a series of inconsistent offspring of phenotypes is obtained（Fig. 8）；

PCR detects the isogenic genotype of ' 5 ' ASR, F3 H, AN2 in self progeny, and DNA is carried out according to above method used in PCR , AN2, HF1（F3’5’H）, the genetic tests the primer sequence such as ASR it is as follows：

F3’5’H-1-F：CCAAATCTCCCTTACCTCC,

F3’5’H-1-R：CATCTATAGCTATGGTACATAAAC；

AN2(axi)-R：CTCCTTCCTGCATGAGGTTGCTC,

AN2(inf)-R；CTTGAGTAAGGCTGCTTTCAGC,

AN2-F：AGTTGCAGACTTAGGTGGTTG；

ASR1/2/3-F：CAGACGCCCATATCATGAATAC,

ASR2-R：CACTGGTCTCCATTATCTGTTG；

PCR system and operation are carried out with reference to aforementioned operation.

The result shows that：

Containing 5 ' H gene of ASR genomes and F3 ', self progeny's pattern is deeper, wherein brighter in corolla and floral tube engaging portion It is aobvious；And stamen has blue phenotype feature；

ASR genomic deletions or the mutation of 5 ' H genes of F3 ', self progeny's pattern is shallower, wherein corolla and floral tube engaging portion compared with Obviously；And stamen does not have blue phenotype feature；

Whether AN2 is mutated, as long as self progeny contains 5 ' H gene of ASR genomes and F3 ', flower is all presented in petal and stamen The phenotypic characteristic of green element accumulation（Fig. 9）；

To sum up it can be determined that：ASR genomic genes play an important role in the synthesis of regulation and control delphinidin, purple in initiative There is preferable application prospect in the flower breeding of color/blue.

