CN112626076B - Application of gene PeNAC67 in breeding of Phalaenopsis miniata - Google Patents
Application of gene PeNAC67 in breeding of Phalaenopsis miniata Download PDFInfo
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- CN112626076B CN112626076B CN202011337754.8A CN202011337754A CN112626076B CN 112626076 B CN112626076 B CN 112626076B CN 202011337754 A CN202011337754 A CN 202011337754A CN 112626076 B CN112626076 B CN 112626076B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
Abstract
The application discloses application of a gene PeNAC67 in breeding of Phalaenopsis miniata, wherein the protein coded by the gene PeNAC67 has a NAM structural domain, and the sequence of the protein coded by the gene PeNAC67 is shown in SEQ ID NO. 3.
Description
Technical Field
The invention relates to the field of biological variety breeding, in particular to application of a gene PeNAC67 in breeding of Phalaenopsis plantulata.
Background
The Phalaenopsis (Phalaenopsis) belongs to the genus Phalaenopsis of the family Orchidaceae of the family Vanda subfamily of the family Vanophora of the subfamily orchidae, is originally produced in tropical rainforests, is epiphytic herbaceous, has developed fleshy roots, short stems, thick and flat leaf texture, bright color and peculiar flower type, can achieve the flowering phase of 90-150 days, is a treasure in tropical orchids, and is a prime name of 'queen in orchid'. It is widely cultivated and planted as one of important ornamental flowers in the world. The Phalaenopsis miniata (Phalaenopsis organization), also known as pink Phalaenopsis and Ji Phalaenopsis, is an orchid family (Orchidaceae) and a Phalaenopsis (Phalaenopsis), is native to the Philippine, is an excellent parent for breeding Phalaenopsis due to rich flower colors. The chromosome set of the plant is consistent with that of most phalaenopsis plants, is diploid (2n =2x = 38), is one of model species for researching the morphological development mechanism of orchids and is the first orchids which issue whole genome sequences, and provides an important basis for researching the diversity and evolution of orchids on the genome level. The flower of the butterfly orchid is similar to the structure of a typical orchid: unique bilateral symmetry structure, including 3 sepals, 2 petals, labial petals and highly evolved synanthic pillar (formed by fusion of male and female reproductive organs, with stamen at the top).
At present, researches on the development of flower organs mainly focus on model plants, namely arabidopsis thaliana and snapdragon, a classical 'ABCDE' model, and the fact that A + E jointly controls the formation of sepals, A + B + E jointly controls the formation of petals, B + C + E controls the formation of stamens, C + E controls the formation of carpels and D + E controls the formation of ovules. This model, although confirmed by the "quartering model", is not entirely applicable to all seed plants. The 'boundary sliding model' and 'boundary attenuation' which are subsequently proposed are complementary to the classical 'ABC' model. Orchid has a highly evolved structure, and none of the molecular models proposed above can reveal the development of orchid. The hypothesis of "orchid code" has been proposed and used by scientists to reveal orchid development, but the above hypothesis focuses mainly on the MADS-box gene.
With the improvement of living standard, plant flowers are more and more concerned, and different preferences of organ phenotypes of phalaenopsis (such as lip-flap traits or petal traits) also endow the phalaenopsis with remarkable economic benefits; however, the traditional breeding method is time-consuming and labor-consuming, and the flower phenotype meeting the market demand cannot be obtained efficiently, so that the technical personnel in the field are dedicated to providing a gene for regulating the flower type development of orchid organs and application thereof in breeding.
Disclosure of Invention
In view of the above defects of the prior art, the technical problem to be solved by the invention is how to efficiently obtain lipped or lipped phalaenopsis miniata.
To achieve the above object, the present invention provides a use of gene peaca 67 in breeding of phalaenopsis miniata, wherein the protein encoded by the gene peaca 67 has a NAM domain.
In certain embodiments, the protein sequence encoded by the gene petac 67 is set forth in SEQ ID No. 3.
In certain embodiments, the coding sequence of the gene petac 67 is obtained by PCR using the primer sequences shown in SEQ ID nos. 4 and 5.
In certain embodiments, the coding sequence of the gene peaca 67 is set forth in SEQ ID No.2.
