CN105132433B

CN105132433B - The cDNA sequence of the MYB transcription factors of positive regulation and control freesia flower anthocyanin synthesis

Info

Publication number: CN105132433B
Application number: CN201510403554.0A
Authority: CN
Inventors: 高翔; 王丽; 李月庆; 单晓彤
Original assignee: Northeast Normal University
Current assignee: Northeast Normal University
Priority date: 2015-07-12
Filing date: 2015-07-12
Publication date: 2018-10-09
Anticipated expiration: 2035-07-12
Also published as: CN105132433A

Abstract

The present invention for the first time the clone from safflower freesia petal obtain two positive regulation and control anthocyanin synthesis myb transcription factors (FhPAP1L1WithFhPAP1L2) cDNA sequence, and speculate obtained its coding amino sequence.The overexpression in arabidopsis and tobaccoFhPAP1L1WithFhPAP1L2The coloring of plant tissue can significantly be enhanced, so that it is determined that it participates in the function of positive regulation and control flower color glycosides synthesis.

Description

The cDNA sequence of the MYB transcription factors of positive regulation and control freesia flower anthocyanin synthesis

Technical field

The invention belongs to plant molecular biotechnologies, and in particular to two isolated tools of safflower freesia petal It is functional, relevant MYB transcription factors are synthesized with flower anthocyanin（FhPAP1L1 and FhPAP1L2) cDNA sequences Row and its amino acid sequence.

Background technology

Influence vegetation color mainly has three categories pigment：Anthocyanin, carotenoid and betanidin.People are for pattern Great interest is entertained in the research of glycosides, this plays very important effect derived from it to plant physiology and human health.Anthocyanin It is by a series of Enzyme catalyzed synthesis, these enzymes are then controlled by corresponding structural gene, research shows that：These structural genes Expression by a complicated transcription regulatory network regulation and control, wherein MYB transcription factors be MBW (MYB-bHLH-WDR) transcriptions because The important member of sub- compound, it plays the effect of specificity adjusting.Plant MYB transcription factor families, which are one, to be had The large family of different adjusting functions, they are for activating the expression of specific downstream gene to play a significant role.According to MYB The difference of albumen conserved domain, MYB can be divided into R2R3 MYB and R3 MYB etc..

Up to the present, the clone in many plants obtains just regulating and controlling the R2R3 MYB that anthocyanin synthesizes people, such as Toad's-mouth, arabidopsis, wild cabbage, capsicum, sweet orange, Herba Epimedii, mangosteen, rough gentian, African Chrysanthemum, sweet potato are led a cow, apple, clover, poplar Plum, tobacco, petunia, European pear, sand pear, flowering peach, tomato, potato and grape etc., but these belong to dicotyledonous plant Object, in unifacial leaf only from lily, iris and corn clone obtained anthocyanin synthesize relevant MYB transcription because Son.Involved in the present invention to FhPAP1L1 and FhPAP1L2 genes clone and obtain from monocotyledon freesia, Coding synthesizes relevant MYB transcription factors with flower color glycosides, and the specifically gene is turn that positive regulation and control flower color is formed The factor is recorded, the synthesis of anthocyanin can be promoted, with pattern row at closely related.

Invention content

The present invention for the first time the clone from safflower freesia petal obtain the MYB transcriptions of two positive regulation and control anthocyanin synthesis because The cDNA sequences of sub (FhPAP1L1 and FhPAP1L2), and speculate and obtained the amino sequence of its coding.In arabidopsis and Overexpression FhPAP1L1 and FhPAP1L2 can significantly enhance the coloring of plant tissue in tobacco, so that it is determined that it is joined The function of being synthesized with positive regulation and control flower color glycosides.

The main object of the present invention is to provide the coding FhPAP1L1's and FhPAP1L1 for having obtained functional verification CDNA sequences and the amino acid sequence thus inferred.

