CN116987168B - Method capable of stably changing leaf color of orchid - Google Patents
Method capable of stably changing leaf color of orchid Download PDFInfo
- Publication number
- CN116987168B CN116987168B CN202311245681.3A CN202311245681A CN116987168B CN 116987168 B CN116987168 B CN 116987168B CN 202311245681 A CN202311245681 A CN 202311245681A CN 116987168 B CN116987168 B CN 116987168B
- Authority
- CN
- China
- Prior art keywords
- myb
- ccr2r3
- gene
- orchid
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 20
- 241000233855 Orchidaceae Species 0.000 title abstract description 29
- 241000196324 Embryophyta Species 0.000 claims abstract description 56
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 230000009261 transgenic effect Effects 0.000 claims abstract description 22
- 241000026010 Dendrobium candidum Species 0.000 claims abstract description 18
- 241000589158 Agrobacterium Species 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 13
- 239000013598 vector Substances 0.000 claims description 20
- 230000009466 transformation Effects 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 5
- 230000002068 genetic effect Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 3
- 230000002018 overexpression Effects 0.000 abstract description 10
- 230000000243 photosynthetic effect Effects 0.000 abstract description 4
- 239000000049 pigment Substances 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 241001523681 Dendrobium Species 0.000 abstract 1
- 230000008635 plant growth Effects 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 235000021466 carotenoid Nutrition 0.000 description 4
- 150000001747 carotenoids Chemical class 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 240000004638 Dendrobium nobile Species 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 229930002868 chlorophyll a Natural products 0.000 description 3
- 229930002869 chlorophyll b Natural products 0.000 description 3
- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003208 gene overexpression Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000004960 subcellular localization Effects 0.000 description 3
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- 241001076416 Dendrobium tosaense Species 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229930003935 flavonoid Natural products 0.000 description 2
- 150000002215 flavonoids Chemical class 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 101150089559 MYB1 gene Proteins 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 241000207746 Nicotiana benthamiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000358848 Orchidantha maxillarioides Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001505935 Phalaenopsis Species 0.000 description 1
- 244000127818 Phalaenopsis amabilis Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 101100368917 Schizosaccharomyces pombe (strain 972 / ATCC 24843) taz1 gene Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 101100186009 Xenopus laevis mybl2 gene Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000005200 bud stage Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- QDOXWKRWXJOMAK-UHFFFAOYSA-N dichromium trioxide Chemical compound O=[Cr]O[Cr]=O QDOXWKRWXJOMAK-UHFFFAOYSA-N 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention belongs to the technical field of plant genetic engineering, and particularly discloses a method capable of stably changing the color character of orchid leaves, which comprises the following steps ofCcR2R3‑MYBGene application in plant leaf color trait improvement, saidCcR2R3‑MYBThe nucleotide sequence is shown as SEQ ID NO. 1, and the invention clones out from She Banyi orchidCcR2R3‑MYBThe gene is subjected to over-expression in the dendrobium candidum, and the dendrobium candidum is transformed through agrobacterium mediation, and experimental results show that the gene is in the over-expression transgenic dendrobium candidumCcR2R3‑MYBThe gene can be expressed normally and transferred into over-expressionCcR2R3‑MYBThe plant growth condition of the gene is yellow-green compared with the control plant, and the plant leaf color is over-expressedCcR2R3‑MYBThe gene can reduce photosynthetic pigment in leaf to obtain new plant leaf color specificity character changing type.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method capable of stably changing the color traits of orchid leaves.
