CN113025623B - Rose fragrance regulatory gene RrTPS1 and application thereof - Google Patents
Rose fragrance regulatory gene RrTPS1 and application thereof Download PDFInfo
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Abstract
The invention relates to a rose fragrance regulatory gene RrTPS1 and application thereof, wherein the nucleotide sequence is shown as SEQ ID NO. 1; the RNA sequence of the mRNA of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO.2, and the amino acid sequence of the expression protein of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO. 3. According to the invention, the expression modes of the RrTPS1 gene at different flowering stages and different parts of the floral organs are detected by a real-time fluorescent quantitative detection technology, and meanwhile, the petunia is transformed by constructing an overexpression vector, so that the petunia is proved to promote the synthesis of aromatic substances, and has important application values in the flower breeding fields of enhancing the synthesis of floral substances, improving the quality and yield of essential oil products and the like.
Description
Technical Field
The invention relates to a rose fragrance regulating gene RrTPS1 and application thereof, belonging to the technical field of plant genetic engineering.
Background
Roses (A), (B)Rosa rugosaThunbThe rose essential oil is an important ornamental and economic dual-purpose plant of rosa of rosaceae, the rose essential oil is obtained by extracting the aromatic substance of the rose through a process, has strong fragrance and high price, is widely applied to high-grade cosmetics and perfume, and the demand of international and domestic markets for the rose and the rose products is increased year by year. The rose essential oil has very complicated chemical components, more than 300, including terpene compounds, alkane, alcohol, lipid, aldehyde and the like, and the components of the Pingyin rose essential oil are qualitatively and quantitatively analyzed by a gas chromatography-mass spectrometry combined technology, wherein the main components are respectively as follows: citronellol (63.02%), geraniol (10.06%), eugenol methyl ether (4.67%), citronellol acetate (4.64%), and monoterpene alcohol as a main component constituting rose fragrance. Terpene synthases are key enzymes in the synthesis of terpenoids, and are also referred to as cyclases because the terpene synthase products are mostly cyclic. The terpenoids are the most abundant compounds in the plant body, and it is the reason for the most abundant compoundsDue to the diversity of terpene synthases. The terpene synthase gene family can be divided into 7 subfamilies according to the sequence correlation by utilizing the clustering analysis of the terpene synthase amino acid sequence, namely TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g and TPS-h, and the gene homology among all the subfamilies is more than 40 percent. Researches find that the action effect of the plant terpene synthase is extremely diverse, not only can adjust the flower fragrance and promote growth regulation, but also has great significance on the defense mechanism of plants. At present, TPS genes for synthesizing monoterpene are not clearly identified in roses, TPS function research is mainly limited to model plants such as arabidopsis thaliana and the like, the plants have no typical aromatic characteristics and do not contain aromatic substances such as citronellol and the like, the TPS action of the plants is inevitably different from that of rose TPS, and a rose terpene synthase gene TPS1 is cloned and utilized, so that the synthesis path of the volatile aromatic substances of the roses is facilitated to be clarified, a molecular tool is provided for genetic engineering breeding of roses with high aromatic substance content, and the potential economic value of improving the yield and quality of rose essential oil is achieved.
Disclosure of Invention
The invention aims to solve the problems and provides a rose fragrance regulatory gene RrTPS1 and application thereof.