Technical scheme of the present invention：

In order to which from for the first time, clone obtains FhPAP1L1 and FhPAP1L2 genes from freesia, the present inventor first with 1 sequence of Arabidopsis thaliana PAP is as bait, to laboratory established safflower freesia blossom period transcript profile library Carry out local Blast, screening and the higher sequence of homology in arabidopsis.Isolated length is 212bp's FhPAP1L1 segments are analyzed the sequence and are shown：This segment and the R2R3 MYB homologys of Torenia fournieri are most Height reaches 67%, and the segment includes ATG initiation codons, and the ends 3' of the gene are cloned followed by RACE methods Sequence obtains terminating comprising TAA close

The length of numeral is the segment of 717bp, this segment and the MYB75-like of Elaeis guineensis are same Source property highest, reaches 47%.Above-mentioned two sequences are spliced, full-length cDNA sequence is obtained, are spent followed by safflower freesia For valve total serum IgE as template, design primer carries out RT-PCR, and clone obtains the overall length FhPAP1L1 genes of 929bp, should The long 702bp of gene open reading frame encodes 233 amino acid, and the molecular weight supposition of protein is 27,023 Da, etc. Electricity point is 6.23.Sequence analysis shows：The MYB75-lke homology highests of this gene and Elaeis guineensis, Reach 52%.In addition, we are also isolated to the FhPAP1L2 segments that length is 755bp, sequence analysis is shown：The piece Section includes ATG initiation codons and TAA terminator codons.Safflower freesia petal total serum IgE is extracted as template, is carried out RT-PCR, clone obtain the overall length FhPAP1L2 genes of 755bp, and the long 729bp of open reading frame encodes 242 ammonia The molecular weight supposition of base acid, protein is 28,076 Da, isoelectric point 5.09.Sequence analysis shows：This gene also with The MYB75-like homology highests of Elaeis guineensis, reach 53%.

Have detected FhPAP1L1's and FhPAP1L2 using Gal4-GUS protoplasts of Arabidopsis thaliana broken by ultrasonic transient transfection systems Transcriptional activity, the results showed that：FhPAP1L1 and FhPAP1L2 is transcription activator.

FhPAP1L1 the and FhPAP1L2 genes that clone obtains are inserted into pZP211 binary expression vectors, and It is transferred in Agrobacterium GV3101 cells, transfects arabidopsis and tobacco.

Respectively with colored method and leaf disk method transfection arabidopsis and tobacco is dipped in, transfer-gen plant character mutation is observed.As a result it shows Show：The synthesis of transfer-gen plant anthocyanin significantly increases, and illustrates that FhPAP1L1 the and FhPAP1L2 genes in freesia promote The synthesis of anthocyanin, preliminary proof we to clone obtained gene be the MYB that positive regulation anthocyanin synthesizes in freesia Transcription factor.

The cDNA sequences of freesia FhPAP1L1 and FhPAP1L2 gene and its amino acid sequence information of coding are such as Under：Wherein 1：The full length nucleotide sequence of FhPAP1L1 genes（SEQ ID NO. 1） ATCCAATATTCCTCCCCCTAATGTCTTATCTTGCATGATTCACAATTATGAAACATCAGTACTGCTCCGAGCT

CCGACCTTCGGGCGTTCAGAATCGGAAAGGGGCATGGAACAAAGATGAAGATGAGCTGCTGAGGAAGTGCATTGAAA AGTATGGAATAGGGAAGTGGAGTACAGTTCCCATGAGGGCAGGACTAAGAAGGTGTCGCAAGAGCTGTCGTCTTCGC TGGCTGAACTATCTATCCCCTGACATCAACCGAGGAACCTTTAACGACGACGAAGACGACCTCATCCTTCGGCTTCA CAAGCTCTTGGGCAATAGGTGGTCCCTAATTGCAGGCAGAATCCCCGGCAGGACGGCGAACGACATCAAGAACTACT GGAACTCTCACTTGGGCAAGAAAGCAGACACCGAGAGAAGGGTACCAGAGAAGACTGACAAGGCTGAGATTGTAAGG CCACAACCTCAAAGACCGACTTCGACGTGGATTTGGCCGAAGATTAATATTAATCCTCATCAAGAAACTGATCAATT AGGAACATCTGCCGTGGAGCCAAAACTGCCAACAGTCTTAGAGGAGGAAGAAGCATGGCTGAATGCTTTGATCACCG ATGATTACAATGAGAATGTGGATGCCGCCTTTTCGGACTTCCATGGCTACGAGATAGGGGGAGGAATATTGGATGAC ACGCCTTTTTTCGAAGGAGTTACAGGATGGGAGGAACTATACCAGAGAACTGAAAATTAAGTTTAATTTTTCATTGT TCTAACAATACTGATATCTATATATATATTTTTTTTTTCCTTCGAAGATCAAAGATAGAAAGAAATGAGACCAGTTC CCCATTGTTGAAAAATAAAGGAATAAGGTTTATGTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCGATGTCG ACTCGAGTC

