A transcription factor regulating flower color formation in Melastoma plants, its encoding gene and application

Abstract

The present invention discloses a transcription factor for regulating the flower color formation of Melastoma plants, a coding gene thereof and an application thereof. The transcription factors include MedPAP1 and MedTT8, the amino acid sequence of MedPAP1 is shown in SEQ ID No.2, and the amino acid sequence of MedTT8 is shown in SEQ ID No.4. The transcription factors were transformed into leaves of Nicotiana benthamiana and petals and sepals of Phalaenopsis alba by transient expression method, and the results showed that the coding genes of transcription factors MedPAP1 and MedTT8 were co-expressed. MedPAP1 and MedTT8 It can make tobacco leaves and Phalaenopsis petals appear red and lavender, and can increase the expression levels of key enzyme genes in the cyanidin synthesis pathway of tobacco and Phalaenopsis, supporting the formation of a complex by transcription factors MedPAP1 and MedTT8 to jointly regulate the color formation of Phalaenopsis flowers.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a transcription factor for regulating the formation of flower color of Melastoma plants, a coding gene thereof and an application thereof. Background Art

[0002] Dioscorea Melastomadodecandrum ) is also known as ground brocade and ground pomegranate, and is a member of the genus Melastomataceae ( Melastoma ) is a perennial creeping subshrub. In my country, the ground sedge is widely distributed in various areas south of the Yangtze River Basin. It has strong adaptability, fast growth, long flowering period, large number of flowers, bright flower colors, and low creeping plants. It can be used as an excellent garden ornamental plant with great development and utilization potential. Flower color is one of the main ornamental traits of ground sedge. In order to further develop the garden application value of ground sedge, the improvement of flower color can be used as a new goal for its variety development.

[0003] Hybrid breeding is currently the main method for breeding new varieties of Melastoma, which usually requires multiple generations of breeding to obtain ideal varieties. The breeding cycle is long and the target traits are unstable. In recent years, molecular directed breeding methods have developed rapidly. Through transgenic technology, the expression level of genes regulating ornamental traits is changed to obtain transgenic plants with new phenotypes, and finally the ideal varieties are obtained. Exploring the key genes regulating ornamental traits can provide important genetic resources and information for the development of molecular breeding.

[0004] Anthocyanins are important secondary metabolites in higher plants and are widely distributed. They can make plant organs appear red, purple, blue, etc., and their biosynthetic pathway has been completely analyzed. The essence of flower color is the accumulation of anthocyanins on petals. The R2R3-MYB transcription factor family and the bHLH transcription factor family of higher plants are involved in regulating a variety of biological processes, including the synthesis of secondary metabolites. These two types of transcription factors play an important role in the regulation of anthocyanin synthesis, and their functions have been verified in both model and non-model plants. For example, Anthurium ( Anthurium andraeanum ) have an interaction between AaMYB6 protein and AabHLH1 protein, regulating the pink color of the spathe; Chinese narcissus ( Narcissus tazetta var. chinensis ) NtMYB8 protein interacts with NtbHLH1 protein, activates the flavonoid biosynthesis pathway, and may promote the synthesis of flavonols in the corona; Chimonanthus praecox ( Chimonanthus praecox ) The bHLH transcription factor CpTT8 is a positive regulatory factor in the synthesis of anthocyanins. It interacts with CpMYB protein to jointly regulate the expression of CpANS gene and thus accumulate anthocyanins in wintersweet.

[0005] At present, there are no reports on the molecular mechanism of the formation of ornamental traits such as flower color in Melastoma plants, and the molecular mechanism of flower color formation is still unclear. In order to further develop and utilize Melastoma and enrich the ornamental variety resources of Melastoma, it is necessary to reveal the molecular mechanism that regulates the formation of flower color in Melastoma and provide important molecular resources for the cultivation of new flower color varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a transcription factor regulating the flower color formation of Melastoma plants and its encoding gene and application, and to clone the encoding genes of the transcription factors MedPAP1 and MedTT8 regulating the flower color formation of Melastoma plants from Melastoma spp. MedPAP1 and MedTT8 , and clarify the application of these two transcription factors, namely, transcription factor MedPAP1 and transcription factor MedTT8 in regulating flower color formation in Melastoma plants and regulating anthocyanin synthesis in Melastoma plants.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention first provides a transcription factor for regulating flower color formation of Melastoma plants. The transcription factor comprises transcription factor MedPAP1 and transcription factor MedTT8. The amino acid sequence of MedPAP1 is shown in SEQ ID No.2, and the amino acid sequence of MedTT8 is shown in SEQ ID No.4.