SEQUENCE LISTING

<110>Henan Academy of Agricultural Sciences

<120>Petunia ASRs genomes and its application

<130> none

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1511

<212> DNA

<213> Petunia inflata

<400> 1

atgaatacta ttgttatctg tacgccgacg ttgggagtga gaaaaggttc atggactgaa 60

gaagaagatt ttcttttgag aaaatgcatt gaaaagtatg gtgaaggaaa gtggcatctt 120

gttcccgtta gagctggtaa acaaactaaa actataagcc atactctttt cctctttata 180

ttcctttaaa tagaagtata tgcatatgtc taatactctc cttctctttt acatatgaaa 240

caggtctgaa tagatgaaga aagagttgca gactaaggtg gttgaattat ctaaggccac 300

atattaagag aggtgacttc tctgaggatg aagtggatct cattttgagg cttcataagc 360

ttctaggcaa caggcaagct tttttctttt taattttttt gaaagctgat attcgtaaca 420

ctgcttcggt taatctgata tcatgccagg ttggcgacat gcaagtgaca actattggcg 480

gagtaaggaa ttaaaataaa gtggattttc aaaaacacta gaatgtcacg gttaactagt 540

taagatttga aattagatcc taaaataata ttttgaatga ggattcaata atctatatgt 600

aacataaaaa aatttatttt tgccctactt ttacagttta atatttcgaa aaattattga 660

gtttgtagtt gaacagatta agaaggagat tatagagagg atagagatgt tacaaagctc 720

aaagagagct catagaagta ggttgtttat tcagcattta ttgtaagtag ttgcgactgt 780

gggaggccta aagttcagtt tggaacttgt agatgctctt taggtttaag gttttggatg 840

taatggtaat catttacagt ttatcaaaaa aaatagtata tatatatttc gacaaaattc 900

atgaattgaa agatctccca aaacagaaac agagggagta atattaagta cacttcaccc 960

acaatattga tttccatgca taaccaagaa catatatatt gagcttgtct cctcttatca 1020

tatgcattcc atgtaggtgg tcacttattg cgggtagact tcctggaaga acagcaaatg 1080

atgtgaaaaa ctattggaac actaactttc gaaggaaatt atctcctcaa agacaaggga 1140

aaaggtgcag atctgccatt aagatcactg agaataccat aataaggcct caacctcgga 1200

tcttttcaag cgcaaagaat gtttctttgg gcagcaacaa gagtatcaca aacagtacta 1260

tccataagga tgaaagcagc aaaaatataa taaggactaa cgagaagcaa atgactgaag 1320

catcgacaga aaatggagtt caatggtggg caagtttatt ggatgatcct aatgaatttg 1380

ataaggaagc agcaactgga agcatacctt cgaattcatg taatatcaac tccaaggaag 1440

aattaccaac attcttgcaa gaaggagaaa ctggctgggg tgatattgac ctctggagtc 1500

ttcttaatta g 1511

<210> 2

<211> 1535

<212> DNA

<213> Petunia inflata

<400> 2

atgaatacta ttcctatctg tacgccgacg ttgggagtga ggaaaggttc atggactgaa 60

gaagaagatt ttcttttgag aaaatgcatt gaaaagtatg gtgaaggaaa gtggcatctt 120

gttcccgtta gtgctggtaa acaaactaaa actataagcc gtactctttt cctctttata 180

ttcctttaaa tagaagtata tgcataggtc taatactctc cttgtctttt atatatgaaa 240

caggtctgaa tagaatgtag aaagagttgc agactaaggt ggttgaatta tctaagacca 300

catattaaga gaggtgattt ctctgaggat gaagtggatc tcattttgag gcttcataag 360

cttctaggca acaggcaagt ttttttcttt tttatttttt tgaaagctga tattcgtaac 420

actgcttcgg ttaatctgat atcatgccag gtaggcgaca tgcaagtgac cactattggc 480

ggagtaagga attaaaataa agaggatttt caaaaacaca cgaaatgtca cggttaacta 540

gttaagattt gaaattagac ctaaataata ttttgaatcc cctttgttac aactgaattt 600

ttgtttacgt aaagaagatt caataatcta tattataaca taaaattttt tatttttgcc 660

ttacttgtac agaataattt ttcggcaaag gttaattaat gaaccccctt ctgtgaatct 720

atctatgccc ctaattacca ccaccaacac attttatatg aaggtatttc actgaataca 780

taatatttaa attttcggtc ttaaacatat cattatagtt gtataaaatt atgtcatcga 840

ggatataatt agattaaagt attctaaacc agtagaggtg tcaatatttt tggaaacgga 900

ttaaaaaaga aagatgtccg aaaatgaaac cagaaagtaa tatttactac actttcaccc 960

acaatcccca gttgatgttg atctccaact aagaacatat gctgccttat gctgcataca 1020

tatattgagt cttgtctgat tttatcatgt acacttcctt taggtggtca cttattgcgg 1080

gtagacttcc cggaagaaca gcaaacgatg tgaaaaacta ttggaacact cactttcaaa 1140

ggaagttatt ttctccacaa atacaaggga aaaagtgcag atctgccgtt aagatcactg 1200

agaataccat aataagacct caacctcgga acttctcaag cacgaagaat gtttctttgg 1260

gcagaaacaa gagtatcaca aacggtacta tacataagga tgaaaccagc aaaacaatta 1320

taaggactaa cgagaagcaa acgactgaag catcaacaga taatggagac cagtggtggg 1380

caagtttatt ggatgatcgt aatgaatttg atgaggaagc agcagctgga aaaacacctt 1440

catgtaagat caactctgag ctagaaacac caacattgct gcaagaagga gaaactggct 1500

ggggtgatat tgacctctgg agtctactta attag 1535

<210> 3

<211> 1503

<212> DNA

<213> Petunia inflata

<400> 3

atgaatacaa ttcctatctg tacgtcgacg ttgggagtga ggaaaggtgc atggactgaa 60

gaagaagatt ttcttttgag aaaatgcatt gaaaagtatg gtgagggaaa gtggcatctt 120

gttcccgtta gagctggtaa acaaacttgt actattaagt actcctcttt atattcgttt 180

aaatatatag aagtatatgc atatgtctaa tactctcctt gtgttttata tgtgaaaata 240

tacaggtctg aatagatgta ggaagagttg cagactgagg tggttgaatt atctaaggcc 300

acatattaag agaggtgact tctctgagga tgaagtggat ctcattttga ggcttcataa 360

gcttctaggc aacaggcgag tttgttaatt ttgttttttt cgctagctga tattcgtaac 420

actgattcga ttattctggt atcttgccag gtaggcgaca tgcaagtgac cactagtggc 480

gggttaggag ttcagatata gagggttatc agaaacacta gaatgtcaca tctaagatat 540

