Dcros1, a DNA demethylase gene from carnation and its use

Abstract

The application provides a DNA demethylase gene DcROS1 of a carnation and application thereof, and belongs to the field of functional gene technology. The DNA demethylase gene DcROS1 of the carnation provided by the application has a nucleotide sequence as shown in SEQ ID NO:2. The application shows that the gene DcROS1 has the function of regulating the aging of flowers, fruits and vegetables through the VIGS technology to silence the expression of the DcROS1 gene and the results of the transient overexpression of the DcROS1 gene, and provides a new method for improving and breeding of long-bottled flower varieties of fresh-cut flowers.

Description

Technical Field

[0001] This invention belongs to the field of functional gene technology, specifically relating to the carnation DNA demethylase gene DcROS1 and its applications. Background Technology

[0002] Cut flowers, having been separated from the parent plant, cannot effectively obtain nutrients and water, and are susceptible to environmental stresses and pathogens, significantly impacting their vase life and ornamental value. Therefore, effectively extending the vase life of cut flowers has become a key scientific issue restricting the development of the cut flower industry. Past methods for extending the post-harvest vase life of carnation cut flowers mainly focused on the artificial application of preservatives, such as cytokinins, citric acid, nano-silver, silver nitrate, glycine, and sucrose. [1，2，3，4，5] Other factors, such as blue light, affect the vase life. [6] Studies on the function of ethylene biosynthetic enzymes and transcription factors in the ethylene signaling pathway during postharvest senescence of carnation cut flowers, etc. [7，8，9，10，11] In addition, epigenetic modifications also participate in regulating various aspects of plant growth and development.

[12] This mainly includes histone methylation modification, histone acetylation modification, and DNA methylation modification. Previous research has shown that histone H3K4me3 is involved in regulating carnation cut flower senescence during ethylene-induced senescence.

[13] .

[0003] However, there are currently no reports on the role of DNA methylation modification in regulating the anti-aging and preservation of flowers, fruits, and vegetables.

[0004] References

[0005] [1] Liu Xuanyuan, Guo Jia, Huang Li, Wang Haimiao, He Shan (2021) Study on dynamic preservation effect of preservative on cut carnation flowers. Modern Horticulture.

[0006] [2] Cheng Guiping, Li Wanping, Li Fang, Huang Xinmin, He Shenggen (2012) Physiological effects of nano-silver fungicide on senescence of carnation cut flowers. Northern Horticulture.

[0007] [3] Wan Zhuzhu, Tan Xiumei, Liu Min, Wu Liang, Dong Cao et al. (2020) Effect of cytokinin treatment on the preservation effect of carnation cut flowers. Northern Horticulture.

[0008] [4] Zhuoma Cairin, Li Yuxiong, Wei Guoliang (2018) Effects of the main components of three preservatives on the preservation effect of carnation cut flowers. Journal of Qinghai University (Natural Science Edition).

[0009] [5] Ouyang Huixian, Zhang Xianfang, Wang Guoli, Chen Ying (2018) Screening study of plant-derived preservatives for fresh-cut carnations. Zhejiang Agricultural Sciences.

[0010] [6]Aalifar M,Aliniaeifard S,Arab M,et al.Blue Light Improves VaseLife of Carnation Cut Flowers Through Its Effect on the Antioxidant DefenseSystem.Front Plant Sci.2020;11:511

[0011] [7]Kenichi Shibuya,et al.Comparison of mRNA levels of three ethylenereceptors in senescing flowers ofcarnation(Dianthus caryophyllus L.),Journalof Experimental Botany,Volume 53,Issue 368,1March 2002,Pages 399-406

[0012] [8]Shibuya,K.,et al.,Role of the gynoeciumin natural senescence ofcarnation(Dianthus caryophyllus L.)flowers.Journal of Experimental Botany,2000.51(353):p.2067-2073.

[0013] [9]Shibuya,K.,et al.,Comparison of mRNA levels of three ethylenereceptors in senescing flowers ofcarnation(Dianthus caryophyllus L.).Journalof Experimental Botany,2002.53(368):p.399-406.

[0014]

[10] Wang T, Sun Z, Wang S, Feng S, Wang R, Zhu C, Zhong L, Cheng Y, Bao M, Zhang F. DcWRKY33 promotes petal senescence in carnation (Dianthus caryophyllus L.) by activating genes involved in the biosynthesis of ethylene and abscisic acid and accumulation of reactive oxygen species. Plant J. 2023 Feb; 113(4): 698-715.

[0015]

[11] Zhu C, Huang Z, Sun Z, Feng S, Wang S, Wang T, Yuan X, Zhong L, Cheng Y, Bao M, Zhang F. The mutual regulation between DcEBF1 / 2 and DcEIL3-1 is involved in ethylene induced petal senescence in carnation (Dianthus caryophyllus L.). Plant J. 2023 May; 114(3): 636-650.

[0016]

[12] Ay N, Janack B, Humbeck K. Epigenetic control of plant senescence and linked processes. J Exp Bot 2014 65(14): 3875-3887.

[0017]

[13] Feng S, Jiang X, Wang R, Tan H, Zhong L, Cheng Y, Bao M, Qiao H, Zhang F. Histone H3K4 methyltransferase DcATX1 promotes ethylene induced petal senescence in carnation. Plant Physiol. 2023 May 2; 192(1): 546-564. SUMMARY OF THE INVENTION

[0018] In view of this, the purpose of this invention is to provide a carnation DNA demethylase gene DcROS1, which has the function of regulating the preservation and anti-aging of flowers, fruits and vegetables.