[0009] The present invention further provides a gene encoding the above-mentioned transcription factor, wherein the gene comprises gene MedPAP1 and genes MedTT8 ,Gene MedPAP1 The nucleotide sequence of gene is shown in SEQ ID No.1. MedTT8 The nucleotide sequence is shown in SEQ ID No.3.

[0010] The present invention also provides a primer for amplifying the above gene, MedPAP1 The primers are:

[0011] MedPAP1_KF: 5'-ATGAGGGTGAAGTCGAGTC-3',

[0012] MedPAP1_KR: 5'-TCACGCGACGGGTTGAGG-3';

[0013] Amplification of genes MedTT8 The primers are:

[0014] MedTT8_KF:5'-ATGGCCGCGCCGCCTAGT-3'

[0015] MedTT8_KR: 5'-TGAGTCCGTATGGGGGATGA-3'.

[0016] The present invention also provides a recombinant vector comprising the above gene.

[0017] The present invention also provides a recombinant bacterium comprising the above gene or the above recombinant vector.

[0018] The present invention also provides the use of the above-mentioned transcription factor and its encoding gene in regulating the flower color formation of Melastoma plants and regulating the anthocyanin synthesis in Melastoma plants. Specifically, the encoding genes of the transcription factors MedPAP1 and MedTT8 are co-expressed in Melastoma plants. MedPAP1 and MedTT8 , which can increase the content of anthocyanins in Melastoma plants, thereby changing the color of the flowers.

[0019] The significant advantages of the present invention are:

[0020] The present invention clones the coding genes of transcription factors MedPAP1 and MedTT8 related to flower color formation of Melastoma plants MedPAP1 and MedTT8 , and further constructed pMDC202- MedPAP1 and pMDC202- MedTT8 The recombinant plant expression vector was used to transform tobacco and white Phalaenopsis, proving that the transcription factor MedPAP1 and the transcription factor MedTT8 interact with each other and jointly participate in regulating the synthesis of anthocyanin substances, thus increasing the content of anthocyanin in Melastoma plants. The transcription factors MedPAP1 and MedTT8 and their encoding genes provide a molecular biological basis for breeding new flower color varieties of Melastoma plants through plant genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shows the color changes of tobacco leaves in different treatment groups.

[0022] Figure 2 The coloring of epidermal cells of tobacco leaves in different treatment groups.

[0023] Figure 3 Shown is the expression of structural genes in the anthocyanin biosynthesis pathway in tobacco leaves under different treatment groups.

[0024] Figure 4 Anthocyanin content in tobacco leaves of different treatment groups.

[0025] Figure 5 The color changes of petals and sepals of white Phalaenopsis orchids in different treatment groups.

[0026] Figure 6 Schematic diagram of the anatomy of petals and sepals of white Phalaenopsis orchids in different treatment groups after transient expression.

[0027] Figure 7 The expression of structural genes in the anthocyanin synthesis pathway of white Phalaenopsis petals in different treatment groups. DETAILED DESCRIPTION

[0028] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0029] Example 1: Transcription factor encoding genes MedPAP1 and MedTT8 Cloning and sequencing

[0030] Separate transcription factor encoding genes from Melastoma sphaerocarpa MedPAP1 and MedTT8 , whose sequences are shown in SEQ ID No.1~2 and SEQ ID No.3~4 respectively. Designed according to the sequence shown in SEQ ID No.1 MedPAP1 Primer pair, designed according to the sequence shown in SEQ ID No.3 MedTT8 Primer Pairs:

[0031] MedPAP1_KF: 5'-ATGAGGGTGAAGTCGAGTC-3',

[0032] MedPAP1_KR: 5'-TCACGCGACGGGTTGAGG-3';

[0033] MedTT8_KF: 5'-ATGGCCGCGCCGCCTAGT-3',

[0034] MedTT8_KR: 5'-TGAGTCCGTATGGGGGATGA-3'.

[0035] The petals of the schisandra chinensis were taken and total RNA was extracted using a plant total RNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and reverse transcription was performed using a reverse transcription kit (purchased from TaKaRa Biotechnology (Beijing) Co., Ltd.) according to the kit's method. The obtained cDNA fragment was used as a template for PCR amplification using MedPAP1_KF / MedPAP1_KR and MedTT8_KF / MedTT8_KR primers.