In certain embodiments, the sequence of the gene petac 67 is set forth in SEQ ID No. 1.
In another aspect, the application also provides application of the protein in breeding of Phalaenopsis miniata, wherein the protein is encoded by a gene PeNAC67, and the sequence of the gene PeNAC67 is shown as SEQ ID No. 1.
In certain embodiments, the protein has the sequence shown in SEQ ID No. 3.
In another aspect, the application also provides a primer pair, the sequences of the primer pair are shown as SEQ ID No.9 and 10, and the primer pair is used for silencing PeNAC67 of the phalaenopsis miniata gene.
In another aspect, the application also provides a primer pair, the sequences of the primer pair are shown as SEQ ID No.9 and 10, and the primer pair is used for silencing the phalaenopsis miniata gene PeNAC67.
In another aspect, the present application also provides a method for lipping a natural mutant of phalaenopsis miniata, wherein the method comprises knocking out or silencing the gene peanc 67 in the natural mutant of phalaenopsis miniata.
In certain embodiments, silencing the gene peaca 67 in the natural mutant of phalaenopsis miniata comprises using a primer pair as set forth in SEQ ID nos. 9 and 10.
In certain embodiments, the protein sequence encoded by the gene peaca 67 is set forth in SEQ ID No. 3.
In certain embodiments, the coding sequence of the gene petac 67 is obtained by PCR using the primer sequences shown in SEQ ID nos. 4 and 5.
In certain embodiments, the coding sequence of the gene petac 67 is shown in SEQ ID No.2.
In certain embodiments, the sequence of the gene petac 67 is set forth in SEQ ID No. 1.
In another aspect, the application also provides a method for promoting the lip development of the butterfly orchid of the small orchid, which comprises introducing a gene PeNAC67 into the butterfly orchid of the small orchid, wherein the protein encoded by the gene PeNAC67 has a sequence shown in SEQ ID NO. 3.
In certain embodiments, the coding sequence of the gene peaca 67 is in a vector that enables its expression in phalaenopsis miniata.
According to the application, through the operation of the gene PeNAC67, the natural mutant gene PeNAC67 of the Phalaenopsis alata is silenced to remove lip valvification so as to obtain Phalaenopsis alata with a petal phenotype different from the existing phenotype, or the gene PeNAC67 is overexpressed in wild Phalaenopsis alata so as to obtain Phalaenopsis alata with different degrees of lip valvification different from the natural mutant, so that different market demands are met, the economic benefit of the Phalaenopsis alata is obviously improved, the method is efficient, rapid and low in cost, and the application prospect is wide.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
Fig. 1 shows a wild type of phalaenopsis miniata and a mutant of phalaenopsis miniata in the present application.
FIG. 2 shows a prediction map of the tertiary structure of the PeNAC67 protein in the present application.
Fig. 3 is a graph showing the results of analysis of the organ expression pattern of petac 67 in the present application in wild-type phalaenopsis miniata.
Fig. 4 is a graph showing the analysis results of different flowering-stage expression patterns of pelac 67 in wild-type phalaenopsis miniata.
FIG. 5 shows graphs analyzing the expression level of different PeNAC67 silencing lines and a control line PeNAC67 in the present application.
FIG. 6 shows a phenotype of a PeNAC67 silencing line with a control line in the present application.
Detailed Description
The present invention will now be further described with reference to the following examples, which are intended to be illustrative only, and the present invention may be embodied in many different forms of embodiments, and the scope of the present invention is not limited to the embodiments set forth herein.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The reagents used are commercially available or publicly available reagents unless otherwise specified.
In the present application, various vectors known in the art, such as commercially available vectors, including plasmids and the like, can be used.
The wild type of phalaenopsis miniata produces a natural mutant (peloric mutant) during natural growth, and compared with the wild type, 2 side petals are characterized in a structure similar to a lip petal (shown in fig. 1). In the application, the wild type of the butterfly orchid and the natural mutant of the butterfly orchid (peloric mutant) are both purchased from the small-city shrimps landscaping service department in east China of Dongguan city.
Example 1 cloning of the Phalaenopsis miniata Pennac 67 Gene
The wild type Oreochromia miniata petal total RNA is extracted by using a commercially available Kit Steadypure Plant RNA Extraction Kit, and the total RNA is reversely transcribed into cDNA by using a commercially available reverse transcription Kit. The reverse transcription cDNA is used as a template, and primer sequences shown as SEQ ID NO.4 and SEQ ID NO.5 are utilized to clone by PCR to obtain a 981bp strip of a gene coding region. The nucleotide sequence is shown as SEQ ID NO.2, the amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO.3, the nucleotide sequence consists of 321 amino acid residues, and the molecular weight is 37.1 kilodaltons. The gene PeNAC67 has a total length of 1352bp, a total-length nucleotide sequence table shown as SEQ ID NO.1, and a gene coding region sequence shown as SEQ ID NO.2.
Example 2 Phalaenopsis miniata PenNAC 67 protein bioinformatics analysis
(1) The pelac 67 protein sequence was placed in the NCBI database and homologous proteins were retrieved.
(2) The conserved domain between PeNAC67 and the homologous amino acid sequence is analyzed on line by applying https:// www.genome.jp/tools-bin/clustalw, and the result shows that the PeNAC67 transcription factor has a typical NAM domain.
(3) And selecting amino acid sequences of different species, applying MEGA5, and analyzing the phylogenetic relationship of the PenNAC 67, wherein the results show that the PenNAC 67 and the Orchidaceae plant, namely Dendrobium officinale, are clustered in the same branch, and belong to a monocotyledon branch.
(4) The tertiary structure of the PenAC67 protein is predicted by using https:// swisssmall.expasy.org/interactive, and as shown in figure 2, the tertiary structure of the PenAC67 protein is predicted to form a homodimer to play a role.
Example 3 analysis of organ expression patterns of wild type Phalaenopsis miniata PenNAC 67 gene
Respectively extracting total RNA in wild butterfly orchid root, flower branch, leaf, sepal, petal, lip and synanthus column, reversely transcribing the total RNA into cDNA by using a reverse transcription kit, and performing Real-time PCR detection by using primers SEQ ID NO.6 and SEQ ID NO.7 as shown in figure 3. The results show that the expression level of the gene in the reproductive organ is obviously higher than that of the vegetative organ.
Example 4 analysis of expression levels of wild type Phalaenopsis miniata PenNAC 67 Gene at different developmental stages of flower bud
Dividing the flower buds of the wild butterfly orchid into 5 periods according to the size of 0-1mm, 1-2mm, 2-5mm and 5-10mm, and marking the periods as B1, B2, B3, B4 and B5. The total RNA in the above 5 periods was extracted and reverse transcribed into cDNA, and Real-time PCR detection was performed using primers SEQ ID NO.6 and SEQ ID NO.7, as shown in FIG. 4. The results show that the expression level of the PeNAC67 is increased along with the development period of the flowers.
Example 5VIGS Down-Regulation of the Phalaenopsis miniata natural mutant (peloric mutant) PeNAC67 Gene restores lipped petals to the petal phenotype
(1) VIGS primers (shown as SEQ ID NO.9 and SEQ ID NO. 10) are designed according to SEQ ID NO.1, a CDS homologous fragment shown as SEQ ID NO.8 is cloned by taking cDNA as a template, and the fragment is connected to a pCymMV (purchased from Vitaceae, successfully university, taiwan, china) viral vector to form a recombinant vector.
(2) The recombinant vector is transformed into agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi Biotechnology Co., ltd.), and a GV3101 bacterial solution is obtained by culturing, and then 300 mu L of the bacterial solution is injected into the inflorescence of the natural mutant of the phalaenopsis miniata to silence the gene of the natural mutant of the phalaenopsis miniata, namely the PeNAC67 gene.
(3) After 4-5 weeks of injection, qPCR is carried out by taking SEQ ID NO.6 and SEQ ID NO.7 as primers to verify the expression condition of the PeNAC67 gene of the natural mutant of Phalaenopsis miniata.
(4) Observing the phenotype characteristics of the flower organs of the phalaenopsis miniata in each group, and taking a picture for recording.
Both steps (3) and (4) were performed in parallel with no injection of the natural mutant (peloric mutant).