Background
Orchid is a monocotyledonous plant with higher evolution degree, is one of the largest flowering families of angiosperms, and belongs to more than about 700 species and 2.5 thousands. Orchid is a plant of Orchidaceae (Orchidaceae), which is mainly distributed in tropical and subtropical areas, and has extremely high ornamental value, cultural value and medicinal value. The orchid has unique plant type, beautiful flower appearance, rich flower color, fragrant flower and long flower period, is an important cut flower and potted flower, and along with the development of technology, the improvement of the human ornamental level, and the demand of people on excellent orchid varieties is also increasing. The orchid is bred for a long time, a plurality of variant varieties are generated, and the variety is divided into two types of flower art and leaf art according to different ornamental positions, wherein She Banyi orchid originates from China and is gradually recognized in recent years. "leaf art" is known horticulturally as She Bucai plaque (variegation) and is an important component of ornamental plant leaf variation. The orchid leaf spot art variety has high ornamental value and is an important characteristic of 'exquisite orchid'. In the Ming dynasty Yong orchid poem, there is a good sentence of looking at the leaf and flower, people pursue the orchid flower art and pursue the beautiful appearance of orchid plants, she Yilan is pursued from ancient times by virtue of the ancient, thick, short and small plant types and various singular leaf forms, and the unique leaf spot property is valued and selected as the cultivation key point. At present, she Yilan is mostly from natural filial generation of wild orchid plants or natural variation plants in a long-term cultivation process, so She Yilan breeding has the bottleneck problems of extremely rare quantity, long breeding time and the like, and the statue is also generally unstable, so that She Yilan directional cultivation is extremely difficult. In recent years, with the cultivation of a new variety of She Yilan and the deep appreciation of She Yilan by the public, new types of different colors and shape variations appear. However, the traditional crossbreeding has the defects of long time consumption, reproductive isolation and the like, and is difficult to meet the current flower market, so that molecular technology is used for cultivating new varieties of orchids with different leaf colors, the breeding period can be shortened, the interspecific limit can be broken, and a convenient way is provided for orchid germplasm innovation and character improvement.
Aiming at the genotype capable of stably changing the leaf color of orchid, no report is available at present.
Disclosure of Invention
The main purpose of the invention is to provide a method for stably changing the leaf color of orchid, which uses plant genetic engineering technology to genetically transform dendrobium candidum, compared with the leaf green of Wild (WT) plant, the leaf color of transgenic plant is yellow-green, the photosynthetic pigment (chlorophyll a, chlorophyll b and carotenoid) content of WT plant leaf is higher than that of transgenic plant, and the difference is very obvious. In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of a leaf spot orchid CcR R3-MYB gene in plant leaf color trait improvement, wherein the CcR R3-MYB nucleotide sequence is shown as SEQ ID NO. 1.
Further, the present invention provides a method for stably changing the leaf color of orchid, comprising the steps of:
(1) Amplifying She Banyi orchid CcR R3-MYB genes, wherein the nucleotide sequence of the CcR R3-MYB genes is shown as SEQ ID NO. 1;
(2) Constructing a recombinant vector;
(3) Agrobacterium-mediated genetic transformation;
(4) And culturing and detecting transgenic plants.
Preferably, in the step (1), the nucleotide sequence of the primer pair used for amplifying the She Banyi orchid CcR2R3-MYB gene is shown as SEQ ID NO. 3-4.
Preferably, in step (2), the starting vector used to construct the recombinant vector is pCAMBIA1301, containing a 35S promoter followed by a CcR R3-MYB gene.
Preferably, in step (3), the agrobacterium is EHA105.
The invention also provides a recombinant vector which contains a nucleotide sequence shown as SEQ ID NO. 1.
The invention also provides a kit which contains a primer pair for amplifying the nucleotide sequence shown as SEQ ID NO. 1.
As one embodiment, the amino acid sequence shown in SEQ ID NO. 4, which is obtained by encoding the CcR R3-MYB nucleotide sequence shown in SEQ ID NO. 2, can be applied to plant leaf color trait improvement.
The invention clones CcR R3-MYB genes from She Banyi orchids by utilizing a degenerate primer design and full-length amplification method, performs over-expression in dendrobium candidum, and genetically transforms Arabidopsis thaliana by using an agrobacterium-mediated inflorescence infiltration method, and experimental results show that the over-expression transgenic dendrobium candidum CcR R3-MYB genes can be normally expressed, the growth condition of the over-expression CcR R3-MYB genes is expressed as yellow green than that of a control plant, and the over-expression CcR R3-MYB genes can greatly reduce the contents of chlorophyll a, chlorophyll b and carotenoid.