[0036] The amplified transcription factor encoding gene MedPAP1 and MedTT8They were connected to 18-T vector (purchased from TaKaRa Biotechnology (Beijing) Co., Ltd.), and positive clones were screened and sequenced to obtain the required CDS sequence. The positive clones with correct sequencing were named MedPAP1-T and MedTT8-T plasmids.

[0037] Example 2: Construction of recombinant plant expression vectors pMDC202-MedPAP1 and pMDC202-MedTT8

[0038] Design recombination primers containing homologous sequences:

[0039] MedPAP1_KLF: 5'-ggagaggacctcgactctagaATGAGGGTGAAGTCGAGTCT-3',

[0040] MedPAP1_KLR: 5'-ctcattttttctaccggtaccTCACGCGACGGGTTGAGG-3';

[0041] MedTT8_KLF: 5'-ggagaggacctcgactctagaATGGCCGCGCCGCCTAGT-3',

[0042] MedTT8_KLR: 5'-ctcattttttctaccggtaccTGAGTCCGTATGGGGGATGA-3'.

[0043] Using the correctly sequenced MedPAP1-T and MedTT8-T vector plasmids as templates, adding recombination primers containing homologous sequences for PCR amplification, and obtaining the CDS of the target gene with the homologous sequence of the expression vector. Using pMDC202-GFP as the expression vector, after double restriction linearization and purification and recovery, the target gene was inserted into the expression vector to complete the construction of the recombinant plasmid, transformed into Escherichia coli DH5α competent cells and propagated, and positive clone detection and sequencing were performed to obtain the recombinant plant expression vector pMDC202- MedPAP1 and pMDC202- MedTT8 .

[0044] Example 3: Preparation of infection solution

[0045] The constructed recombinant plant expression vector pMDC202- MedPAP1 and pMDC202- MedTT8Transform Agrobacterium GV3101 competent cells respectively, and screen positive transformants by PCR. Dip the Agrobacterium containing the recombinant plant expression vector with an inoculation loop and inoculate it into 5 mL YEB liquid medium (containing 50 mg / L gentamicin, 50 mg / L kanamycin and 25 mg / L rifampicin), shake culture at 28 ℃ until the logarithmic growth phase, take 2 mL of activated bacterial solution and inoculate it into 10 mL YEB liquid medium (containing 50 mg / L gentamicin, 50 mg / L kanamycin and 25 mg / L rifampicin), shake culture at 28 ℃ for 10 h, at which time the bacterial solution is turbid and orange-yellow, and set aside.

[0046] Weigh 1.9524 g of morpholineethanesulfonic acid (MES) and dissolve it in 10 mL of sterile water to obtain MES solution. Weigh 0.1962 g of acetosyringone (AS) and dissolve it in 10 mL of dimethyl sulfoxide (DMSO) to obtain AS solution. Weigh 2.03 g of magnesium chloride (MgCl2·6H2O) and dissolve it in 10 mL of sterile water to obtain MgCl2 solution. Weigh 4.34 g of MS medium powder and 5 g of sucrose and dissolve them in sterile water and make up to 1 L. Adjust the pH value to 5.7 to obtain MS medium. Take 100 mL of MS medium, sterilize it at 121 ℃ for 20 min, add 1 mL of MES solution, 1 mL of MgCl2 solution, and 100 μL of AS solution, mix well, and use as infection buffer for later use.

[0047] Place the turbid and orange-yellow bacterial solution obtained by the above culture into a centrifuge and centrifuge at 8,000 rpm for 8 minutes, discard the supernatant; rinse the Agrobacterium at the bottom of the tube with the prepared infection buffer, mix it by pipetting, centrifuge it at 8,000 rpm for 8 minutes, and discard the supernatant; rinse the Agrobacterium at the bottom of the tube with the prepared infection buffer, mix it by pipetting, centrifuge it at 8,000 rpm for 8 minutes, and discard the supernatant. Using a UV spectrophotometer, add an appropriate amount of infection buffer to the centrifuge tube and measure the OD value of the bacterial solution. 600 The values ​​were adjusted to about 0.4 and 0.8 respectively, and then placed in a 28 ℃ incubator away from light for 3 h to serve as the infective bacterial solution.