As shown in FIG. 5, the expression of the Phalaenopsis miniata PeNAC67 gene was successfully reduced by VIGS, wherein pCymMV-PeNAC67-1 and pCymMV-PeNAC67-2 are experimental repeats. The VIGS-injected phalaenopsis miniata was observed, the plants grew well, and two differential phenotypes were found: petals become smaller (fig. 6C), and lipped petal sections restore the petal phenotype (fig. 6D, D). The result shows that the PenAC67 gene positively regulates the development of the lip valve of the butterfly orchid.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Shanghai university of Master
Application of <120> gene PeNAC67 in breeding of Phalaenopsis miniata
<130> CN015-20012PICN
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 1352
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PenAC67 full-Length sequence
<400> 1
ttctgcaacc atcctccccg cttactttga aacagggagg cctgccactc ttccatctac 60
accatgtcga acacctcacc ttcactcccg cctggcttcc gcttccaccc taccgacgag 120
gagctcatcc ttgattacct ctgcaagcga gcagcatcag ctccctgccc tgtcgccata 180
atcgctgaca tcaacatata caagttcaac ccatgggatc tcccagctaa agctaagttt 240
ggggagaggg aatggtattt cttcaccccc cgtgaccgca agtaccccaa tggagtccgg 300
ccgaaccggt ctgcagggtc tggctactgg aaagcaaccg gaacagacaa accgatcaca 360
acgagccgat ggaatgagaa cattggcgtt aagaaggctc tggtgttcta caaggggaag 420
cctcccaagg gcgtgaagac taactggatc atgcacgagt atagacttgc tgaaactctt 480
cacagcaatc aaaccaaaca gctgaagctt agagaaggat ctatgagact ggatgactgg 540
gttctctgcc gaatctatca gaaaaccaga catcattctt cggttgatcg cgagcaagac 600
gacgatgcag gaaccatgaa attcgcaaac tcgaacaaga ttcacaagtc tttctctgtt 660
tctgagttcc tggtcgagga tgctgatttc tctctgttat cagggttgct ggaaacaccc 720
atgaatgtgc cgttaaccaa tgatgatccc ttttctaact cgattttgaa ccattcattc 780
gtaaacgata tgggcaacag caatggcggc tgctacggca cgcaaccaaa tcaatcgcaa 840
gcttcagtat tggctgcaga acaaagcctg aagcgacaaa aaacggcgga taattgctat 900
tttgaggatt gtggaggtca actgaatcat tcgaagaagc tgaggaattt cagcattttt 960
gcccaattta acaaccagtt tgacatctct caacctttct tcaaccagca actgcagctg 1020
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ggacgttgat taaagagcaa attgctcatt ttatgagtct ctcacatact taatgaaaag 1140
gacttgtgca aggattttga gaggaaaaat ggatggaaaa ttgtttagct gcttcagttg 1200
tgccccaagg aaaagttgag ctggaaattg gagggtggga tttgcatgat taatcatgct 1260
atgtattttc taattatagt ttattttaat ttcttattcc ctttaaatta tctgtttctt 1320
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gctgacatca acatatacaa gttcaaccca tgggatctcc cagctaaagc taagtttggg 180
gagagggaat ggtatttctt caccccccgt gaccgcaagt accccaatgg agtccggccg 240
aaccggtctg cagggtctgg ctactggaaa gcaaccggaa cagacaaacc gatcacaacg 300
agccgatgga atgagaacat tggcgttaag aaggctctgg tgttctacaa ggggaagcct 360
cccaagggcg tgaagactaa ctggatcatg cacgagtata gacttgctga aactcttcac 420
agcaatcaaa ccaaacagct gaagcttaga gaaggatcta tgagactgga tgactgggtt 480
ctctgccgaa tctatcagaa aaccagacat cattcttcgg ttgatcgcga gcaagacgac 540
gatgcaggaa ccatgaaatt cgcaaactcg aacaagattc acaagtcttt ctctgtttct 600
gagttcctgg tcgaggatgc tgatttctct ctgttatcag ggttgctgga aacacccatg 660
aatgtgccgt taaccaatga tgatcccttt tctaactcga ttttgaacca ttcattcgta 720
aacgatatgg gcaacagcaa tggcggctgc tacggcacgc aaccaaatca atcgcaagct 780
tcagtattgg ctgcagaaca aagcctgaag cgacaaaaaa cggcggataa ttgctatttt 840
gaggattgtg gaggtcaact gaatcattcg aagaagctga ggaatttcag catttttgcc 900
caatttaaca accagtttga catctctcaa cctttcttca accagcaact gcagctgaat 960
tctcaattgg gattgcagtg a 981
<210> 3
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<223> protein sequence encoding PeNAC67
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Met Ser Asn Thr Ser Pro Ser Leu Pro Pro Gly Phe Arg Phe His Pro
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Thr Asp Glu Glu Leu Ile Leu Asp Tyr Leu Cys Lys Arg Ala Ala Ser
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Ala Pro Cys Pro Val Ala Ile Ile Ala Asp Ile Asn Ile Tyr Lys Phe