The purpose of the invention is realized by that, a rose fragrance regulating gene RrTPS1 is characterized in that: the nucleotide sequence is shown as SEQ ID NO. 1;
SEQ ID NO.1 is:
ATGCAGGCAGAATGTTATCGAATTCAGATTCAGAAGCTAATAGAAGATGTTAAGGATTTGTTTGGTGAATGTAAGGAATTGGGTTTACTAGCTAAGTTGGAGCTGATTGACAGCATCCAAAAACTAGGCCTGAACAGCTACTTTGAGAAGGAAATCAAGGAAACCCTAGACACCATAGCATCTGTCGAACTGAAAAACAACAGCAATCCTTTCATTAGTTCAGAGGGTGATCTCTACGCTACTGCACTACTCTTTAAGATTCTGAGGCAGCATGGTTATAAAGTTTCACAAGGTATAGACGCAGATGTACTAATTAATTAGTTGAACATTGCTAGGTGTATAACTTGTTTGGTACCACATGCATCCCATTTTATGTGGCAGTTGATGTAGCACAATCACGTAATTTAATGAAATCCTCTGATAGATTGGGTATTGTATATGCTACATCTACAACCACATAAGATGGGATACAGGTGGTGCCCAAAAATGTGGTCTAATTTATCTTCTAACTAATCTAACGTACTAATCCAGTTTACTAAACTTTTCAGATGTATTTGGTGTTTTCATAGACGAAATTGGTACATTAAAGAAGAGCACATTTGGGGATGTGAAAGGAATGCTCGAACTTTTGGAGGCCTCAAACTTAGCTTTAGAAGGTGAAAACATCCTAGATGAGGCTAAAGCTTTCTTAATGGTCACTCTCAGAGATACCAATACCATGTGTGATGATATAGACCATTACATTTCCAAGCATGTGGCCTATGCCTTGAAGCTTTCATCACAAAGGAGAGTCCAGTGGTTTAATGTGAAATGA；
the RNA sequence of the mRNA of the rose fragrance regulating gene RrTPS1 is shown as SEQ ID NO.2, and the amino acid sequence of the expression protein of the rose fragrance regulating gene RrTPS1 is shown as SEQ ID NO. 3;
SEQ ID NO.2 is:
AUGCAGGCAGAAUGUUAUCGAAUUCAGAUUCAGAAGCUAAUAGAAGAUGUUAAGGAUUUGUUUGGUGAAUGUAAGGAAUUGGGUUUACUAGCUAAGUUGGAGCUGAUUGACAGCAUCCAAAAACUAGGCCUGAACAGCUACUUUGAGAAGGAAAUCAAGGAAACCCUAGACACCAUAGCAUCUGUCGAACUGAAAAACAACAGCAAUCCUUUCAUUAGUUCAGAGGGUGAUCUCUACGCUACUGCACUACUCUUUAAGAUUCUGAGGCAGCAUGGUUAUAAAGUUUCACAAGAUGUAUUUGGUGUUUUCAUAGACGAAAUUGGUACAUUAAAGAAGAGCACAUUUGGGGAUGUGAAAGGAAUGCUCGAACUUUUGGAGGCCUCAAACUUAGCUUUAGAAGGUGAAAACAUCCUAGAUGAGGCUAAAGCUUUCUUAAUGGUCACUCUCAGAGAUACCAAUACCAUGUGUGAUGAUAUAGACCAUUACAUUUCCAAGCAUGUGGCCUAUGCCUUGAAGCUUUCAUCACAAAGGAGAGUCCAGUGGUUUAAUGUGAAAUGA；
SEQ ID NO.3 is:
MQAECYRIQIQKLIEDVKDLFGECKELGLLAKLELIDSIQKLGLNSYFEKEIKETLDTIASVELKNNSNPFISSEGDLYATALLFKILRQHGYKVSQDVFGVFIDEIGTLKKSTFGDVKGMLELLEASNLALEGENILDEAKAFLMVTLRDTNTMCDDIDHYISKHVAYALKLSSQRRVQWFNVK。
contains a rose fragrance regulatory gene RrTPS1 plant overexpression vector.
The vector is assembled with an overexpression box of the RrTPS1 gene, the 5' end of the expression box is assembled with an overexpression promoter P35S to ensure that the RrTPS1 gene is overexpressed in a transgenic plant, the vector is assembled with an HPT gene expression box as a screening marker, and the screening of transgenic plant materials is carried out by hygromycin.
Application of rose fragrance regulating gene RrTPS1 in promoting synthesis of floral fragrance substances.