[0019] This invention provides a carnation DNA demethylase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0020] This invention provides a carnation DNA demethylase gene DcROS1, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0021] The present invention provides a recombinant expression vector containing the carnation DNA demethylase gene DcROS1.

[0022] The present invention provides a recombinant engineered bacterium containing the carnation DNA demethylase gene DcROS1 or the recombinant expression vector.

[0023] Preferably, the host bacterium is Agrobacterium.

[0024] This invention provides the application of the carnation DNA demethylase, the carnation DNA demethylase gene DcROS1, the recombinant expression vector, or the recombinant engineered bacteria in the preservation of flowers, fruits, and vegetables.

[0025] Preferably, the preservation of flowers, fruits and vegetables includes slowing down the aging process of flowers, fruits and vegetables.

[0026] Preferably, the flowers in the flowers and fruits include fresh cut flowers.

[0027] Preferably, the fruits in the flowers and vegetables include tropical fruits and / or temperate fruits.

[0028] Preferably, the vegetables in the flower and fruit vegetable category include at least one of the following: leafy vegetables, stem vegetables, flower bud vegetables, and legume vegetables.

[0029] This invention provides a method for preserving flowers, fruits, and vegetables, comprising the following steps:

[0030] Increase the expression level of the carnation DNA demethylase gene DcROS1 or its encoded protein in flowers, fruits and vegetables.

[0031] This invention provides the application of the carnation DNA demethylase, the carnation DNA demethylase gene DcROS1, the recombinant expression vector, or the recombinant engineered bacteria in the improvement or breeding of long-vase-period flower varieties.

[0032] The carnation DNA demethylase gene DcROS1 provided by this invention has the nucleotide sequence shown in SEQ ID NO:2. This invention utilizes VIGS technology and transient overexpression technology to silence and overexpress the DcROS1 DNA demethylase gene in carnation flowers. Results show that transient silencing of the DcROS1 gene in carnation petals can shorten the vase life of carnations by about 5 days, and transient silencing can also promote the senescence of the flower head and petals. This indicates that the DcROS1 gene plays an important role in the senescence of carnation petals. Transient overexpression of the DcROS1 gene can extend the vase life of carnations by about 4 days, and transient overexpression can also significantly slow down the senescence rate of the flower head and petals. Therefore, the carnation DNA demethylase gene DcROS1 provided by this invention has the ability to slow down the senescence rate of flowers and fruits, thus playing a biological role in the preservation of flowers and fruits, and also providing a new means for the improvement or breeding of flower varieties with long vase life. Attached Figure Description

[0033] Figure 1 The results show the effects of transient overexpression and silencing of DcROS1 on carnation flowers. A represents the effect of transient overexpression and silencing of DcROS1 in petals on flower senescence; B represents the statistical results of flowering period; C represents the expression results of the DcROS1 gene in petals; and D represents the effect of transient overexpression and silencing of DcROS1 in the flower head on the senescence of the flower head.

[0034] Figure 2 The results show the effects of transient overexpression of DcROS1 on carnation flowers. A represents the effect of transient overexpression of DcROS1 in petals on flower senescence; B represents the statistical results of flowering period; C represents the expression results of the DcROS1 gene in petals; and D represents the effect of transient overexpression of DcROS1 in the flower head on flower head senescence. Detailed Implementation

[0035] The present invention provides a carnation DNA demethylase, whose amino acid sequence is set forth in SEQ ID NO:1 (MIEIQEDRDGKVAGPIIDSMPDKVITAENDIPEKGNQVIDLNDTPQKKPR RRRYQPKVIRENKPKRTPKPNTSKPTEAQSKRKYNRKTKSSTPSNPIPEEATEPKPTKPTPTEPQPAEPEQTEPEVNETPAKSCKRSLNFDLNGQVTEDNDELVGKTSMSAYDLNLAQNHELSEYISLPVMETLDNSRTDNNLSVGEKVKSSESLSGKKTESSSIKNPTSAQSPNSSPCASSSDVAQTRGLKRSVSYAAETTLIDVEIGTITEYRPWALSHTASLNTHSDEPRQSFTPEICKRKKVETVETSCSQSTSAVNASEDISQLGKDHGQPQVELTASKYYCCNTTIGISGMSSNYAALVEISENRHQLIKTWADLSRLKKKKRTGPTRVRDLAPPSGIASGANSQTKVESESGSHPSHTTSNGVVSCTDGLASETKTTPKSRRRLKKQDYVNMNWDIVLYDNNGGAAREATGSLETFPWRKRRCTIDGLTRLLERLDINREKRKKSRKQNRSIVPYRGKKQEQNALVLYEKDGSIVPFEGPFNPISKRRQRARVDLDDETTRVWRLLLENIENEGINGIDEEKAQWWENERSVFRGRVDSFIARMRLVQGDRRFIPWKGSVLDSVIGVFLTQNVSDHLSSSAFMSMAARFPLKPETSGSTVSEEATSGLFVEPEISIVEPEERITWNLKIIEEPTMEKNSRAVFYFDHSEEKEVVNSGERCGKADPVGSTQHFREACRKDSSYSRGYDANSQTTETETVYPQGEDKARDDATSSLQSVISSQNSINSLNQQTVETMSTFTESNTRTQVVTSSSKFDCLDHTASFMGLLKMAEGNIHHQYNSQAQVDHLDGIKHATEYYRAAPGVFSSNYQLNLEAGFHLQDTQGLDMLEVESRSSDMSRKDENSPPTEQSNLTSEIADQEKSPDSFPRAAQNMSSCYFLEGDTMIVQSQRKEVKESFDTIASSSRAEAIPNCKEVNVPILSQEMADVIESTRTLERTRNICKGEEQIHGHNYPGVASGVTDNNSAKTAKGKPGKGKKNEFDWDSLRREAHVNGKPERPAHNRDSLDWEAVRHASANDIAETIKERGMNNVLAGRIKDLLDRLVKDHGSIDMEWLRDIPPDKAKEYLLSFRGLGLKSVECVRLLTLHHLAFPVDTNVGRIAVRLGWVPLQPLPESLQLHLLELYPILESIQQYLWPRLCKLDQKILYELHYHMITFGKVFCTKRQPNCNACPLRGECRHFASAFASARFALPGPEERSIMPASGNTAPQVTPSPGTNLLLSTAPWDSHNPHSNSGSWDGHNPHSNSENYASAIVQGPSLPLSGPLPQTVHLERKSINSKCEPIVEVPASPEQEPEHEQALCDIEDAFYEDPNEIPTINLNMKEFTETLQNFMLQETGMSNALVALTAEAASIPTPKLKNINRLRTEHHVYELPDSHPLLQGLEKREPDDPCSYLLAIWIPGETADSIDPPARRCSFDDPRMLCNEETCCYCSSQREANSQIVRGTLLIPTRTAMRGSFPLNGTYFQVNEVFADHDSSLKPIAVPRSWLWNLPRRTVYFGTSIPTIFKGLNTEDIQHCFWRGYVCVRGFDQKTRAPRPLMARLHFPASRIQRAKGKAYDE) is shown as above.