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Asn Pro Trp Asp Leu Pro Ala Lys Ala Lys Phe Gly Glu Arg Glu Trp
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Tyr Phe Phe Thr Pro Arg Asp Arg Lys Tyr Pro Asn Gly Val Arg Pro
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Asn Arg Ser Ala Gly Ser Gly Tyr Trp Lys Ala Thr Gly Thr Asp Lys
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Pro Ile Thr Thr Ser Arg Trp Asn Glu Asn Ile Gly Val Lys Lys Ala
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Leu Val Phe Tyr Lys Gly Lys Pro Pro Lys Gly Val Lys Thr Asn Trp
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Ile Met His Glu Tyr Arg Leu Ala Glu Thr Leu His Ser Asn Gln Thr
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Lys Gln Leu Lys Leu Arg Glu Gly Ser Met Arg Leu Asp Asp Trp Val
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Leu Cys Arg Ile Tyr Gln Lys Thr Arg His His Ser Ser Val Asp Arg
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Glu Gln Asp Asp Asp Ala Gly Thr Met Lys Phe Ala Asn Ser Asn Lys
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Ile His Lys Ser Phe Ser Val Ser Glu Phe Leu Val Glu Asp Ala Asp
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Phe Ser Leu Leu Ser Gly Leu Leu Glu Thr Pro Met Asn Val Pro Leu
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Thr Asn Asp Asp Pro Phe Ser Asn Ser Ile Leu Asn His Ser Phe Val
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Asn Asp Met Gly Asn Ser Asn Gly Gly Cys Tyr Gly Thr Gln Pro Asn
245 250 255
Gln Ser Gln Ala Ser Val Leu Ala Ala Glu Gln Ser Leu Lys Arg Gln
260 265 270
Lys Thr Ala Asp Asn Cys Tyr Phe Glu Asp Cys Gly Gly Gln Leu Asn
275 280 285
His Ser Lys Lys Leu Arg Asn Phe Ser Ile Phe Ala Gln Phe Asn Asn
290 295 300
Gln Phe Asp Ile Ser Gln Pro Phe Phe Asn Gln Gln Leu Gln Leu Asn
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Ser Gln Leu Gly Leu Gln
325
<210> 4
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<213> Artificial Sequence (Artificial Sequence)
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atgtcgaaca cctcaccttc actc 24
<210> 5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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tcactgcaat cccaattgag aattcag 27
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<400> 6
<210> 7
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<213> Artificial Sequence (Artificial Sequence)
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<223> reverse primer 2
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gagatgtcaa actggttgtt aaat 24
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<211> 406
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CDS homologous sequence
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gcgtgaagac taactggatc atgcacgagt atagacttgc tgaaactctt cacagcaatc 60
aaaccaaaca gctgaagctt agagaaggat ctatgagact ggatgactgg gttctctgcc 120
gaatctatca gaaaaccaga catcattctt cggttgatcg cgagcaagac gacgatgcag 180
gaaccatgaa attcgcaaac tcgaacaaga ttcacaagtc tttctctgtt tctgagttcc 240
tggtcgagga tgctgatttc tctctgttat cagggttgct ggaaacaccc atgaatgtgc 300
cgttaaccaa tgatgatccc ttttctaact cgattttgaa ccattcattc gtaaacgata 360
tgggcaacag caatggcggc tgctacggca cgcaaccaaa tcaatc 406
<210> 9
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Forward primer 3
<400> 9
ggggacaagt ttgtacaaaa aagcaggctg cgtgaagact aactggatca tgc 53
<210> 10
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> reverse primer 3
<400> 10
ggggaccact ttgtacaaga aagctgggtg attgatttgg ttgcgtgccg tag 53
Claims (5)
1. GenePeNAC67Application in breeding of Phalaenopsis miniata, wherein the genePeNAC67Has a NAM domain, and the genePeNAC67The sequence is shown as SEQ ID NO. 1; the application refers to knocking out or silencing genes in natural mutants of the phalaenopsis miniataPeNAC67And (4) lip-removing of the natural mutant of the butterfly orchid of the small orchid.