The invention is advanced and scientific, and the DNA sequence of the rose fragrance regulatory gene RrTPS1 is shown in SEQ ID NO. 1. The RNA sequence of the mRNA of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO.2, and the amino acid sequence of the expression protein of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO. 3. An overexpression vector containing the rose fragrance regulatory gene RrTPS 1. Application of a rose fragrance regulating gene RrTPS1 in enhancing synthesis of plant fragrance metabolites.
Has the beneficial effects that: the invention takes a main rose cultivar 'Pingyin I' as a material, and obtains the RrTPS1 gene by homologous cloning. The expression modes of the RrTPS1 gene at different flowering stages and different parts of a flower organ are detected by a real-time fluorescent quantitative detection technology, and meanwhile, the petunia is transformed by constructing an overexpression vector, so that the petunia is proved to promote the synthesis of aromatic substances, and has important application values in the flower breeding fields of enhancing the synthesis of flower fragrance substances, improving the quality and yield of essential oil products and the like.
Drawings
FIG. 1 is the monoterpene floral material content (a) and the RrTPS1 gene expression pattern (b) during rose flowering;
FIG. 2 is the plant overexpression vector pNC-1304-35S: RrTPS1 plasmid map;
figure 3 is a graph of RrTPS1 overexpressing petunia and wild type petunia phenotypes at anthesis (a) and corresponding major floral material content (b).
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
1. Through transcript homology alignment, the RrTPS1 gene is identified and cloned
Based on a conserved region of plant TPS genes, carrying out homologous alignment on rose transcripts, identifying the rose RrTPS1 transcript, comprising a complete Open Reading Frame (ORF), cloning a DNA sequence and an ORF sequence (cDNA sequence) of RrTPS1 by respectively taking total RNA and DNA of a rose organ as templates, cloning into a T-vector, carrying out positive screening on an inserted fragment, carrying out sequencing, and determining that the sequencing result sequence is the complete RrTPS1 gene by comparing a near-source species of rose and strawberry.
I. Primer design
RrTPS1 The ORF primer pair is ORF-F: 5 'ATGCAGGCAGAATGTTATCG 3'And is ORF-R: 5 'TCATTTCACATTAAACCACTG 3';
cloning of ORF sequences
(1) Reverse transcription;
the reverse transcription reaction system is as follows: mu.g of Total RNA, 1. mu.L of dNTP Mix, 1. mu.L of Oligo dT Primer, 4. mu.L of 10 XT Buffer, 0.5. mu.L of RNase Inhibitor, 1. mu. L M-MLV Reverse Transcriptase, Nuclean-free Water to 20. mu.L. The reaction procedure is as follows: reacting at 42 ℃ for 60 min and at 70 ℃ for 15 min to obtain a cDNA template.
（2）PCR；
The PCR reaction system is as follows: PrimeSTAR Max Premix 25 ul, ORF-F primer 1ul, ORF-Rpirmer 1ul, cDNA 5 ul, Water to 50 μ L; the reaction procedure is as follows: 3min at 98 ℃; 10sec at 98 ℃, 5sec at 60 ℃, 5sec at 72 ℃ and 35 cycles; PCR products were obtained at 72 ℃ for 7 min.
(3) Connecting a cloning vector;
and (3) connecting with a pMD19-T simple vector cloning vector, wherein a connection reaction system (5 mu L) comprises: 2.2. mu.L of recovered PCR product, 0.3. mu.L of pMD-19 Simple Vector, 2.5. mu.L of Solution I. The reaction conditions are as follows: incubating at 16 ℃ for 30min to obtain a ligation product
(4) E, transforming escherichia coli;
adding 5 μ L of ligation product into 100 μ L of Escherichia coli competent cells, mixing gently, ice-cooling for about 10 min, water-bathing at 42 deg.C for 60 sec, and rapidly placing on ice for 3-5 min; adding 800 μ L LB liquid culture medium, resuscitating at 37 deg.C &100rmp for 1 h; centrifuging at 4000rmp for 3min, sucking off 600 μ L of culture medium on the upper layer, mixing the rest bacterial liquid, uniformly coating on an LB screening culture plate containing Amp, and performing inverted culture at 37 ℃ overnight.
(5) Screening and sequencing positive clones;
selecting a single colony from an overnight culture plate, inoculating the single colony in an LB liquid culture medium, carrying out overnight culture at 37 ℃ and 250rmp to obtain a bacterial liquid, carrying out PCR detection on a recombinant transformant by taking the bacterial liquid as a template, wherein the PCR reaction system is as follows: t5 PCR Mix (2X) 10 ul, M13F primer 1ul, M13R primer 1ul, bacterial liquid 2 ul; the reaction procedure is as follows: 3min at 98 ℃; 10sec at 98 ℃, 10sec at 55 ℃, 5s at 72 ℃, 25 cycles; and (4) detecting the positive electrophoresis at the temperature of 72 ℃ for 1 min. The bacteria solution which is detected to be positive is sent to the Biotech company (Shanghai) for sequencing.
Cloning of DNA sequences
Extracting rose genome DNA by a radix asparagi polysaccharide polyphenol plant genome DNA extraction kit. By using DNA as templateRrTPS1 The steps of PCR amplification, cloning vector connection, Escherichia coli transformation, positive clone screening and sequencing by using ORF-F/-R as a primer are the same as the above steps.
The result of the analysis and the sequencing shows that,RrTPS1the DNA of (1) has a length of 814bp, and the sequence of the DNA is shown as SEQ ID NO.1,RrTPS1the ORF of (1) has the length of 558bp, the sequence of which is shown as SEQ ID NO.2, and the corresponding RrTPS1 protein sequence of 185 amino acid residues, and the sequence of which is shown as SEQ ID NO. 3.
Example 2
Exploration by fluorescent quantitative PCR techniqueRrTPS1Spatio-temporal expression patterns of genes based onRrTPS1Fluorescent quantitative PCR forward primer 5'-AGGCCTCAAACTTAGCTTTAGAAGGTG-3' and reverse primer 5'-ACCACTGGACTCTCCTTTGTGATGA-3' are designed in the exon region of the gene, and reference primer forward primer 5'-TGAGGCCATTTACGACAT-3' and reverse primer 5'-AGATCACAGGAGCATAGGAG-3' are designed based on the Actin gene. The number of cycles until the fluorescence threshold value is reached by the quantitative PCR was obtained using TaKaRa Premix Taq (Takara Co.) and a quantitative PCR fluorescence instrument CFX96TM (Bio-RAD Co.) with reference to the PCR system and procedure of the manufacturer's instructions, and the number of cycles until the fluorescence threshold value is reached was calculatedRrTPS1The relative expression amount of the gene in three flowering period petals, and simultaneously, GC-MS measures the contents of four main rose monoterpene floral substances such as citronellol and the like in corresponding periods.
As shown in figure 1, with the blooming of flowers, the contents of main terpenoid aromatic components citronellol, citronellyl acetate, geranyl acetate and neryl acetate in the roses are increased continuously (figure 1 a), the expression level of RrTPS1 is also increased continuously (figure 1 b), and RrTPS1 is in positive correlation with the synthesis and accumulation of the rose terpenoid floral substances.
Example 3
RrTPS1Constructing an overexpression vector and carrying out genetic transformation on petunia;
I. vector construction
Using NC seamless cloning kitRrTPS1The ORF of (1) was inserted into the vector pNC-1304-35S (FIG. 2), and 35S-containing DNA was obtained RrTPS1And (3) excessively expressing the recombinant plasmid of the frame, and transforming the recombinant plasmid into agrobacterium EHA105 competence by a liquid nitrogen freeze-thawing method to obtain a positive strain.
Pretreatment of leaves
Selecting a robust petunia sterile plant with the growth height of about 5cm, selecting leaves with the length and width of more than 1.5cm from the petunia plant in a superclean bench, cutting the leaves into 1cm on sterilized filter paper 2 The periphery of each leaf of the square is provided with a notch, the front of each leaf faces upwards and is placed on the petunia pre-culture medium, and dark culture is carried out for 2d at 25 ℃.
Preparation of the staining solution
Inoculating 1 mL of positive strain to 50mL of YEB liquid medium (containing Rif and Kan), culturing at 28 deg.C and 200 rpm under reduced pressure to OD 600 Centrifuging at 4 deg.C and 5000 rpm for 10 min at 0.8-1.0 deg.C, removing supernatant, adding precooled infection solution, and diluting to OD 600 To 0.3-0.5.
Leaf Co-culture
Placing petunia hybrida leaves cultured in dark for 2 days in an infection solution, uniformly and slowly shaking for infection for 5 minutes, taking out the leaves, sucking bacterial liquid on the surfaces of the leaves by using filter paper, placing the leaves on a co-culture medium with the leaf surfaces upward, adding a piece of sterilized pollution-free filter paper on the co-culture medium before placing the leaves so as to inhibit the large-area amplification of agrobacterium on the culture medium, and performing dark culture at 25 ℃ for 3 days.
V. differential screening culture
Taking out petunia leaf cultured for 3 days, washing with sterile water to remove Agrobacterium on leaf surface, sucking with sterile filter paper to remove sterile water and bacterial liquid on leaf surface, transferring onto screening culture medium with leaf surface facing upwards, culturing at 25 deg.C and illumination intensity of 2000 LUX. The culture medium is replaced once in two weeks, and after the leaves are differentiated to obtain larger callus, the callus and the leaves are separated and are independently placed in a differentiation and screening culture medium for culture.
Screening and culturing for rooting
Cutting off bud plants differentiated from the positive callus, transferring the bud plants into a rooting culture medium, placing 1-2 plants in each bottle, transplanting petunia plants from the culture medium into sterile nutrient soil added with vermiculite and perlite when the petunia grows to form robust roots and the height of the plants reaches 5cm, and placing the petunia plants into a climatic box for culture, wherein the culture environment of the climatic box is set as follows: culturing the transgenic plant and the wild plant to the flowering stage under the same condition at 25 ℃ and 2000LUX for 16 h.
Flower organs at full bloom stage are picked, the flower fragrance components of a wild type (2 biological repeats, figure 3 a) and a transgenic petunia (2 biological repeats, figure 3 a) are detected by adopting a GC-MS technology and are compared and analyzed, and as a result, the content of main fragrance components such as isoeugenol, methyl benzoate and benzyl benzoate in transgenic petunia plants is obviously increased and is about 2.1 times higher than that of the wild type on average (figure 3 b), which shows thatRrTPS1The gene has the function of promoting the synthesis of floral substances. Of rosesRrTPS1The gene has important application values in enhancing the synthesis of floral substances of flowers and improving the quality and yield of essential oil.