[0036] The present invention provides a carnation DNA demethylase gene DcROS1, whose nucleotide sequence is set forth in SEQ ID NO: 2 (ATGATCGAGATTCAGGAGGACCGTGACGGGAAAGTT GCAGGCCCCATCACGGATTCTATGCCTGATAAAGTCATAACGGCGGAGAACGACATCCCTGAGAAGGGCAACCAAGTTATTGACCTCAACGACACACCTCAAAAGAAACCACGAAGGAGAAGGTATCAACCCAAGGTGATTAGAGAAAACAAGCCTAAACGCACCCCAAAACCTAACACCTCAAAACCCACTGAAGCTCAAAGTAAAAGGAAGTATAACCGAAAAACCAAATCAAGCACTCCATCAAATCCAATCCCCGAAGAAGCGACCGAACCAAAACCAA

[0037] CCAAACCGACACCAACTGAACCACAGCCAGCAGAACCAGAACAAAC

[0038] AGAGCCAGAAGTAAACGAAACCCCAGCTAAATCCTGTAAAAGATCCCT

[0039] AAACTTTGACTTGAATGGTCAAGTAACAGAGGATAACGACGAATTAGT

[0040] TGGCAAAACATCCATGAGTGCCTATGACCTTAACCTCGCTCAGAATCAC

[0041] GAACTGAGCGAGTATATCTCATTGCCTGTTATGGAAACTCTCGACAACT

[0042] CACGGACTGATAATAATTTGTCAGTTGGAGAGAAAGTGAAAAGCTCAG

[0043] AAAGTTTGAGTGGGAAAAAGACCGAGAGTTCCTCAATAAAAAACCCC

[0044] ACTTCAGCACAAAGTCCCAACTCCAGCCCTTGCGCAAGTTCCAGTGAT

[0045] GTTGCACAAACAAGGGTTAAAAAGGAGTGTTTCGTATGCAGCTGA

[0046] AACAACACTTATTGTTGAAATTGGGACCATCACAGAATATCGCCCT

[0047] TGGGCATTAAGTCACACCGCTTCCCTGAACACACATAGTGATGAACCC

[0048] CGCCAAAGTTTCACTCCAGAAATTTGCAAAAGAAAGAAAGTTGAAAC

[0049] AGTAGAAACGTCTTGCTCACAAAGCACATCAGCTGTCAATGCCTCTGA

[0050] AGATATCAGCCAGTTAGGTAAAGATCATGGTCAACCACAAGTCGAACT

[0051] CACTGCATCGAAATACTACTGTTGCAATACCACTATCGGAATCAGTGGA

[0052] ATGAGTAGTAATTATGCAGCGCTTGTTGAAATATCCGAGAATAGGCATC

[0053] AGCTAATTAAGACATGGGCTGACCTTTCAAGGTTAAAAAGAAAAAGA

[0054] GAACAGGTCCAACTCGGGTTCGTGATCTAGCTCCGCCATCCGGGATTG

[0055] CATCAGGAGCAAATAGTCAGACGAAAGTCGAAAGTGAGTCTGGTTCTC

[0056] ATCCTTCCCATACAACATCAAATGGTGTCGTCTCTTGCACAGATGGACT

[0057] AGCTTCAGAAACTAAAACAACCCCGAAGTCAAGAAGGAGATTAAAGA

[0058] AACAAGATTATGTAAACATGAACTGGGACATTGTTCTGTATGACAATAA

[0059] TGGTGGTGCTGCAAGGGAGGCAACAGGTTCCCTTGAGACATTCCCATG

[0060] GAGAAAGAGGCGCTGCACAATTGACGGTTTAACCAGGTTACTTGAGA

[0061] GATTAGATATCAACAGAGAAAAACGTAAGAAATCAAGAAAACAAAATA

[0062] GATCGATTGTCCCTTATCGAGGGAAAAAGCAAGAGCAGAATGCACTTG

[0063] TTCTCTATGAGAAAGATGGCTCAATAGTTCCGTTTGAGGGCCCTTTCAA

[0064] TCCAATTAGCAAACGGCGTCAAAGGGCGAGGGTTGACCTTGATGATGA

[0065] GACCACGCGAGTGTGGAGGCTTCTTCTTGAGAACATCGAAAATGAAG

[0066] GCATCAATGGAACTGATGAGGAAAAAGCACAATGGTGGGAAAACGAA

[0067] AGAAGTGTCTTTCGAGGACGGGTGGACTCATTCATAGCACGCATGCGT

[0068] CTTGTTCAAGGGGATAGACGCTTCACCCCATGGAAAGGCTCTGTGTTA

[0069] GACTCGGTTATTGGAGTTTTTCTCACTCAGAATGTCTCAGACCATCTCT

[0070] CCAGCTCTGCATTCATGTCAATGGCTGCGCGCTTTCCTCTCAAGCCAGA

[0071] AACATCTGGAAGTACTGTGTCGGAGGAGGCAACAAGTGGTTTATTCGT

[0072] AGAGCCAGAGATCAGTATAGTTGAACCTGAAGAAAGAATCACTTGGAA

[0073] CTTGAAGATAATAGAAGAACCAACTATGGAGAAGAACTCGAGGGCAG

[0074] TTTTTTACTTTGACCACAGTGAAGAGAAAGAGGTTGTCAATAGCGGAG

[0075] AGCGTTGTGGAAAAGCTGACCCGGTTGGCTCTACACAACACTTCAGA

[0076] GAAGCATGTCGAAAGGACTCCAGCTATTCGAGGGGTTACGATGCAAAC

[0077] AGCCAGACTACAGAGACAGAAACAGTCTACCCCCAAGGAGAAGATAA

[0078] AGCAAGGGATGATGCAACTTCATCACTGCAGTCCGTTATCTCATCACAG

[0079] AACTCCATCAACTCGTTGAACCAGCAGACAGTCGAAACAATGAGCACT

[0080] TTCACAGAAAGCAACACAAGAACACAGGTCGTGACATCCAGCTCCAA

[0081] ATTCGATTGTCTTGATCATACCGCTTCTTTCATGGGGCTACTAAAGATGG

[0082] CAGAGGGAAATATCCACCACCAATATAACAGCCAAGCACAAGTTGATC

[0083] ACCTTGATGGTATAAAGCATGCCACAGAGTATTATCGAGCAGCACCTGG

[0084] TGTTTTCTCAAGTAATTACCAATTGAACTTAGAAGCAGGATTTCATTTAC

[0085] AAGATACCCAGGGTCTTGATATGCTTGAAGTAGAGAGCAGATCTTCTGA

[0086] TATGTCGAGGAAAGACGAGAACAGCCCACCAACAGAACAAAGCAATC

[0087] TTACCTCAGAAATAGCAGATCAAGAGAAAAGTCCAGACAGCTTTCCTA

[0088] GAGCTGCTCAGAATATGTCCTCGTGCTATTTTCTTGAAGGAGATACAAT