Drawings
FIG. 1 shows a gel electrophoresis display of CcR R3-MYB gene before and after amplification (M: maker2000bp; M: DL2000,1-4: PCR product);
FIG. 2 shows a graph of homology alignment of CcR R3-MYB with R2R3-MYB of other plants (CcR R3-MYB: she Banyi blue; dhMYB1: dendrobium nobile; dsp.MYB1: dendrobium nobile variety; dcMYBML1: dendrobium nobile; cemyb-related: jian blue; peMYB16: phalaenopsis, taMYB16: wheat; taMYB 77):
wheat; pemyb-related: small orchid butterfly orchid; atMYB106: arabidopsis thaliana);
FIG. 3 is a chart of subcellular localization of CcR2R3-MYB genes (GFP: fluorescence image; DIC: differential interference contrast;
MERGE: merging the images; scale = 50 μm);
FIG. 4 CcR2R3—MYB gene colony PCR (M: DL2000, 1: PCR product);
FIG. 5 shows an electrophoresis chart of CcR2R3-MYB gene overexpression positive plants (M: DL2000, -: negative control, +: positive control, A:1-8: PCR products (primers: hpt557-F and hpt 557-R), B:1-4:
PCR products (primer: ccR R3-MYB-F and CcR R3-MYB-R);
FIG. 6 is a diagram of genetic transformation process;
fig. 7 ccr2r3—myb gene expression levels (××representing p < 0.0001);
FIG. 8 transgenic plant Total Flavonoids content (different lower case letters indicate significance of difference (p < 0.05), no
The same capital letters indicate the difference is extremely significant (p < 0.01));
FIG. 9 comparison of transgenic plants (WT: wild type Dendrobium officinale leaf; ccR R3-MYB transgenic plant leaf; ccR R3-MYB transgenic plant);
FIG. 10 photosynthetic pigment content (different capital letters indicate differential extreme significance (p < 0.01));
FIG. 11 is a diagram of a CcR2R3-MYB gene overexpression vector (A: PC1300nu-CcR2R 3-MYB).
Detailed Description
In order to make the technical spirit and advantages of the present invention more clearly understood, the applicant will now make a detailed description by way of example, but the description of the examples is not intended to limit the scope of the invention, and any equivalent transformation made merely in form, not essentially, according to the inventive concept should be regarded as the scope of the technical solution of the present invention.
In order to avoid unnecessary detail, known techniques will not be described in detail in the following embodiments. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.
Example 1
She Banyi orchid CcR R3-MYB gene has a nucleotide sequence shown as SEQ ID NO. 1, ORF Finder analysis shows that CcR R3-MYB gene has a longer and complete open reading frame, CDS is 1101bp, 367 amino acids are encoded altogether, the amino acid sequence is shown as SEQ ID NO. 2, and the total average hydrophilic coefficient of protein is-0.778 after ExPASy analysis, so that CcR R3-MYB protein is presumed to be an unstable hydrophilic protein, and CcR R3-MYB protein has NO transmembrane structure.
1. The recombinant vector pCAMBIA1300-35S-CcR R3-MYB (FIG. 11) was constructed by the following method:
(1) Amplification of She Banyi orchid CcR R3-MYB Gene
RNA was extracted from leaves of She Banyi orchid (step reference to RNA extraction kit) and its concentration and purity were detected by a nucleic acid concentration detector, and reverse transcribed into cDNA according to kit instructions. Blast is respectively carried out on She Banyi blue CcR R3-MYB gene EST sequences to respectively obtain a gene dendrobe hybrid DhMYB1 (accession number: JX 846911.1) with highest homology, specific primers are respectively designed and synthesized by using Primer 5.0 software, target bands are amplified by a PCR technology (figure 1), and the bioinformatics analysis results show that: the genes all contained MYB-DNA-binding and 2 SANT conserved domains, which were presumed to belong to the R2R3-MYB family (FIG. 2).