2. The use of claim 1, wherein the gene isPeNAC67The coding sequence of (A) is shown in SEQ ID NO.2.
3. Application of protein in breeding of Phalaenopsis miniata, wherein the protein is formed by genesPeNAC67Code, the genePeNAC67The sequence is shown as SEQ ID NO.1, and the application refers to knocking out or silencing genes in the natural mutant of phalaenopsis miniataPeNAC67And (4) lip-removing of the natural mutant of the butterfly orchid of the small orchid.
4. A method for lip-lipping a natural mutant of butterfly orchid of mini orchid, wherein the method comprises knocking out or silencing a gene in the natural mutant of butterfly orchid of mini orchidPeNAC67，The genePeNAC67The sequence is shown as SEQ ID NO. 1.
5. The method of claim 4, wherein the silencing of the gene in the natural mutant of butterfly orchid is performed in the Yuan orchidPeNAC67The method comprises the following steps:
(1) Designing VIGS primers according to SEQ ID NO.1, wherein the VIGS primers are shown as SEQ ID NO.9 and SEQ ID NO.10, cloning CDS homologous fragments shown as SEQ ID NO.8 by taking cDNA as a template, and connecting the CDS homologous fragments to a pCymMV viral vector to form a recombinant vector;
(2) Transforming the recombinant vector into agrobacterium tumefaciens GV3101, culturing to obtain GV3101 bacterial liquid, injecting 300 mu L of the bacterial liquid into the inflorescence of the natural mutant of the phalaenopsis miniorcum, and silencing the natural mutant of the phalaenopsis miniorcumPeNAC67A gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011337754.8A CN112626076B (en) | 2020-11-25 | 2020-11-25 | Application of gene PeNAC67 in breeding of Phalaenopsis miniata |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011337754.8A CN112626076B (en) | 2020-11-25 | 2020-11-25 | Application of gene PeNAC67 in breeding of Phalaenopsis miniata |
Publications (2)
| Publication Number | Publication Date |
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| CN112626076A CN112626076A (en) | 2021-04-09 |
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| CN113789318B (en) * | 2021-08-31 | 2023-10-27 | 上海师范大学 | Gene PeFtsH5 and application thereof in improvement of pathogenic bacteria infection resistance of butterfly orchid |
| CN114774428B (en) * | 2022-03-30 | 2023-09-29 | 上海师范大学 | Gene PeKAN2 for regulating and controlling organ development of small orchid butterfly orchid and application thereof |
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| CN101200722A (en) * | 2007-10-18 | 2008-06-18 | 复旦大学 | Butterfly orchid ORAP11 gene coded sequence and uses thereof |
| CN101935342A (en) * | 2009-07-02 | 2011-01-05 | 复旦大学 | Moth orchid development B gene-PhAP3 coded sequence and application thereof |
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| WO2005112608A2 (en) * | 2004-05-21 | 2005-12-01 | Michigan State University | Modified plant transcription factors |
| CN101200722A (en) * | 2007-10-18 | 2008-06-18 | 复旦大学 | Butterfly orchid ORAP11 gene coded sequence and uses thereof |
| CN101935342A (en) * | 2009-07-02 | 2011-01-05 | 复旦大学 | Moth orchid development B gene-PhAP3 coded sequence and application thereof |
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