[0089] GATTGTACAGTCACAAAGGAAAGAAGTCCAAAGAATCATTTGATACTAT

[0090] TGCTTCATCTTCCCGAGCAGAGGCAATTCCAAACTGTAAAGAGGTAAAA

[0091] TGTACCAATTTTATCTCAAGAAATGGCTGATGTTATAGAAAGCACCAGA

[0092] ACATTGGAAAGAACAAGAAATATCTGCAAAGGGGAAGAGCAAATCCA

[0093] TGGGCATAACTATCCAGGTGTGGCATCTGGCGTGACCGACAATAATTCT

[0094] GCCAAACAGCAAAGGAAAGCCGGGAAAGGGAAAGAAAAAATGAAT

[0095] TTGACTGGGACAGCCTTCGAAGAGAGGCACATGTTAACGGAAAACCA

[0096] GAGAGGCCAGCCCACAACAGGGACTCATTAGACTGGGAAGCAGTTAG

[0097] GCATGCTAGGCGCGAACGATATTGCAGAGACCATAAAGAGCGGGGAAT

[0098] GAACAATGTGCTTGCAGGCCGTATTAAGGACCTCCTGGACAGACTCGT

[0099] CAAGGATCATGGCAGCACTGACATGGAATGGCTAAGAGATATTCCACC

[0100] AGACAAAGCAAAGGAATACCTGCTAAGTTTCAGAGGTTTGGCTTGAA

[0101] GAGCGTGGAGTGTGTCCGCCTTTTGACTCTGCATCATCTTGCATTCCCA

[0102] GTTGACACTAATGTTGGCCGCATTGCTGTTCGACTAGGATGGGTTCCAC

[0103] TTCAACCATTACCAGAGTCACTTCAACTGCATCTTTTAGAACTGTACCC

[0104] AATTTTGGAGTCGATTCAGCAGTACTTATGGCCACGCTTGTGCAAGCTT

[0105] GATCAAAAGACATTGTATGAGTTGCACTATCACATGATCACATTTGGCA

[0106] AGGTATTTTGCACCAAAAGACAGCCAAACTGCAATGCTTGCCCCTTAA

[0107] GAGGAGAGTGCAGACACTTTGCCAGTGCTTTCGCTAGTGCCAGGTTTG

[0108] CACTTCCGGGGCCAGAAGAAAGAAGCATCATGCCAGCAAGTGGGAAC

[0109] ACTGCTCCGCAAGTTACTCCGAGTCCTGGCACTAATCTTTTGCTTTCTA

[0110] CAGCCCCATGGGACAGTCATAATCCACATTCGAACTCGGGTTCATGGG

[0111] ACGGCCACAATCCTCATTCAAACTCCGAAAACTATGCCTCCGCAATAGT

[0112] TCAGGGCCCATCTCTCCCGTTGTCAGGACCCTTGCCACAAACTGTCCAT

[0113] TTAGAAAGGAAATCCATAAACAGTAAATGTGAACCAATTGTTGAAGTA

[0114] CCAGCATCACCAGAGCAGGAACCAGAACATGAACAAGCATTATGCGA

[0115] CATCGAAGATGCATTCTATGAAGATCCTAATGAAATCCCAACAATCAAC

[0116] CTCAACATGAAGGAATTCACTGAGACTTTGCAGAATTTTATGTTGCAAG

[0117] AGACAGGAATGTCAAATGCCTTGGTAGCTCTAACAGCTGAAGCTGCTT

[0118] CGATACCTACACCAAAGCTGAAAAACATAAACCGTTTAAGAACAGAGC

[0119] ATCATGTTTATGAACTTCCAGACAGTCACCCTCTGCTCCAAGGGTTGGA

[0120] GAAAAGGGAGCCTGATGATCCATGCTCTTACCTACTCGCAATTTGGACC

[0121] CCAGGTGAAACGGCAGACTCAATCGATCCTCCAGCGAGAAGATGTAGT

[0122] TTTGATGATCCAAGGATGCTCTGCAATGAGGAGACATGCTGTTACTGCA

[0123] GCAGTCAGCGGGAAGCAAACTCCCAAATAGTACGAGGAACACTTCTG

[0124] ATACCAACCAGAACAGCCATGAGAGGTAGCTTCCCACTCAATGGAACA

[0125] TACTTCCAAGTTAACGAGGTATTTGCTGACCATGACTCAAGCCTTAAAC

[0126] CAATAGCAGTACCCAGGTCTTGGCTGTGGAACCTTCCAAGGCGAACTG

[0127] TTTACTTTGGAACATCGATACCGACCATATTTAAAGGTCTGAACACTGA

[0128] GGATATTCAACATTGCTTCTGGAGAGGATACGTCTGTGTAAGAGGGTTT

[0129] GACCAGAAAACTAGAGCGCCTCGTCCGCTCATGGCTAGATTGCATTTC

[0130] As shown in CCAGCCAGCAGGATTCAAAGAGCGAAAGGTAAGGCATATGATGAGTAG).

[0131] The present invention provides a recombinant expression vector containing the carnation DNA demethylase gene DcROS1.

[0132] In this invention, the backbone vector of the recombinant expression vector is preferably pCAMBIA1300, and the cloning site is preferably digested with HandIII and KpnI enzymes. The construction method of the recombinant expression vector preferably utilizes a first primer pair to amplify the carnation DNA demethylase gene DcROS1. The amplified product is cloned into the backbone vector via homologous recombination, and the vector successfully inserting the DcROS1 gene is verified. The first primer pair preferably includes a forward primer with the nucleotide sequence shown in SEQ ID NO:3 and a reverse primer with the nucleotide sequence shown in SEQ ID NO:4. The amplification reaction program is preferably: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 5 min, 35 cycles; 72℃ for 5 min. This