[0048] Example 4: Transient expression in tobacco leaves

[0049] Use a 1 mL disposable medical syringe to draw an appropriate amount of the prepared infection solution (OD 600=0.4), select relatively tender leaves of Nicotiana benthamiana, gently scratch the back tissue of the leaves with a needle, and inject the liquid into the leaves. The injection volume is about 100~200 μL per time, and 3~4 injections are made on the same leaf. After injection, traces of liquid infection in the leaves can be observed, indicating that the injection is successful. The experimental design is shown in Table 1: Each pot of tobacco is numbered 1~9, and 4 leaves are selected from each pot, numbered Y1~Y4, and 3 biological replicates are set. Among them, tobacco No. 1~3: Y1~Y3 leaves are injected with pMDC202- MedPAP1 The Y4 leaf was injected with the infection solution containing the empty pMDC202; for tobacco No. 4-6: the Y1-Y3 leaf was injected with the infection solution containing pMDC202- MedTT8 The Y4 leaf was injected with the infection solution containing the empty pMDC202; for tobacco No. 7-9: the Y1-Y3 leaf was injected with the infection solution containing pMDC202- MedPAP1 and pMDC202- MedTT8 The infection solution (containing pMDC202- MedPAP1 The infection solution was mixed with pMDC202- MedTT8 The infection solution containing the empty vector pMDC202 was mixed in equal volumes of the infection solution of Y4 leaves.

[0050] Table 1 Tobacco transient expression experiment design

[0051]

[0052] After injection, the leaves were placed in an artificial climate box for culture under the following conditions: 22°C for 16 h of light, 8 h of darkness, and 70% humidity. After 4 days of culture, the color changes of tobacco leaves were observed, and the epidermal cells of tobacco leaves were observed using an optical microscope imaging system and photographed. The anthocyanin content of tobacco leaves was determined by spectrophotometry. In addition, total RNA of tobacco leaves was extracted, and the expression of structural genes related to the anthocyanin pathway in tobacco leaves was analyzed by qRT-PCR after reverse transcription.

[0053] from Figure 1 As can be seen, pMDC202- MedTT8 The leaves of the tobacco group did not turn red, pMDC202- MedPAP1 Some irregular red spots formed on the surface of tobacco leaves instead of flake coloration, pMDC202- MedPAP1+ pMDC202- MedTT8 Group of tobacco leaves turning red.

[0054] from Figure 2 As can be seen, pMDC202- MedTT8 The epidermal cells of tobacco leaves in the pMDC202 group and the pMDC202 group did not show any coloration. Compared with the control group pMDC202 group, the pMDC202- MedPAP1A small part of the epidermal cells of tobacco leaves in the group showed darker and redder colors; compared with the control group pMDC202 group, pMDC202- MedTT8 Group and pMDC202- MedPAP1 Compared with the pMDC202- MedPAP1+ pMDC202- MedTT8 Most of the cells in the leaves of group A tobacco turned red and darker, and the area was relatively large.

[0055] from Figure 3 It can be seen that compared with the control group pMDC202 group, pMDC202- MedPAP1+ pMDC202- MedTT8 Group of tobacco leaves NbCHS , NbCHI , NbF3H The expression of structural genes such as pMDC202- MedPAP1 The structural genes of tobacco leaves were also expressed to a certain extent, indicating that MedPAP1 and MedTT8 Synergistic regulation of anthocyanin synthesis in radix erythrinae, and transient expression MedPAP1 Compared with the leaves of tobacco transiently expressing empty vector, the expression of structural genes was slightly upregulated, but not significantly.

[0056] from Figure 4 It can be seen that compared with the control group pMDC202 group, pMDC202- MedPAP1 Group with pMDC202- MedPAP1+ pMDC202- MedTT8 Trace amounts of anthocyanins were detected in the leaves of tobacco in the pMDC202 group and pMDC202- MedTT8 Almost no anthocyanin was detected in the leaves of tobacco. MedPAP1 and MedTT8 It is a key gene that regulates the formation of flower color in Euphorbia milii.

[0057] Example 5: Transient expression in white Phalaenopsis orchids

[0058] The prepared infection solution (OD 600 =0.8), respectively, injected into the petals and sepals of Phalaenopsis, set up 3 biological replicates, and the experimental design is shown in Table 2. Each Phalaenopsis was numbered 1 to 9, and the petals of each Phalaenopsis were numbered H1 (petal on the left), H2 (petal on the right), and H3 (sepals on the top). Among them, No. 1 to 3 white Phalaenopsis: H2 to H3 were injected with pMDC202- MedPAP1 of infection solution, H1 was injected with infection solution containing empty pMDC202; No. 4~6 white Phalaenopsis: H2~H3 were injected with infection solution containing pMDC202- MedTT8Infectious bacteria solution, H1 was injected with the infection solution containing empty pMDC202; No. 7~9 white Phalaenopsis: H2~H3 were injected with the infection solution containing pMDC202- MedPAP1 and pMDC202- MedTT8 The infection solution (containing pMDC202- MedPAP1 The infection solution was mixed with pMDC202- MedTT8 H1 was injected with the infection solution containing empty pMDC202.

[0059] Table 2 Design of transient expression experiment in Phalaenopsis leucophylla

[0060]

[0061] After injection, the plants were placed in an artificial climate chamber for culture under the following conditions: 24°C, 16 h of light, 8 h of darkness, and 70% humidity. After 3 days of culture, the phenotypic changes were observed and photographed. The expression of structural genes of the anthocyanin pathway in Phalaenopsis petals was analyzed using qRT-PCR technology.

[0062] from Figure 5 and Figure 6 As can be seen, pMDC202- MedTT8 Group and pMDC202- MedPAP1 The petals and sepals of the white Phalaenopsis orchids in the group were not colored, while the pMDC202- MedPAP1+ pMDC202- MedTT8 The petals and sepals of the white Phalaenopsis orchid have a lavender-red area, indicating MedPAP1 and MedTT8 There is an interactive relationship between them, and they jointly participate in regulating the synthesis of anthocyanins.

[0063] from Figure 7 As can be seen from the figure, compared with the Phalaenopsis petals of the transiently expressed empty pMDC202 group, the pMDC202- MedPAP1 + pMDC202- MedTT8 The expression of structural genes in the anthocyanin biosynthesis pathway of Phalaenopsis petals was significantly upregulated, indicating MedPAP1 and MedTT8 It is a key gene that regulates the formation of flower color in Euphorbia milii.

[0064] The above shows that the present invention provides two transcription factors and their encoding genes MedPAP1 and MedTT8 The ability to regulate the synthesis of anthocyanin substances in transgenic plants proves that the present invention is feasible and effective.

[0065] The above only takes tobacco and Phalaenopsis as examples. The corresponding transcription factors and their encoding genes can also be applied to other closely related species to regulate the synthesis of anthocyanin substances. The present invention provides genetic resources for the study of ornamental traits such as flower color of wild peony plants, and has the potential to cultivate new flower color varieties of Melaleuca and other wild peony plants.

[0066] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein. SEQUENCE LISTING <110> Fujian Agriculture and Forestry University <120> A transcription factor regulating flower color formation in Melastoma plants, its encoding gene and application <130> <160> 12 <170> PatentIn version 3.3 <210> 1 <211> 732 <212> DNA <213> Melastoma dodecandrum <400> 1 atgagggtga agtcgagtct cgggttgagg aaaggttctt ggactgagga ggaggatgca 60 ctactccgga agtgcgtgca gcgccacggc gaagggaggt ggcacctcgt tccttgccga 120 gcagggttga accggtgccg gaagagctgc cggctgaggt ggttgaatta ccttaagccg 180 aatataaaga ggggcgagtt ccaggaagac gaggtcgaca ttattatcag actccacaac 240 ctcctgggca atcggtggtc cctgatcgcg ggaagaatcc cggggaggac cgcgaatgac 300 gtgaagaact attggaacac ccaccttgct aagaagacca cattcaaggg cgaaaatccg 360 aaagaacagc ctgaaaaaat agtcaaagtt accgccttta ggccgcgcgc gcgaaccttc 420 tctaagagct tggcttggct cagtggccgg gcaacattga tcacttcgtc cgcccttcga 480 ccagacaaca acatcaccaa taaccacgac tcgcgatctc cagtaatgga agagccttgg 540 tgggaagagt tgttgggcaa cacggacgaa tttgccctag caatcccctc gggagaggat 600 ccgtcggcaa agacagccga agaagcatgc gtcgaggcgc ctgacagggt aaacggtgaa 660 cggtgggacg atctttcctt ggatgtggat ctttggcagt tcttgggcaa cgcacctcaa 720 cccgtcgcgt ga 732 <210> 2 <211> 243 <212> PRT <213> Melastoma dodecandrum <400> 2 Met Arg Val Lys Ser Ser Leu Gly Leu Arg Lys Gly Ser Trp Thr Glu 1 5 10 15 Glu Glu Asp Ala Leu Leu Arg Lys Cys Val Gln Arg His Gly Glu Gly 20 25 30 Arg Trp His Leu Val Pro Cys Arg Ala Gly Leu Asn Arg Cys Arg Lys 35 40 45 Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Lys Pro Asn Ile Lys Arg 50 55 60 Gly Glu Phe Gln Glu Asp Glu Val Asp Ile Ile Ile Arg Leu His Asn 65 70 75 80 Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Ile Pro Gly Arg 85 90 95 Thr Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr His Leu Ala Lys Lys 100 105 110 Thr Thr Phe Lys Gly Glu Asn Pro Lys Glu Gln Pro Glu Lys Ile Val 115 120 125 Lys Val Thr Ala Phe Arg Pro Arg Ala Arg Thr Phe Ser Lys Ser Leu 130 135 140 Ala Trp Leu Ser Gly Arg Ala Thr Leu Ile Thr Ser Ser Ala Leu Arg 145 150 155 160 Pro Asp Asn Asn Ile Thr Asn Asn His Asp Ser Arg Ser Pro Val Met 165 170 175 Glu Glu Pro Trp Trp Glu Glu Leu Leu Gly Asn Thr Asp Glu Phe Ala 180 185 190 Leu Ala Ile Pro Ser Gly Glu Asp Pro Ser Ala Lys Thr Ala Glu Glu 195 200 205 Ala Cys Val Glu Ala Pro Asp Arg Val Asn Gly Glu Arg Trp Asp Asp 210 215 220 Leu Ser Leu Asp Val Asp Leu Trp Gln Phe Leu Gly Asn Ala Pro Gln 225 230 235 240 Pro Val Ala <210> 3 <211> 2106 <212> DNA <213> Melastoma dodecandrum <400> 3 atggccgcgc cgcctagtag ccggctacaa ggcatgttgc aagcggcagt ccagtcagtg 60 aagtggactt acagcctctt ctggcaaatc tgtccccaac aagggatatt ggtatgggga 120 gacgggtact acaacggacc gataaagacg aggaagactg tgcagccgat ggaggtgacc 180 gcggaggagg cttccctcca gaggagtcaa cagcttcgcg agctttacga gtcattgtcg 240 gccggggaga cgaaccagcc ccctagacgg ccgtgcgtgg ccctctctcc agaggacttg 300 acagagacgg agtggttcta cctcatgtgc gtgtccttct cgttccctcc cggggtaggg 360 ttgcccggaa aggctcttgc gaggaggcag cacgtatggc agacaggagc taacgaagcg 420 gatagcaaga cattttctag agctatccta gctaagagcg ctcgaatcca gactgtggtt 480 tgcatacctt tactcgacgg cgtggtggaa ttcggctcga cggacaaggt ttccgaagat 540 gtttcattca tcaaccacgt gaagaccttc ttcgtcgacc accacccccc gcaccctccc 600 aagccggccc tctccgaaca ctcaacctca aacccagcgt catccgaaca ccccaggttt 660 cagtccccgg tccaaccccc