(1) PCR primer
Designing a primer:
CcR2R3-MYB-F（SEQ ID NO:3）：
AGGTCGACTCTAGAGGATCCATGGGCCGTTCCCCCTG；
CcR2R3-MYB-R（SEQ ID NO:4）：
ATTCGAGCTGGTCAGAGCTCCTAGAACACAGGTGATGGTGAATT；
hygromycin (Hyg) primer sequence:
hpt557-F：ACACTACATGGCGTGATTTCAT
hpt557-R：TCCACTATCGGCGAGTACTTCT
the primer sequence of the dendrobium candidum GAPDH internal reference gene:
GAPDH-F：GGCGACTCCCCTCACTACTA
GAPDH-R：CAGGCATCTCATTGCCCAGA
(2) PCR was performed using the above reverse transcription product, and the system was as follows: 10xPCR buffer 5.0 ul dNTP mix (10 mM) 1.0ul, primer F (10. Mu.M) 1.0ul, primer R (10. Mu.M) 1.0ul, DNA 1.0ul, KOD (1U/. Mu.L) 1.0ul and ddH2O 40.0 ul, total capacity 50ul;
(3) the PCR reaction procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 98℃for 30 sec, annealing at 56℃for 30 sec, extension at 68℃for 2min,32 cycles; the reaction was terminated by extension at 68℃for 5min and at 4 ℃.
(2) Construction of CcR R3-MYB Gene plant overexpression vector pCAMBIA1300-35S-CcR R3-MYB
The PCR products were ligated to PC1300nu vector (Hyg tag with 557 bp), and the pCAMBIA1300-35S vector was digested with BamHI/SalI and recovered for purification, and reacted at 37℃for 30 min. The vector after enzyme digestion is respectively mixed with PCR amplification products of CcR R3-MYB, and a pCAMBIA1300-35S-CcR R3-MYB vector is constructed by using a seamless cloning method and reacted for 30 min at 37 ℃.
(1) The carrier enzyme digestion system is as follows: pCAMBIA1300-35S 16 ul, 10X FastDigest buffer 2 ul, fastDiget BamHI 1ul, fastDiget SalI 1ul, total capacity 20 ul;
(2) the PCR product and the enzyme digestion carrier are used for seamless connection, and the reaction system is as follows: PCR product 4 ul, cut vector 4 ul, assembly Enzyme 1ul, 10x Assembly Buffer 1ul, total capacity 10 ul;
transforming the plant over-expression vector into escherichia coli, screening out recombinants through enzyme digestion identification, constructing a recombinant vector pCAMBIA1300-35S-CcR R3-MYB expressed by the plant, obtaining a recombinant plasmid, enabling a target gene fragment to be clear, enabling the size to be consistent with an expected result (1101 bp), and successfully constructing the over-expression vector without non-specific amplified bands; FIG. 4 is a graph of colony detection of CcR R3-MYB gene overexpression vectors.
3. R2R3-MYB gene subcellular localization
The subcellular localization results show that fluorescence is mainly distributed on plasma membranes and cytoplasm in tobacco leaf cells with empty vector containing GFP fluorescent protein as a control, and the constructed fusion expression vector is transformed into Nicotiana benthamiana leaves, so that green fluorescence is found to coincide with autofluorescence of cell nuclei, and CcR R3-MYB genes (shown in figure 3) are all localized on the cell nuclei.
4. Genetic transformation of Agrobacterium and detection of transformants
Competent cells of Agrobacterium were prepared, the plant expression vector pCAMBIA1300-35S-CcR2R3-MYB constructed above was transferred into Agrobacterium (EHA 105) by electric pulse method, and colonies of transformants were selected on plates with hygromycin. The lysate of agrobacterium colony is used as template for PCR reaction, specific primer CcR R2R3-MYB-BamHI F and CcR R3-MYB-SalI R of CcR R3-MYB gene are used as PCR detection, and the transformant colony confirmed by colony PCR is used for transforming plant.
5. Transformation of plants with agrobacterium overexpressed by plants containing CcR R3-MYB gene
The single colony of agrobacterium carrying plasmid pCAMBIA1300-35S-CcR2R3-MYB is selected and inoculated in a liquid culture medium for culture, the thalli are collected by centrifugation and then suspended by an MS liquid culture medium. The plant tissue which is easy to differentiate is infected by suspended agrobacterium, then the transgenic seedling is obtained by tissue culture, and then the transgenic plant is obtained by screening with antibiotics, so that the positive plant or protocorm is obtained, and the genetic transformation process diagram is shown in figure 6.