SEQ ID NO.1
RrTPS1Gene DNA sequence
ATGCAGGCAGAATGTTATCGAATTCAGATTCAGAAGCTAATAGAAGATGTTAAGGATTTGTTTGGTGAATGTAAGGAATTGGGTTTACTAGCTAAGTTGGAGCTGATTGACAGCATCCAAAAACTAGGCCTGAACAGCTACTTTGAGAAGGAAATCAAGGAAACCCTAGACACCATAGCATCTGTCGAACTGAAAAACAACAGCAATCCTTTCATTAGTTCAGAGGGTGATCTCTACGCTACTGCACTACTCTTTAAGATTCTGAGGCAGCATGGTTATAAAGTTTCACAAGGTATAGACGCAGATGTACTAATTAATTAGTTGAACATTGCTAGGTGTATAACTTGTTTGGTACCACATGCATCCCATTTTATGTGGCAGTTGATGTAGCACAATCACGTAATTTAATGAAATCCTCTGATAGATTGGGTATTGTATATGCTACATCTACAACCACATAAGATGGGATACAGGTGGTGCCCAAAAATGTGGTCTAATTTATCTTCTAACTAATCTAACGTACTAATCCAGTTTACTAAACTTTTCAGATGTATTTGGTGTTTTCATAGACGAAATTGGTACATTAAAGAAGAGCACATTTGGGGATGTGAAAGGAATGCTCGAACTTTTGGAGGCCTCAAACTTAGCTTTAGAAGGTGAAAACATCCTAGATGAGGCTAAAGCTTTCTTAATGGTCACTCTCAGAGATACCAATACCATGTGTGATGATATAGACCATTACATTTCCAAGCATGTGGCCTATGCCTTGAAGCTTTCATCACAAAGGAGAGTCCAGTGGTTTAATGTGAAATGA
SEQ ID NO.2
RrTPS1Gene mRNA sequence
AUGCAGGCAGAAUGUUAUCGAAUUCAGAUUCAGAAGCUAAUAGAAGAUGUUAAGGAUUUGUUUGGUGAAUGUAAGGAAUUGGGUUUACUAGCUAAGUUGGAGCUGAUUGACAGCAUCCAAAAACUAGGCCUGAACAGCUACUUUGAGAAGGAAAUCAAGGAAACCCUAGACACCAUAGCAUCUGUCGAACUGAAAAACAACAGCAAUCCUUUCAUUAGUUCAGAGGGUGAUCUCUACGCUACUGCACUACUCUUUAAGAUUCUGAGGCAGCAUGGUUAUAAAGUUUCACAAGAUGUAUUUGGUGUUUUCAUAGACGAAAUUGGUACAUUAAAGAAGAGCACAUUUGGGGAUGUGAAAGGAAUGCUCGAACUUUUGGAGGCCUCAAACUUAGCUUUAGAAGGUGAAAACAUCCUAGAUGAGGCUAAAGCUUUCUUAAUGGUCACUCUCAGAGAUACCAAUACCAUGUGUGAUGAUAUAGACCAUUACAUUUCCAAGCAUGUGGCCUAUGCCUUGAAGCUUUCAUCACAAAGGAGAGUCCAGUGGUUUAAUGUGAAAUGA
RrTPS1 protein amino acid sequence
MQAECYRIQIQKLIEDVKDLFGECKELGLLAKLELIDSIQKLGLNSYFEKEIKETLDTIASVELKNNSNPFISSEGDLYATALLFKILRQHGYKVSQDVFGVFIDEIGTLKKSTFGDVKGMLELLEASNLALEGENILDEAKAFLMVTLRDTNTMCDDIDHYISKHVAYALKLSSQRRVQWFNVK
Sequence listing
<110> Yangzhou university
<120> rose fragrance regulatory gene RrTPS1 and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 814
<212> DNA
<213> Rose (Rosa rugosa)
<400> 1
atgcaggcag aatgttatcg aattcagatt cagaagctaa tagaagatgt taaggatttg 60
tttggtgaat gtaaggaatt gggtttacta gctaagttgg agctgattga cagcatccaa 120
aaactaggcc tgaacagcta ctttgagaag gaaatcaagg aaaccctaga caccatagca 180
tctgtcgaac tgaaaaacaa cagcaatcct ttcattagtt cagagggtga tctctacgct 240
actgcactac tctttaagat tctgaggcag catggttata aagtttcaca aggtatagac 300
gcagatgtac taattaatta gttgaacatt gctaggtgta taacttgttt ggtaccacat 360
gcatcccatt ttatgtggca gttgatgtag cacaatcacg taatttaatg aaatcctctg 420
atagattggg tattgtatat gctacatcta caaccacata agatgggata caggtggtgc 480
ccaaaaatgt ggtctaattt atcttctaac taatctaacg tactaatcca gtttactaaa 540
cttttcagat gtatttggtg ttttcataga cgaaattggt acattaaaga agagcacatt 600
tggggatgtg aaaggaatgc tcgaactttt ggaggcctca aacttagctt tagaaggtga 660
aaacatccta gatgaggcta aagctttctt aatggtcact ctcagagata ccaataccat 720
gtgtgatgat atagaccatt acatttccaa gcatgtggcc tatgccttga agctttcatc 780
acaaaggaga gtccagtggt ttaatgtgaa atga 814
<210> 2
<211> 558
<212> RNA
<213> Rose (Rosa rugosa)