gctaatgaat tttcgtttat gttcaaagaa ttcaatatat agtctatacc taatgtaaca 600

aagtttcctt ttgccctaca tgtacaatgt agggcaaaga aagactacga acctcgttaa 660

ttctacgaac ctcgctatac ccatagttac cactaccctc aacactatgt ggagatgttt 720

tactgagcaa gttgtttaaa aaaagaaaga cttttggtac ttgtggtctt gacatattaa 780

tttgtgtaaa atcatgtcat tgaggataaa agaattatct aaataaattg ttcaaatatt 840

atatagaggt gtcaatcttt ctggaaacag atttaaaaga aagagtccca aaaatgaaac 900

agagggatta atatttacta ccctttcacc caccatcccc agttgatctc catgcataac 960

caagaacata tgctgcataa atatatatgg agtcttgtct gactttatca tataaagtac 1020

atttccttta ggtggtcact tattgcgggt agaattccag gaagaacagc aaacgacgtg 1080

aaaaactatt ggaacactca ctatcgaagg aagttaattt ctcttcaaag acaagggaaa 1140

aagtgcagat ctgccattaa gatcactgaa aataccatta taagacctca acctcggagc 1200

ttctcaagca caaagaatgt ttctttgggc agcaacaaga gtatcacaaa cagtacaata 1260

cataaggatg aaagcagcaa aagagttgta aggactaacg agaaacaaac gactgaagca 1320

ttgacagaca atggatttca atggtgggct agtctattgg atgatcctaa tgaatttgat 1380

gaggaagcag cagctggaag cacagctttc tgtaagatca actccgagga agaaacacca 1440

acatcgttgc aagaaggaga aactggctgg ggtgatattg acctctggag tctacttaat 1500

tag 1503

Claims (8)

1. a petunia ASRs genome, which is characterized in that the genome includes tri- genes of ASR1, ASR2 and ASR3, is compiled The transcription factor of code R2R3-MYB types is related to anthocyanidin synthesis regulation respectively；

The base sequence of the ASR1, including 1511bp, base sequence is as shown in SEQ ID NO.1；

The base sequence of the ASR2, including 1535 bp, base sequence is as shown in SEQ ID NO.2；

The base sequence of the ASR3, including 1503bp, base sequence is as shown in SEQ ID NO.3.

2. application of the petunia ASRs genomes described in claim 1 in pattern regulation and control, which is characterized in that the genome Tri- genes of ASR1, ASR2 and ASR3 are related to anthocyanidin synthesis regulation respectively.

3. application of the petunia ASRs genomes as claimed in claim 2 in pattern regulation and control, which is characterized in that the genome Tri- genes of ASR1, ASR2 and ASR3 are related to regulation and control delphinidin synthesis respectively.

4. utilizing the overexpression recombinant vector constructed by petunia ASRs genomes described in claim 1, which is characterized in that utilize Tri- genetic transformation of ASR1, ASR2 and ASR3 of ASRs genomes are entered overexpression vector by gateway technologies respectively In pK2GW2.0/rfa, obtained to build.

5. the PCR amplification method of petunia ASRs genomes described in claim 1, which is characterized in that contain ASRs genomes The genome of the petunia kind of gene carries out PCR amplifications to three genes respectively as PCR amplification template, when PCR amplification Specific primer sequence is designed as：

Primer is when expanding ASR1 genes：

ASR1-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTGTTATCTGTAC,

ASR1-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGAAGACTCCAGAGGTCA；

Primer is when expanding ASR2 genes：

ASR2-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACTATTCCTATCTGTAC,

ASR2-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTCTAATTAAGTAGACTCCAGAGG；

Primer is when expanding ASR3 genes：

ASR3-Fw：GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAATACAATTCCTATCTGTAC,

ASR3-Rv：GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGTAGACTCCAGAGG.

6. the PCR amplification method of petunia ASRs genomes as claimed in claim 5, which is characterized in that described to contain ASRs bases Because the petunia kind of group gene is：Petunia M1 or V30 kind.

7. a kind of method of adjustment petunia pattern, which is characterized in that using the method for hybridization recombination, to contain ASRs genomes The petunia kind of gene is maternal or male parent, is male parent or mother with other petunia kinds without ASRs genomic genes This, is hybridized, and hybridizes petunia variety design so as to adjust filial generation；

Or technique for gene engineering is utilized, ASRs genomes are overexpressed to accumulate anthocyanidin, or carry out gene mistake Living, gene silencing or gene knockout to reduce anthocyanin accumulation, or the conversion of ASRs genomes are entered and is free of ASRs genes In the plant of group, so as to adjust pattern.

8. the method for adjustment petunia pattern as claimed in claim 7, which is characterized in that described containing ASRs genomic genes Petunia kind is petunia M1, V30 kind；The petunia kind without ASRs genomic genes be petunia R27, W115 kinds.