6. Insertion condition of CcR R3-MYB gene in transgenic plant and detection of expression quantity
To confirm that the transgenic plants obtained by the selection did contain CcR R3-MYB genes, the transgenic plants selected were further identified using the 2XT5 Direct PCR Kit (Plant) kit. Setting untreated wild dendrobium candidum plant as negative control, over-expression vector (glycerinum) as positive control, designing primer amplification fragment with hygromycin sequence and target gene sequence. The result of gel electrophoresis photographing shows that (FIG. 5), the negative control (-) has no band, the positive control (+) has bands (hpt 557-F and hpt557-R:557 bp, ccR2R3-MYB-F and CcR R3-MYB-R:1638 bp), the positions of the bands of the PCR products are consistent with those of the positive control bands, the bands are clear, and the CcR R2R3-MYB gene is successfully transformed into dendrobium candidum.
And selecting cluster buds of wild dendrobium candidum (WT) and CcR R3-MYB gene over-expressed dendrobium candidum as materials, and detecting the expression quantity of the genes. The GAPDH is taken as an internal reference gene (primers: GAPDH-F and GAPDH-R), and a specific primer (CcR R3-MYB-F and CcR R3-MYB-R) is used for qRT-PCR (SYBR Premix Ex Taq Kit), so that the identification is that the expression quantity of the GAPDH is positive, and the identification result is shown in FIG. 7, wherein the expression quantity of CcR R3-MYB genes in a transgenic plant is extremely obviously higher than that of 210% of homologous genes of wild dendrobium officinale (WT). The result shows that the CcR R3-MYB genes are successfully transferred into the dendrobium candidum, and reach higher expression level in the plant cluster bud stage.
7. Experiment of variation of morphology and physiological index of transgenic plant
The transgenic and control wild type protocorms are respectively cultivated in a culture medium to form seedlings, and the plant dendrobium candidum plants which are over-expressed with CcR R3-MYB genes are subjected to measurement of various physiological indexes, so that measurement results of figures 8, 9 and 10 are obtained, and experimental results show that CcR R3-MYB genes in the transgenic over-expressed dendrobium candidum can be normally expressed, and compared with the control plants (non-transgenic wild type, WT), the growth condition of the transgenic CcR R3-MYB genes is extremely higher than that of the control plants, and the expression quantity of CcR R3-MYB genes in the transgenic plants is extremely higher than that of 210 percent of WT plants; the total flavone content of the WT plant is extremely higher than that of a CcR R3-MYB plant by 37.4 percent. The leaf color of the transformed CcR R3-MYB plant is Yellow-Green (Strong Yellow Green A), the leaf color of the WT plant is Green (Green Group, deep Yellowish Green A), and the leaf colors are obviously different. The content of photosynthetic pigment (chlorophyll a, chlorophyll b and carotenoid) of the WT plant is higher than that of the CcR R3-MYB plant, and the difference is very remarkable. In conclusion, the She Banyi blue CcR R3-MYB genes are shown to successfully express the dendrobium candidum in a heterologous manner, the expression quantity is high, and the genes can be involved in chlorophyll, carotenoid and flavonoid metabolism, so that the color development of plant leaves is affected.
Claims (8)
1. She Banyi orchidCcR2R3-MYBThe application of the gene in the improvement of the leaf color character of dendrobium candidum is characterized in thatCcR2R3-MYBThe nucleotide sequence is shown as SEQ ID NO. 1.
2. The dendrobium candidum leaf color character improvement method is characterized by comprising the following steps of:
(1) She Banyi orchidCcR2R3-MYBAmplification of genes, saidCcR2R3-MYBThe nucleotide sequence of the gene is shown as SEQ ID NO. 1;
(2) Constructing a recombinant vector;
(3) Agrobacterium-mediated genetic transformation;
(4) Transgenic plant cultivation and detection.
3. The method according to claim 2,characterized in that in the step (1), she Banyi orchidCcR2R3-MYBThe nucleotide sequence of the primer pair used for amplifying the gene is shown as SEQ ID NO. 3-4.
4. The method according to claim 2, wherein in step (2), the starting vector for constructing the recombinant vector is pCAMBIA1301, which comprises a 35S promoter followed immediately thereafterCcR2R3-MYBAnd (3) a gene.