cgtgtacgtc ccacaagaac ccccggccga tgcgggcaac 720 agggttgagg acgatgaagt agacgaggac aacgacgacg acgaggacga agacgacgac 780 gaggacgaag acgatgattc agactgcgag gccaagacgg gcaggaacag caacaggcag 840 gggttggacc caaacccagg agcggcaact gctgaaccga gcgagtttga gatgtcggag 900 gacataaggc tggggtcacc agacgacggg tcaaacaacc tcgactcgga cttccccatg 960 ctaagcataa accatcaaac ggggagggcg gcggccacgg gtgaccggca aaggttggca 1020 gagtcataca gggcggagtc gacgaggatg tggcaggcgg cagggcagga acaggcggcg 1080 aggagcggcg gcctccattc cccaccttca ggcccgccgc cgcaacctga gctgtcgcac 1140 gaggacgcac actactccca gacggtgtcg accatcctgc agtgccagcc cggtcgatgg 1200 gctgagtcgt cgtcgaatag ctatgcgggc tactcgtccc agtctgcctt ctcgaagtgg 1260 tccgcccgtg ctgatgcagt cctcagcgtg ccctccgagg ggaactcgca gtggctcctc 1320 aagtatatcc tgttcagcgt ccccctgctc cacagccagt gcagggatga gaactccccc 1380 aagtccaggg atggcggaaa cggggcggcg gccaggttta ggaaagggac ccctgcggat 1440 gagctgagcg cgaaccatgt cctggctgag cgacgcaggc gcgagaagct aaacgagagg 1500 ttcatcatcc tgaggtcgct tgtgccgttt gttaccaaga tggacaaggc ctcaatcctc 1560 ggtgacacga tcgagtacgt gaagcagctg aggaagaaga tccaggacct cgaggcaaag 1620 aaccggcaga tggaggccaa gagccggtcg agtccgggag gagaccttca gcgttccacc 1680 agcatgaagg agctgagaac actggcgggc gccagctcgg ggactgaccg gggatccgtg 1740 ggccaggaca agcggaagat gaggatcgtg gaggcaggct ccaaacccaa ggcagtggag 1800 ccgccgctgc cttctcctcc tccaccgcaa ccgacaccca cagagacgag cgtgcaggtc 1860 tcgataatag agagcgatgc gttgctcgag cttcagtgcc cgcacaggga agggctgttg 1920 ctcgacgtga tgcagatgct gagggagcac cggatagagg tcacggctgt ccagtcatcg 1980 ttgacgaacg ggttcttctc cgcggaactg agagcaaagg tgaaggagaa cacgaacgga 2040 aagaaggcga gcatcgtaga ggtgaagaga gcgatccagc agatcatccc ccatacggac 2100 tcatag 2106 <210> 4 <211> 701 <212> PRT <213> Melastoma dodecandrum <400> 4 Met Ala Ala Pro Pro Ser Ser Arg Leu Gln Gly Met Leu Gln Ala Ala 1 5 10 15 Val Gln Ser Val Lys Trp Thr Tyr Ser Leu Phe Trp Gln Ile Cys Pro 20 25 30 Gln Gln Gly Ile Leu Val Trp Gly Asp Gly Tyr Tyr Asn Gly Pro Ile 35 40 45 Lys Thr Arg Lys Thr Val Gln Pro Met Glu Val Thr Ala Glu Glu Ala 50 55 60 Ser Leu Gln Arg Ser Gln Gln Leu Arg Glu Leu Tyr Glu Ser Leu Ser 65 70 75 80 Ala Gly Glu Thr Asn Gln Pro Pro Arg Arg Pro Cys Val Ala Leu Ser 85 90 95 Pro Glu Asp Leu Thr Glu Thr Glu Trp Phe Tyr Leu Met Cys Val Ser 100 105 110 Phe Ser Phe Pro Pro Gly Val Gly Leu Pro Gly Lys Ala Leu Ala Arg 115 120 125 Arg Gln His Val Trp Gln Thr Gly Ala Asn Glu Ala Asp Ser Lys Thr 130 135 140 Phe Ser Arg Ala Ile Leu Ala Lys Ser Ala Arg Ile Gln Thr Val Val 145 150 155 160 Cys Ile Pro Leu Leu Asp Gly Val Val Glu Phe Gly Ser Thr Asp Lys 165 170 175 Val Ser Glu Asp Val Ser Phe Ile Asn His Val Lys Thr Phe Phe Val 180 185 190 Asp His His Pro Pro His Pro Pro Lys Pro Ala Leu Ser Glu His Ser 195 200 205 Thr Ser Asn Pro Ala Ser Ser Glu His Pro Arg Phe Gln Ser Pro Val 210 215 220 Gln Pro Pro Val Tyr Val Pro Gln Glu Pro Pro Ala Asp Ala Gly Asn 225 230 235 240 Arg Val Glu Asp Asp Glu Val Asp Glu Asp Asn Asp Asp Asp Glu Asp 245 250 255 Glu Asp Asp Asp Glu Asp Glu Asp Asp Asp Ser Asp Cys Glu Ala Lys 260 265 270 Thr Gly Arg Asn Ser Asn Arg Gln Gly Leu Asp Pro Asn Pro Gly Ala 275 280 285 Ala Thr Ala Glu Pro Ser Glu Phe Glu Met Ser Glu Asp Ile Arg Leu 290 295 300 Gly Ser Pro Asp Asp Gly Ser Asn Asn Leu Asp Ser Asp Phe Pro Met 305 310 315 320 Leu Ser Ile Asn His Gln Thr Gly Arg Ala Ala Ala Thr Gly Asp Arg 325 330 335 Gln Arg Leu Ala Glu Ser Tyr Arg Ala Glu Ser Thr Arg Met Trp Gln 340 345 350 Ala Ala Gly Gln Glu Gln Ala Ala Arg Ser Gly Gly Leu His Ser Pro 355 360 365 Pro Ser Gly Pro Pro Pro Gln Pro Glu Leu Ser His Glu Asp Ala His 370 375 380 Tyr Ser Gln Thr Val Ser Thr Ile Leu Gln Cys Gln Pro Gly Arg Trp 385 390 395 400 Ala Glu Ser Ser Ser Asn Ser Tyr Ala Gly Tyr Ser Ser Gln Ser Ala 405 410 415 Phe Ser Lys Trp Ser Ala Arg Ala Asp Ala Val Leu Ser Val Pro Ser 420 425 430 Glu Gly Asn Ser Gln Trp Leu Leu Lys Tyr Ile Leu Phe Ser Val Pro 435 440 445 Leu Leu His Ser Gln Cys Arg Asp Glu Asn Ser Pro Lys Ser Arg Asp 450 455 460 Gly Gly Asn Gly Ala Ala Ala Arg Phe Arg Lys Gly Thr Pro Ala Asp 465 470 475 480 Glu Leu Ser Ala Asn His Val Leu Ala Glu Arg Arg Arg Arg Glu Lys 485 490 495 Leu Asn Glu Arg Phe Ile Ile Leu Arg Ser Leu Val Pro Phe Val Thr 500 505 510 Lys Met Asp Lys Ala Ser Ile Leu Gly Asp Thr Ile Glu Tyr Val Lys 515 520 525 Gln Leu Arg Lys Lys Ile Gln Asp Leu Glu Ala Lys Asn Arg Gln Met 530 535 540 Glu Ala Lys Ser Arg Ser Ser Pro Gly Gly Asp Leu Gln Arg Ser Thr 545 550 555 560 Ser Met Lys Glu Leu Arg Thr Leu Ala Gly Ala Ser Ser Gly Thr Asp 565 570 575 Arg Gly Ser Val Gly Gln Asp Lys Arg Lys Met Arg Ile Val Glu Ala 580 585 590 Gly Ser Lys Pro Lys Ala Val Glu Pro Pro Leu Pro Ser Pro Pro Pro 595 600 605 Pro Gln Pro Thr Pro Thr Glu Thr Ser Val Gln Val Ser Ile Ile Glu 610 615 620 Ser Asp Ala Leu Leu Glu Leu Gln Cys Pro His Arg Glu Gly Leu Leu 625 630 635 640 Leu Asp Val Met Gln Met Leu Arg Glu His Arg Ile Glu Val Thr Ala 645 650 655 Val Gln Ser Ser Leu Thr Asn Gly Phe Phe Ser Ala Glu Leu Arg Ala 660 665 670 Lys Val Lys Glu Asn Thr Asn Gly Lys Lys Ala Ser Ile Val Glu Val 675 680 685 Lys Arg Ala Ile Gln Gln Ile Ile Pro His Thr Asp Ser 690 695 700 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <400> 5 atgagggtga agtcgagtc 19 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <400> 6 tcacgcgacg ggttgagg 18 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <400> 7 atggccgcgc cgcctagt 18 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8 tgagtccgta tgggggatga 20 <210> 9 <211> 41 <212> DNA <213> Artificial sequence <400> 9 ggagaggacc tcgactctag aatgagggtg aagtcgagtc t 41 <210> 10 <211> 39 <212> DNA <213> Artificial sequence <400> 10 ctcatttttt ctaccggtac ctcacgcgac gggttgagg 39 <210> 11 <211> 39 <212> DNA <213> Artificial sequence <400> 11 ggagaggacc tcgactctag aatggccgcg ccgcctagt 39 <210> 12 <211> 41 <212> DNA <213> Artificial sequence <400> 12 ctcatttttt ctaccggtac ctgagtccgt atgggggatg a 41

Claims

1. The use of transcription factors MedPAP1 and MedTT8 in promoting the synthesis and accumulation of orchid cyanidin in Nicotiana benthamiana and Phalaenopsis alba, characterized by: The amino acid sequence of the transcription factor MedPAP1 is shown in SEQ ID No. 2, and the amino acid sequence of the transcription factor MedTT8 is shown in SEQ ID No.

4.

2. Genes MedPAP1 and genes MedTT8 The application of the invention in promoting the synthesis and accumulation of cyanidin in Nicotiana benthamiana and Phalaenopsis alba is characterized by: Gene MedPAP1 The nucleotide sequence of gene is shown in SEQ ID No.

1. MedTT8 The nucleotide sequence is shown in SEQ ID No.3.

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