Wherein 2：The protein sequence speculated by FhPAP1L1 gene nucleotide series（SEQ ID NO. 2） MKHQYCSELRPSGVQNRKGAWNKDEDELLRKCIEKYGIGKWSTVPMRAGLRRCRKSCRLRWLNYLSPDINR

GTFNDDEDDLILRLHKLLGNRWSLIAGRIPGRTANDIKNYWNSHLGKKADTERRVPEKTDKAEIVRPQPQRPTSTW IWPKININPHQETDQLGTSAVEPKLPTVLEEEEAWLNALITDDYNENVDAAFSDFHGYEIGGGILDDTPFFEGVTGW EELYQRTEN.

Wherein 3：The full length nucleotide sequence of FhPAP1L2 genes（SEQ ID NO. 3）

AATGGATCTCAAAGCTTCAGCCCTTCGAAAAGGCGCATGGAGCAAAGAGGAAGACGACCTGCTAAGGAATTGT ATCGATAAGTATGGAGAAGGGAAGTGGAGTTCCGTACCCGAACGTGCAGGGCTTAAAAGGTGTCGAAAGAGCTGTCG ACTTAGATGGCTGAACTATCTTTCGCCGAAGATCAACCGAGGCAAGTTTTCGGATGACGAGATCGACCTCCTTATCA GGCTTCACAAGCTTCTAGGGAACAGGTGGTCGCTGATCGCGGGCAGAATCCCCGGTAGGACAGCGAACGACATCAAG AATTATTGGAACTCTCACCTGAGCAAGAGAGTGGAAGCAACAAAATGTAGGAGCAAAACTATAGATGCTCAAGTCGC GAGGCCAAATCCTGAGAGAGATTCCGCCAACTGGAGTTGGCCAACAAATATAGGAACAAATTATGGGAACAACTTGG AAGCAGAAGAAACCGAAATGCCGAAACTACGAACAAGTCAAGAGAATCAGGGAGAACGGTCAAATGATTTGATCATG GAGAATGGTTGGGAAGAGTTGATGCTGCATACTTCTGACGTGAGCTTACAGAATTTTCAGATAGACTTCAAGACACC GGAAATGGAGGAACTAACTGAGATGGAGGAACTAACTGAGGAACCAATCTCTCTGGGAGATGAAGGGAATGATTGGA CTGCCATGGAAAACTATTTTTTTGATGAGCTCATTAGTTAAGAAAAAAATATATACTAAAATGCGG

Wherein 4：The protein sequence speculated by FhPAP1L2 gene nucleotide series（SEQ ID NO.4） MDLKASALRKGAWSKEEDDLLRNCIDKYGEGKWSSVPERAGLKRCRKSCRLRWLNYLSPKINRGKFSDDEI

DLLIRLHKLLGNRWSLIAGRIPGRTANDIKNYWNSHLSKRVEATKCRSKTIDAQVARPNPERDSANWSWPTNIGTN YGNNLEAEETEMPKLRTSQENQGERSNDLIMENGWEELMLHTSDVSLQNFQIDFKTPEMEELTEMEELTEEPISLGD EGNDWTAMENYFFDELIS.

Description of the drawings：

Fig. 1 FhPAP1L1 and FhPAP1L2 conserved structure domain analysis；

Fig. 2 FhPAP1L1 and FhPAP1L2 phylogenetic analysis；

The transcriptional activation activity of Fig. 3 FhPAP1L1 and FhPAP1L2 are analyzed；

Fig. 4 FhPAP1L1 transgenic arabidopsis character mutations；

Fig. 5 FhPAP1L1 transgene tobacco character mutations；

Fig. 6 FhPAP1L2 transgenic arabidopsis character mutations；

Fig. 7 FhPAP1L2 transgene tobacco character mutations.

Specific implementation mode：

The present invention is further illustrated in following specific embodiment, this does not limit the scope of the invention.

The clone of 1 FhPAP1L1 full length genes of embodiment

The acquisition of [embodiment 1-1] target fragment：According to arabidopsis PAP1 sequences, local Blast is carried out.Sieve Choosing and the higher sequence of arabidopsis PAP1 homologys, and bioinformatic analysis is carried out, obtain the piece that length is 212bp Section, and include initiation codon ATG.

The acquisition of [embodiment 1-2] FhPAP1L1 gene 3' sequences：According to transcript profile sequencing sequence, design is special Property sense primer, i.e. 5'-GGCAGGACTAAGAAGGTGTCG-3'；The polyA structure designs guarded according to mRNA are special Different downstream primer：5'-AAAAAAAAAAAAAAAAATCGATGTCGACTCGAGTC-3'.It is extracted with Trizol kits The total serum IgE in safflower freesia blossom period, reverse transcription generate first chain of cDNA, are carried out in this, as template The PCR products of RACE, obtained 717bp.

The acquisition of [embodiment 1-3] FhPAP1L1 full length gene sequences：According to above-mentioned sequencing obtain as a result, design Special sense primer, i.e. 5'-CCTCCCCCTAATGTCTTATCTTG-3' and 5'- ATATATAGATATCAGTATTGTTA-3'

Downstream primer.The total serum IgE of safflower freesia petal is extracted with Trizol kits, reverse transcription generates cDNA First

Chain carries out RT-PCR in this, as template, obtains FhPAP1L1 full length gene sequences.

The clone of 2 FhPAP1L2 full length genes of embodiment

The acquisition of [embodiment 2-1] target fragment：According to arabidopsis PAP1 sequences, local Blast is carried out.Sieve Choosing and the higher sequence of arabidopsis PAP1 homologys, and bioinformatic analysis is carried out, obtain the FhPAP1L2 of 755bp Segment, sequence analysis show that the segment includes ATG initiation codons and TAA terminator codons.

The acquisition of [embodiment 2-2] FhPAP1L2 full length gene sequences：According to transcript profile sequencing sequence, design is special Property sense primer, i.e. 5'-AATGGATCTCAAAGCTTCAG-3' and 5'-CCGCATTTTAGTATATATTTTTTT-3' Downstream

Primer.The total serum IgE in safflower freesia blossom period is extracted with Trizol kits, reverse transcription generates First chain of cDNA carries out RT-PCR, the full length gene sequence of obtained FhPAP1L2 in this, as template.

The transcriptional activity of embodiment 3 FhPAP1L1 and FhPAP1L2 detects

It is that reporter gene is intended using Gal4-GUS to verify the transcriptional activity of FhPAP1L1 and FhPAP1L2 Southern mustard protoplast transiently transfects, using GD-VP as positive control.The opening of FhPAP1L1 and FhPAP1L2 are read respectively Frame and GD

（The combined areas Gal4 DNA）Fusion, 35S promoters driving, is cloned on pUC19 carriers, is built into GD The protein sequence of label.Plasmid DNA, the arabidopsis leaf trip of growth about surrounding are extracted using except the big extraction reagent kit of endotoxin plasmid Protoplast is separated out, effector GD-FhPAP1L1 or GD-FhPAP1L2 are total with reporter gene Gal4-GUS respectively Protoplasts of Arabidopsis thaliana broken by ultrasonic is transfected, room temperature dark is incubated 20-22h, detection GUS activity.The result shows that：FhPAP1L1 and FhPAP1L2 is transcription activator.

Expression of the 4 FhPAP1L1 and FhPAP1L2 genes of embodiment in arabidopsis and tobacco

In order to verify FhPAP1L1 and FhPAP1L2 protein functions, using the obtained overall length FhPAP1L1 of clone and FhPAP1L2 genes are utilized respectively the primer with Nde I and Cla I restriction enzyme sites and carry out PCR expansions as template Increase.Then the DNA segments obtained using two kinds of above-mentioned amplifications of digestion with restriction enzyme of Nde I and Cla I, and by its Clone enters on improved plasmid pUC19, then is building up on plasmid pZP211 after I single endonuclease digestions of EcoR.Such structure The recombinant plasmid built is respectively designated as pZP211-FhPAP1L1 and pZP211- FhPAP1L2, and imports Agrobacterium GV3101 In cell.

The culture transformant, respectively with dipping in colored method and leaf disk method arabidopsis thaliana transformation Col and tobacco K326.Through Kan screening transgenic plant, and observe character mutation.

<110>Northeast Normal University

Northeast Normal University, Gao Xiang

<120>The cDNA sequences of the MYB transcription factors of two kinds of positive regulation and control freesia flower anthocyanin synthesis

<130> -

<140> -

<141> 2015-04-29

<160> 4

<170> PatentIn version 3.3

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atccaatatt cctcccccta atgtcttatc ttgcatgatt cacaattatg aaacatcagt 60

actgctccga gctccgacct tcgggcgttc agaatcggaa aggggcatgg aacaaagatg 120

aagatgagct gctgaggaag tgcattgaaa agtatggaat agggaagtgg agtacagttc 180

ccatgagggc aggactaaga aggtgtcgca agagctgtcg tcttcgctgg ctgaactatc 240

tatcccctga catcaaccga ggaaccttta acgacgacga agacgacctc atccttcggc 300

ttcacaagct cttgggcaat aggtggtccc taattgcagg cagaatcccc ggcaggacgg 360

cgaacgacat caagaactac tggaactctc acttgggcaa gaaagcagac accgagagaa 420

gggtaccaga gaagactgac aaggctgaga ttgtaaggcc acaacctcaa agaccgactt 480

cgacgtggat ttggccgaag attaatatta atcctcatca agaaactgat caattaggaa 540

catctgccgt ggagccaaaa ctgccaacag tcttagagga ggaagaagca tggctgaatg 600

ctttgatcac cgatgattac aatgagaatg tggatgccgc cttttcggac ttccatggct 660

acgagatagg gggaggaata ttggatgaca cgcctttttt cgaaggagtt acaggatggg 720

aggaactata ccagagaact gaaaattaag tttaattttt cattgttcta acaatactga 780

tatctatata tatatttttt ttttccttcg aagatcaaag atagaaagaa atgagaccag 840

ttccccattg ttgaaaaata aaggaataag gtttatgttt taaaaaaaaa aaaaaaaaaa 900

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aatggatctc aaagcttcag cccttcgaaa aggcgcatgg agcaaagagg aagacgacct 60

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agggcttaaa aggtgtcgaa agagctgtcg acttagatgg ctgaactatc tttcgccgaa 180

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caagaattat tggaactctc acctgagcaa gagagtggaa gcaacaaaat gtaggagcaa 360

aactatagat gctcaagtcg cgaggccaaa tcctgagaga gattccgcca actggagttg 420

gccaacaaat ataggaacaa attatgggaa caacttggaa gcagaagaaa ccgaaatgcc 480

gaaactacga acaagtcaag agaatcaggg agaacggtca aatgatttga tcatggagaa 540

tggttgggaa gagttgatgc tgcatacttc tgacgtgagc ttacagaatt ttcagataga 600

cttcaagaca ccggaaatgg aggaactaac tgagatggag gaactaactg aggaaccaat 660

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cattagttaa gaaaaaaata tatactaaaa tgcgg 755

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Met Asp Leu Lys Ala Ser Ala Leu Arg Lys Gly Ala Trp Ser Lys Glu

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Glu Asp Asp Leu Leu Arg Asn Cys Ile Asp Lys Tyr Gly Glu Gly Lys

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Trp Ser Ser Val Pro Glu Arg Ala Gly Leu Lys Arg Cys Arg Lys Ser

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Cys Arg Leu Arg Trp Leu Asn Tyr Leu Ser Pro Lys Ile Asn Arg Gly

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Lys Phe Ser Asp Asp Glu Ile Asp Leu Leu Ile Arg Leu His Lys Leu

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Glu Ala Thr Lys Cys Arg Ser Lys Thr Ile Asp Ala Gln Val Ala Arg

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Gly Thr Asn Tyr Gly Asn Asn Leu Glu Ala Glu Glu Thr Glu Met Pro

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Lys Leu Arg Thr Ser Gln Glu Asn Gln Gly Glu Arg Ser Asn Asp Leu

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Ile Met Glu Asn Gly Trp Glu Glu Leu Met Leu His Thr Ser Asp Val

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Ser Leu Gln Asn Phe Gln Ile Asp Phe Lys Thr Pro Glu Met Glu Glu

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Leu Thr Glu Met Glu Glu Leu Thr Glu Glu Pro Ile Ser Leu Gly Asp

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Ile Ser

Claims

1. the just cDNA sequence of the myb transcription factor of regulation and control freesia flower anthocyanin synthesis, it is characterized in that：Described The full length nucleotide sequence of cDNA is as shown in sequence table SEQ ID NO.1 or full length nucleotide sequence such as sequence table SEQ ID Shown in NO.3.

2. the just myb transcription factor albumen of regulation and control freesia flower anthocyanin synthesis, it is characterised in that：The transcription factor Shown in the amino acid sequence that albumen is speculated by nucleotide sequence such as sequence table SEQ ID NO. 2 or nucleotide sequence speculates The amino acid sequence arrived is as shown in sequence table SEQ ID NO. 4.