5. The method of claim 2, wherein in step (3), the agrobacterium is EHA105.
6. The application of the recombinant vector in the improvement of the leaf color character of dendrobium candidum is characterized in that the recombinant vector contains a nucleotide sequence shown as SEQ ID NO. 1.
7. The application of the kit in improvement of leaf color traits of dendrobium candidum is characterized in that the kit contains a primer pair for amplifying a nucleotide sequence shown as SEQ ID NO. 1.
8. The application of the protein shown in SEQ ID NO. 2 in improving the leaf color character of dendrobium candidum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311245681.3A CN116987168B (en) | 2023-09-26 | 2023-09-26 | Method capable of stably changing leaf color of orchid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311245681.3A CN116987168B (en) | 2023-09-26 | 2023-09-26 | Method capable of stably changing leaf color of orchid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116987168A CN116987168A (en) | 2023-11-03 |
| CN116987168B true CN116987168B (en) | 2023-12-12 |
Family
ID=88525110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311245681.3A Active CN116987168B (en) | 2023-09-26 | 2023-09-26 | Method capable of stably changing leaf color of orchid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116987168B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001050A2 (en) * | 2003-06-06 | 2005-01-06 | Arborgen, Llc. | Transcription factors |
| CN105132433A (en) * | 2015-07-12 | 2015-12-09 | 东北师范大学 | cDNA sequence of MYB transcription factors for positively regulating anthocyanin synthesis |
| EP2984179A1 (en) * | 2013-04-08 | 2016-02-17 | Malaysian Palm Oil Board | Gene controlling fruit color phenotype in palm |
| CN106749578A (en) * | 2017-01-26 | 2017-05-31 | 中国热带农业科学院热带作物品种资源研究所 | Dendrobium myb transcription factor and its encoding proteins and application |
| CN107723297A (en) * | 2017-11-17 | 2018-02-23 | 河南省农业科学院 | Iris R3 MYBx1 genes and its application in pattern regulation |
| CN110628781A (en) * | 2019-10-17 | 2019-12-31 | 中国林业科学研究院林业研究所 | R2R3MYB transcription factor for promoting anthocyanin formation in orchid |
| CN111778261A (en) * | 2020-07-31 | 2020-10-16 | 东北师范大学 | Clone and application of rice salt-tolerant stress gene OsMYB106 |
| CN114230651A (en) * | 2021-12-30 | 2022-03-25 | 中国热带农业科学院热带作物品种资源研究所 | Method for instantaneously changing color of dendrobium nobile by using DhMYB2 gene |
| WO2022072335A2 (en) * | 2020-09-30 | 2022-04-07 | Pioneer Hi-Bred International, Inc. | Rapid transformation of monocot leaf explants |
| CN116200424A (en) * | 2022-08-09 | 2023-06-02 | 云南农业大学 | Application of She Banyi orchid CcMYB24 gene |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140041073A1 (en) * | 2012-08-03 | 2014-02-06 | Mendel Biotechnology, Inc. | Trait improvement in plants expressing myb-related proteins |
| US9284537B2 (en) * | 2013-04-15 | 2016-03-15 | The United States Of America, As Represented By The Secretary Of Agriculture | Cytochrome P450 enzymes from Sorghum bicolor |
-
2023
- 2023-09-26 CN CN202311245681.3A patent/CN116987168B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005001050A2 (en) * | 2003-06-06 | 2005-01-06 | Arborgen, Llc. | Transcription factors |
| EP2984179A1 (en) * | 2013-04-08 | 2016-02-17 | Malaysian Palm Oil Board | Gene controlling fruit color phenotype in palm |
| CN105132433A (en) * | 2015-07-12 | 2015-12-09 | 东北师范大学 | cDNA sequence of MYB transcription factors for positively regulating anthocyanin synthesis |
| CN106749578A (en) * | 2017-01-26 | 2017-05-31 | 中国热带农业科学院热带作物品种资源研究所 | Dendrobium myb transcription factor and its encoding proteins and application |
| CN107723297A (en) * | 2017-11-17 | 2018-02-23 | 河南省农业科学院 | Iris R3 MYBx1 genes and its application in pattern regulation |
| CN110628781A (en) * | 2019-10-17 | 2019-12-31 | 中国林业科学研究院林业研究所 | R2R3MYB transcription factor for promoting anthocyanin formation in orchid |