<400> 2
augcaggcag aauguuaucg aauucagauu cagaagcuaa uagaagaugu uaaggauuug 60
uuuggugaau guaaggaauu ggguuuacua gcuaaguugg agcugauuga cagcauccaa 120
aaacuaggcc ugaacagcua cuuugagaag gaaaucaagg aaacccuaga caccauagca 180
ucugucgaac ugaaaaacaa cagcaauccu uucauuaguu cagaggguga ucucuacgcu 240
acugcacuac ucuuuaagau ucugaggcag caugguuaua aaguuucaca agauguauuu 300
gguguuuuca uagacgaaau ugguacauua aagaagagca cauuugggga ugugaaagga 360
augcucgaac uuuuggaggc cucaaacuua gcuuuagaag gugaaaacau ccuagaugag 420
gcuaaagcuu ucuuaauggu cacucucaga gauaccaaua ccauguguga ugauauagac 480
cauuacauuu ccaagcaugu ggccuaugcc uugaagcuuu caucacaaag gagaguccag 540
ugguuuaaug ugaaauga 558
<210> 3
<211> 185
<212> PRT
<213> Rose (Rosa rugosa)
<400> 3
Met Gln Ala Glu Cys Tyr Arg Ile Gln Ile Gln Lys Leu Ile Glu Asp
1 5 10 15
Val Lys Asp Leu Phe Gly Glu Cys Lys Glu Leu Gly Leu Leu Ala Lys
20 25 30
Leu Glu Leu Ile Asp Ser Ile Gln Lys Leu Gly Leu Asn Ser Tyr Phe
35 40 45
Glu Lys Glu Ile Lys Glu Thr Leu Asp Thr Ile Ala Ser Val Glu Leu
50 55 60
Lys Asn Asn Ser Asn Pro Phe Ile Ser Ser Glu Gly Asp Leu Tyr Ala
65 70 75 80
Thr Ala Leu Leu Phe Lys Ile Leu Arg Gln His Gly Tyr Lys Val Ser
85 90 95
Gln Asp Val Phe Gly Val Phe Ile Asp Glu Ile Gly Thr Leu Lys Lys
100 105 110
Ser Thr Phe Gly Asp Val Lys Gly Met Leu Glu Leu Leu Glu Ala Ser
115 120 125
Asn Leu Ala Leu Glu Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe
130 135 140
Leu Met Val Thr Leu Arg Asp Thr Asn Thr Met Cys Asp Asp Ile Asp
145 150 155 160
His Tyr Ile Ser Lys His Val Ala Tyr Ala Leu Lys Leu Ser Ser Gln
165 170 175
Arg Arg Val Gln Trp Phe Asn Val Lys
180 185
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggcctcaaa cttagcttta gaaggtg 27
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
accactggac tctcctttgt gatga 25
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgaggccatt tacgacat 18
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Claims (4)
1. A rose fragrance regulatory gene RrTPS1, which is characterized in that: the nucleotide sequence is shown as SEQ ID NO.1, the mRNA sequence of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO.2, and the amino acid sequence of the expression protein of the rose fragrance regulatory gene RrTPS1 is shown as SEQ ID NO. 3.
2. A plant overexpression vector comprising the floral rose regulator gene RrTPS1 of claim 1.
3. The carrier of claim 2, wherein: the vector is assembled with an overexpression box of the RrTPS1 gene, the 5' end of the expression box is assembled with an overexpression promoter P35S to ensure that the RrTPS1 gene is overexpressed in a transgenic plant, the vector is assembled with an HPT gene expression box as a screening marker, and the transgenic plant material is screened through hygromycin.
4. Use of the floral rose regulating gene RrTPS1 according to claim 1 for promoting the synthesis of floral rose substances.
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