invention does not impose any special limitations on the cloning method; any cloning method well-known in the art can be used, such as enzyme digestion and ligation. The verification method preferably involves transforming the cloning product into competent E. coli cells, culturing, and then performing bacterial PCR amplification and sequencing on the bacterial culture. Obtaining the target fragment indicates successful construction of the recombinant expression vector. The recombinant expression vector enters plant cells under the mediation of Agrobacterium, upregulating the expression level of the carnation DNA demethylase gene.

[0133] In this invention, to provide a vector for downregulating the carnation DNA demethylase gene in cells, a gene silencing vector TRV-DcROS1 is constructed. The preferred construction method includes the following steps: amplifying the carnation DNA demethylase gene fragment using a second primer pair, cloning the amplified fragment into the EcoR1 multiple cloning site of the TRV2 vector, and verifying the result to obtain the gene silencing vector TRV-DcROS1. The second primer pair is the forward primer shown in SEQ ID NO:5 and the reverse primer with the nucleotide sequence shown in SEQ ID NO:6. The preferred amplification reaction program is: 95℃ for 5 min, 95℃ for 15 s, 60℃ for 15 s, 72℃ for 5 min, 35 cycles; 72℃ for 5 min. This invention does not impose any special limitations on the cloning method; any cloning method well-known in the art, such as enzyme digestion and ligation, can be used. The verification method preferably involves transforming the cloning product into competent *E. coli* cells, culturing the cells, performing bacterial PCR amplification and sequencing, and obtaining the target fragment, indicating successful construction of the gene silencing vector.

[0134] The present invention provides a recombinant engineered bacterium containing the carnation DNA demethylase gene DcROS1 or the recombinant expression vector.

[0135] In this invention, the host bacterium of the recombinant engineered bacteria is preferably Agrobacterium. In an embodiment of this invention, the strain of Agrobacterium is GV3101.

[0136] The present invention does not impose any special restrictions on the preparation method of the recombinant engineered bacteria; the transformation scheme of Agrobacterium, which is well known in the art, can be used.

[0137] This invention provides the application of the carnation DNA demethylase, the carnation DNA demethylase gene DcROS1, the recombinant expression vector, or the recombinant engineered bacteria in the preservation of flowers, fruits, and vegetables.

[0138] This invention provides a method for preserving flowers, fruits, and vegetables, comprising the following steps:

[0139] Increase the expression level of the carnation DNA demethylase gene DcROS1 or its encoded protein in flowers, fruits and vegetables.

[0140] In this invention, the method for increasing the expression level of carnation DNA demethylase or gene DcROS1 in flowers, fruits, and vegetables is preferably carried out using transgenic methods. In embodiments of this invention, the carnation DNA demethylase gene DcROS1 or the recombinant expression vector is introduced into cells under the mediation of Agrobacterium. The preferred method for cell entry in this invention is vacuum infection.

[0141] In this invention, the preservation of flowers, fruits, and vegetables preferably includes slowing down their senescence. In this invention, the flowers among the flowers, fruits, and vegetables preferably include fresh cut flowers. This invention does not have any particular limitation on the type of fresh flowers; any flower type well-known in the art can be used. In an embodiment of this invention, carnations are used as an example to illustrate the role of the DNA demethylase gene DcROS1 in regulating the senescence rate of fresh flowers. The fruits among the flowers, fruits, and vegetables preferably include tropical fruits and / or temperate fruits. The vegetables among the flowers, fruits, and vegetables include at least one of the following: leafy vegetables, stem vegetables, budding vegetables, and legumes.

[0142] In this embodiment of the invention, silencing the expression of the carnation DNA demethylase gene DcROS1 shortens the vase life of cut carnations and promotes the aging of the carnation flower head and petals. Transient overexpression of the carnation DNA demethylase gene DcROS1, however, can prolong the vase life of carnations and delay the aging of the carnation flower head and petals, thereby preserving the freshness of cut carnations and extending their ornamental time.

[0143] Therefore, the present invention provides the application of the carnation DNA demethylase, the carnation DNA demethylase gene DcROS1, the recombinant expression vector, or the recombinant engineered bacteria in the improvement or breeding of long-vase-period flower varieties.

[0144] The following detailed description, in conjunction with embodiments, illustrates the DcROS1 carnation DNA demethylase gene and its applications provided by this invention, but these should not be construed as limiting the scope of protection of this invention.

[0145] Example 1

[0146] The full-length cDNA sequence (SEQ ID NO:2) of the carnation DNA demethylase gene DcROS1 was obtained using carnation petal transcriptome data. The homolog of AtROS1 was obtained by sequence alignment and phylogenetic analysis with AtROS1 in Arabidopsis thaliana and named DcROS1.1, or DcROS1 for short (SEQ ID NO:1).

[0147] Example 2

[0148] I. A method for constructing a gene silencing expression vector

[0149] 1. Materials and Methods

[0150] 1.1 Test materials

[0151] The experimental material was carnation (Dianthus caryophyllus L.) 'Master'. Grade A cut flowers ordered from Yunnan were cut at 5cm lengths and placed in a bucket containing tap water for cultivation. The water was changed daily, and 3cm of the stem was trimmed each day. Cultivation conditions: 23℃, 14h / d light. Agrobacterium infection experiments were conducted after the flowers were fully open.

[0152] The experimental bacterial culture was Agrobacterium GV3101.

[0153] 1.2 Amplification of the target gene DcROS1 (Dca30799)

[0154] The full-length DcROS1 is 4845 bp (SEQ ID NO:2), encoding 1614 amino acids (SEQ ID NO:1). Transient silencing was performed on a 339 bp fragment at the initiation site, and the primer sequences are as follows:

[0155] Forward primer: 5'-gtgagtaaggttaccgaattcATGATCGAGATTCAGGAGGACCG-3' (SEQ ID NO: 5);

[0156] Reverse primer: 5'-tggaggccttctagagaattcTTCAGTTGGTGTCGGTTTGGT-3' (SEQ ID NO:6).

[0157] Amplification system: cDNA 5μL, ddH2O 14μL, 2×PhantaMax Buffer 25μL, dNTP Mix (10mM each) 1μL, forward primer 2μL, reverse primer 2μL, Phanta Mix Super-Fidelity DNA Polymerase 1μL.

[0158] Amplification program: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0159] 1.3 Carrier linearization processing

[0160] The TRV2 vector was digested with EcoR1 at 37℃ for 30 min. The digestion system consisted of 1 μg TRV2, 2 μl 10×cut smartbuffer, 1 μl EcoR1, and ddH2O to a final volume of 20 μl.

[0161] 1.4 Cloning Methods

[0162] Using the homologous recombinase of vinozine The ligation was performed using the IIOne Step Cloning Kit at 37℃ for 30 min. The ligation system consisted of 2 μl of 5×CE II Buffer, 1 μl of Exnase II, 290 ng of linearized TRV, 30 ng of PCR product, and ddH2O to a final volume of 10 μl.

[0163] The ligation product was transformed into *E. coli* DH5α, incubated on ice for 15 min, heat-shocked at 42°C for 1 min, and then shaken at 37°C for 1 h. The resulting product was then plated onto solid LB agar containing 100 μM kanamycin. Positive colonies were picked and sent for sequencing. The correctly sequenced recombinant vector was then used.

[0164] II. Transformation of Agrobacterium GV3101

[0165] Take 10 μl of GV3101, add 2 μl of the recombinant vector constructed above, incubate on ice for 5 min, in liquid nitrogen for 5 min, and heat shock at 37°C for 5 min. Spread the mixture onto a solid LB agar containing 50 μM rifampicin and 100 μM kanamycin, pick positive colonies for expansion culture, and obtain Agrobacterium GV3101 containing the TRV-DcROS1 vector.

[0166] Example 3

[0167] Method for silencing the DcROS1 gene in carnations using VIGS technology

[0168] 1. Flowerpot Inoculation Method

[0169] Agrobacterium GV3101 carrying vectors 1300-DcROS1, TRV1, TRV2, and TRV-DcROS1, respectively, were incubated overnight at 28°C with 100 μM kanamycin and 50 μM rifampin until OD was reached. 600 0.8-1.0. Then resuspend in a resuspension buffer (resuspension buffer formulation: 200 μM AS (acetylsyleugenone), 1 mM MES (2-(N-morpholino)ethanesulfonic acid), 1 mM MgCl2), and then measure the OD of the Agrobacterium resuspension. 600 Set the concentration to 1.0 and let it stand at 28℃ for 2 hours. Take the outermost petals of a fully opened carnation and use a punch to uniformly punch the petals into flower discs with a diameter of 0.6 mm. Place them in a petri dish and pour in the settled resuspension (TRV-DcROS1 and TRV1 mixed in a 1:1 molar ratio). Use a vacuum pump to evacuate to 0.7 MPa one to two times. A control group was also set up, in which Agrobacterium GV3101 carrying TRV1+TRV2 (1:1) or 1300 were used for flower disc infection experiments. All other steps were the same.

[0170] 2. Whole-flower inoculation method

[0171] The preparation method of the resuspension is the same as that of the flower disc infection method. Pour 500 ml of resuspension into a beaker, immerse the whole flower in the resuspension, and use a vacuum pump to evacuate to 1.2 MPa once or twice until the Agrobacterium resuspension fills all the petals.

[0172] 3. Ion leakage rate analysis

[0173] Ion leakage rate was determined according to existing techniques (Wu et al., 2017). The ion leakage rate was determined by measuring the electrolyte leakage from the carnation flower heads. Twenty carnation flower heads from each treatment were immersed in 25 mL of 0.4 M mannitol at room temperature with gentle shaking for 3 hours. The initial conductivity of the solution was first measured using a conductivity meter (ST3100C). Then, after incubation at 85°C for 20 min, the total conductivity was measured, and the ion leakage rate was calculated.

[0174] The ion leakage rate (%) is the percentage of initial conductivity to total conductivity.

[0175] 4. Real-time quantitative PCR detection

[0176] The specific steps of real-time quantitative PCR are as follows:

[0177] use Total RNA was extracted from the sample using reagents (Invitrogen). 1 μg of total RNA was converted to cDNA using the HiScript First-Strand cDNA Synthesis Kit (Vazyme). RNA extraction and reverse transcription were performed according to the manufacturer's instructions. RT-qPCR reactions were performed on a BIO-RAD CFX Connect real-time system. Quantitative PCR was conducted using Hieff™ qPCR SYBR & Green Master Mix (Yeasen), and amplification procedures were performed according to the manufacturer's instructions. DcUbq3-7 was selected as an internal control. 2 -ΔΔCT The relative expression level was calculated using this method.

[0178] The primers used for the qPCR reaction are as follows:

[0179] Primer sequences:

[0180] pDcROS1-qF: 5'-TTCTCATCCTTCCCATACAACATC-3' (SEQ ID NO: 7); pDcROS1-qR: 5'-GAACCTGTTGCCTCCCTTGC-3' (SEQ ID NO: 8);

[0181] Internal reference primer sequence:

[0182] pUbq3-7-qF: 5'-GTTGTTGGTTTCAGGGCTGGTTTG-3' (SEQ ID NO: 9);

[0183] pUbq3-7-qR: 5'-CTACGGTAATTGAGAATTCACACCGAAATG-3' (SEQ ID NO: 10).

[0184] The qPCR reaction system is as follows:

[0185]

[0186] The qPCR reaction procedure is shown in Table 1:

[0187] Table 1 qPCR reaction procedure

[0188]

[0189] 5. Statistical Analysis

[0190] 5.1 For the results of the flower heads, simply observe the time it takes for the color of each flower head to fade. Flower heads that age earlier will show signs of fading sooner, and at this time, it is necessary to take a picture to record the result.

[0191] 5.2 The flowering period of the whole flower needs to be photographed every day, and at least six sets of data should be collected for statistical analysis. The results should be presented in a bar chart.

[0192] like Figure 1 As shown, transient silencing of the DcROS1 gene in carnation petals can shorten the vase life of carnations by about 5 days. Figure 1 (A, B). Real-time quantitative PCR results showed that transient silencing did indeed reduce the expression of the DcROS1 gene. Figure 1 C) indicates that transient silence is effective. Furthermore, transient silence can also promote the aging of the flower head. Figure 1 (D). In conclusion, the DcROS1 gene plays an important role in the senescence of carnation petals. Transient silencing of DcROS1 accelerates ethylene-induced senescence of carnation petals and flower heads.

[0193] Example 4

[0194] I. A method for constructing a recombinant expression vector for transient overexpression of the carnation DNA demethylase gene DcROS1

[0195] 1. Materials and Methods

[0196] 1.1 Test materials

[0197] The experimental material was carnation (Dianthus caryophyllus L.) 'Master'. Grade A cut flowers ordered from Yunnan were cut at 5cm lengths and placed in a bucket containing tap water for cultivation. The water was changed daily, and 3cm of the stem was trimmed each day. Cultivation conditions: 23℃, 14h / d light. Agrobacterium infection experiments were conducted after the flowers were fully open.

[0198] The experimental bacterial culture was Agrobacterium GV3101.

[0199] 1.2 The target gene was DcROS1 (Dca30799), 4845 bp in length, encoding 1614 amino acids. The primer sequences used for overexpression are as follows:

[0200] pDcROS1-F: 5'-caaatcgactctagaaagcttATGGAAACTCAGAGAAAAGAAGGC-3' (SEQ IDNO: 3);

[0201] pDcROS1-R: 5'-gcccttgctcaccatggtaccCTCATCATAGCCTTACCTTTCGC-3' (SEQ IDNO: 4);

[0202] Amplification system: cDNA 5μL, ddH2O 14μL, 2×PhantaMax Buffer 25μL, dNTP Mix (10mMeach) 1μL, forward primer 2μL, reverse primer 2μL, Phanta Mix Super-Fidelity DNA Polymerase 1μL.

[0203] Amplification program: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 5 min, 35 cycles; 72℃ for 5 min.

[0204] The pCAMBIA1300 vector was digested with Hand III and Kpn I enzymes at 37℃ for 30 min. The digestion system was as follows: 1 μg pCAMBIA1300, 2 μl 10×cut smartbuffer, 1 μl Hand III, 1 μl Kpn I, and ddH2O to make up to 20 μl.

[0205] Using the homologous recombinase of vinozine The ligation was performed using the IIOne Step Cloning Kit at 37℃ for 30 min. The ligation system was as follows: 2 μl of 5×CE II Buffer, 1 μl of Exnase II, 90 ng of linearized pCAMBIA1300, 30 ng of PCR product, and ddH2O to a final volume of 10 μl.

[0206] The ligation product was transformed into *E. coli* DH5α, incubated on ice for 15 min, heat-shocked at 42°C for 1 min, and then shaken at 37°C for 1 h. The resulting inoculum was then plated onto LB agar containing 100 μM kanamycin. Positive colonies were picked and sequenced. The correctly sequenced vector was used for subsequent construction of recombinant *Agrobacterium*.

[0207] II. Preparation of Recombinant Agrobacterium

[0208] Take 10 μl of GV3101, add 2 μl of the recombinant expression vector constructed above, incubate on ice for 5 min, in liquid nitrogen for 5 min, and heat shock at 37°C for 5 min. Spread the mixture onto a solid LB agar containing 50 μM rifampicin and 100 μM kanamycin, pick positive colonies for expansion culture, and obtain recombinant Agrobacterium.

[0209] Example 5

[0210] A method for enhancing DcROS1 gene expression in carnations using transient overexpression technology

[0211] 1. Flowerpot Inoculation Method

[0212] Agrobacterium GV3101 carrying the recombinant expression vector DcROS1 was incubated overnight at 28°C with 100 μM kanamycin and 50 μM rifampin in a medium containing these two vectors until the OD value was reached. 600 0.8-1.0. Then resuspend in a resuspension buffer (resuspension buffer formulation: 200 μM AS (acetylsyleugenone), 1 mM MES (2-(N-morpholino)ethanesulfonic acid), 1 mM MgCl2), and then measure the OD of the Agrobacterium resuspension. 600 Set the temperature to 1.0 and let it stand at 28℃ for 2 hours. Take the outermost petals of a fully bloomed carnation and use a punch to make the petals into uniform flower discs with a diameter of 0.6 mm. Place them in a petri dish, pour in the settled resuspension (Agrobacterium GV3101 carrying the recombinant expression vector of DcROS1), and use a vacuum pump to evacuate to 0.7 MPa once or twice.

[0213] 2. Whole-flower inoculation method

[0214] The preparation method of the resuspension is the same as that of the flower disc infection method. Pour 500 ml of resuspension into a beaker, immerse the whole flower in the resuspension, and use a vacuum pump to evacuate to 1.2 MPa once or twice until the Agrobacterium resuspension fills all the petals.

[0215] 3. Ion leakage rate analysis

[0216] Ion leakage rate was determined according to existing techniques (Wu et al., 2017). The ion leakage rate was determined by measuring the electrolyte leakage from the carnation flower heads. Twenty carnation flower heads for each treatment were immersed in 25 mL of 0.4 M mannitol at room temperature with gentle shaking for 3 hours. The initial conductivity of the solution was first measured using a conductivity meter (ST3100C). The total conductivity was then measured after incubation at 85°C for 20 min.

[0217] Ion leakage rate is the percentage of initial conductivity to total conductivity.

[0218] 4. Real-time quantitative PCR detection

[0219] The real-time fluorescence quantitative PCR detection procedure is the same as described in Example 3.

[0220] 5. Statistical Analysis

[0221] 5.1 For the results of the flower heads, simply observe the time it takes for the color of each flower head to fade. Flower heads that age earlier will show signs of fading sooner, and at this time, it is necessary to take a picture to record the result.

[0222] 5.2 The flowering period of the whole flower needs to be photographed every day, and at least six sets of data should be collected for statistical analysis. The results should be presented in a bar chart.

[0223] Depend on Figure 2It was found that transient overexpression of the DcROS1 gene in carnation petals could extend the vase life of carnations by about 4 days. Figure 2 Real-time quantitative PCR results (A and B) showed that the expression level of the DcROS1 gene was indeed significantly increased in petals overexpressing DcROS1. Figure 2 The result (C) indicates that transient overexpression is effective. Furthermore, transient overexpression can significantly slow down the senescence rate of the flower head. Figure 2 (D). In conclusion, the DcROS1 gene plays an important role in the senescence of carnation petals. Transient overexpression of DcROS1 can delay the senescence of the flower head and petals of cut carnations.

[0224] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A carnation DNA demethylase, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A carnation DNA demethylase gene DcROS1, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, It includes the carnation DNA demethylase gene DcROS1 as described in claim 2.

4. A recombinant engineered bacterium, characterized in that, It contains the carnation DNA demethylase gene DcROS1 as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The host bacterium is Agrobacterium.

6. The application of the carnation DNA demethylase of claim 1, the carnation DNA demethylase gene DcROS1 of claim 2, the recombinant expression vector of claim 3, or the recombinant engineered bacteria of claim 4 or 5 in flower preservation, wherein the flower is a carnation.

7. The application according to claim 6, characterized in that, The purpose of flower preservation is to slow down the aging process of flowers.

8. The application according to claim 6 or 7, characterized in that, The flowers in question are fresh cut flowers.

9. A method for preserving carnations, characterized in that, Includes the following steps: Increase the expression level of the carnation DNA demethylase gene DcROS1 or its encoded protein as described in claim 2 in carnations.

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