| CN111778261A (en) * | 2020-07-31 | 2020-10-16 | 东北师范大学 | Clone and application of rice salt-tolerant stress gene OsMYB106 |
| WO2022072335A2 (en) * | 2020-09-30 | 2022-04-07 | Pioneer Hi-Bred International, Inc. | Rapid transformation of monocot leaf explants |
| CN114230651A (en) * | 2021-12-30 | 2022-03-25 | 中国热带农业科学院热带作物品种资源研究所 | Method for instantaneously changing color of dendrobium nobile by using DhMYB2 gene |
| CN116200424A (en) * | 2022-08-09 | 2023-06-02 | 云南农业大学 | Application of She Banyi orchid CcMYB24 gene |
Non-Patent Citations (4)
| Title |
|---|
| An investigation of the roles of MYB transcription factors in orchid dendrobium hybrida flower development;Lau Su Ee;FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR;第1-112页 * |
| R2R3-MYB transcription factor [Dendrobium hybrid cultivar];GenBank DataBase;GenBank DataBase;ACCESSION NO.AFX97765 * |
| R2R3-MYB转录因子响应植物抗逆机制研究进展;王浩田等;分子植物育种;"3展望"部分 * |
| 小兰屿蝴蝶兰R2R3-MYB转录因子分析;王雪霁等;林业科学研究;第31卷(第3期);第104-113页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116987168A (en) | 2023-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9309527B2 (en) | Protein IPA1 related to plant architecture, its coding genes and uses | |
| US20150267219A1 (en) | Heat-resistance rice gene oszfp, screening marker and separation method thereof | |
| CN109111514B (en) | Method for cultivating transgenic wheat with resistance to sheath blight and root rot and related biological material thereof | |
| CN110938617A (en) | Lilium regale LrPAL-1 gene and application thereof | |
| CN112457380A (en) | Protein for regulating and controlling content of fruit shape and/or fruit juice of plant, related biological material and application thereof | |
| EP1457564B1 (en) | Production of plants having improved rooting efficiency and vase life using stress-resistance gene | |
| JP2016533179A (en) | Corn regulatory elements and uses thereof | |
| CN110204600B (en) | BnSPL14 gene, protein and application thereof in controlling cabbage type rape plant type | |
| CN116200424B (en) | Application of She Banyi orchid CcMYB24 gene | |
| CN116987168B (en) | Method capable of stably changing leaf color of orchid | |
| CN114836437B (en) | Application of peony PsMYB4 gene in changing color and size of plant petal color spots | |
| CN113881685B (en) | Gene PpHSP20-like1 for promoting plant organ to produce red color and application thereof | |
| CN102477092A (en) | Protein used for controlling anthocyanidin content, coding gene thereof, and application thereof | |
| JP2016536978A (en) | Maize (Zea mays) metallothionein-like regulatory elements and uses thereof | |
| JP2016536979A (en) | Corn regulatory elements and uses thereof | |
| US11390877B2 (en) | Blue-grained genes in wheat and application thereof | |
| US8143478B2 (en) | Peptide transporting to chromoplasts in petals and method of constructing plant having yellowish petals by using the same | |
| CN114524868B (en) | Sweet potato leaf development and flavonoid enhancement related protein IbBBX29 and coding gene and application thereof | |
| CN114807160B (en) | Gene RcGT for regulating and controlling petal color of China rose, protein, recombinant vector, recombinant transformant, application and method | |
| JP4472344B2 (en) | Plastid transformation method using microbial recombination enzyme | |
| JP4505628B2 (en) | Promoter with leaf-specific expression activity | |
| CN116514942A (en) | Protein for regulating anthocyanin synthesis and fruit size in peony and encoding gene thereof | |
| CN116042692A (en) | Genetic transformation method of hemerocallis and application thereof | |
| CN116462744A (en) | Protein for regulating flower and fruit traits by peony response to optical signals, coding gene PsCIP7 and application | |
| CN104975025B (en) | The identification and application of plant anther specific expression promoter pTaASG033 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |