CN105820224A - Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof - Google Patents
Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof Download PDFInfo
- Publication number
- CN105820224A CN105820224A CN201610293357.2A CN201610293357A CN105820224A CN 105820224 A CN105820224 A CN 105820224A CN 201610293357 A CN201610293357 A CN 201610293357A CN 105820224 A CN105820224 A CN 105820224A
- Authority
- CN
- China
- Prior art keywords
- plant
- protein
- sequence
- msmyb111
- gene
- 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.)
- Pending
Links
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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses anthocyanin-regulation protein MsMYB111 from a functional apple and an encoding gene and application thereof. The protein is obtained from the apple and named as the protein MsMYB111, and is (a1) or (a2) as follows: (a1) is the protein composed of the amino acid sequences shown in sequence 1 in a sequence table, and (a2) is the protein obtained in a mode of substitution and/or deletion and/or addition of one or more amino acid residues in the amino acid sequence in sequence 1, related to the content of plant anthocyanin and derived from sequence 1. The gene (gene MsMYB111) for encoding the protein MsMYB111 also belongs to the protective range of the invention. The invention further discloses application of the protein MsMYB111. The application is (b1) or (b2) as follows: (b1) regulates the anthocyanin content of a plant and (b2) reduces the anthocyanin content of the plant. The protein MsMYB111 and the functions thereof are found, and the protein MsMYB111 can be used for cultivating transgenic plants with changed anthocyanin contents and has great application prospects in plant breeding.
Description
Technical field
The present invention relates to a kind of anthocyanin modulin MsMYB111 available from functional type Fructus Mali pumilae and encoding gene thereof and application.
Background technology
" doctor's food homology " is developing direction.Fructus Mali pumilae storage property is good, supply cycle is long, it it is worldwide fruit, especially fruit contain higher proportion, human body be easier absorb free polyphenol, there is good antioxidation, antitumor, prevention cardiovascular and cerebrovascular disease and the effect such as protect the liver, healthy nutritive value is high, has " one day Fructus Mali pumilae, doctor is away from me " (Anappleadaykeepsthedoctoraway！) good reputation, the most considerable country is all classified as major consumers fruit and is recommended energetically.But finding in recent years shows, on the one hand, at more than 1000 apple variety being bred as in the past few decades both at home and abroad, 80% being the hybridization of kinds such as ' Jin Shuai ', grow directly from seeds or bud selection offspring, this " inbreeding " often brings the problems such as the hereditary basis of kind is narrow and resistance goes down；On the other hand, characteristic, multi-resistance and multiformity fruit become the important directions of industry development.
Malus sieversii and red meat modification (Malussieversiif.neidzwetzkyana) thereof are ancestors' kinds of world's Cultivated apple, not only genetic diversity is the abundantest, and rich in the functions such as flavonoid, health-care components, it is by degeneration-resistant and quality breeding precious gene bank.But because of reasons such as the farmland reclamation of wastelands, the genetic diversity of Malus sieversii is just being seriously damaged, endangered；China is apple production maximum in the world and country of consumption, wherein within 2012, produces Fructus Mali pumilae 39,500,000 tons, is mainly used in eating raw, and nearly 70% is Fuji's kind that Flavonoid Content is relatively low.
nullTherefore，Around " scientific conservation of Malus sieversii resource and Sustainable and highly-efficient use、Cultivar hereditary basis is expanded、Apple Industry transition and upgrade promotes with Practices Sustainable Increasing Income of Farmers and level of human health to side structure reform together "，Carry out research cooperation and integration and demonstration，The present inventor cooperates with the professor of Cornell Univ USA，Worldwide 97 parts of apple resources such as Malus sieversii and European Forest Fructus Mali pumilae carry out genome resurvey sequence and bioinformatic analysis，Construct Xinjiang red meat Fructus Mali pumilae and the apple variety first generation of hybrid and backcross one、Secondary segregating population，Research specify that Malus sieversii population genetic variations and genetic diversity feature、The technical parameter that Core Germplasms builds、The hereditary variation feature of the character such as Flavonoid Content and development mechanism，Propose concept and " the wide row high level cadre of " functional type Fructus Mali pumilae "、Inter-row green covering、Give grass fertilising、Fertilize the soil support root " Modern orchard management philosophy，Create the Fructus Mali pumilae high-efficient breeding technique system that conventional hybridization organically combines with biotechnology，A collection of new varieties and excellent germplasm are formulated，Have developed Variety of Apple highly effective matched cultivation technical system.At present, authorized and declared patent of invention 10 remainder, field planting hybrid seedling more than 40,000 strain, be bred as new varieties (being) 16；Delivering correlational study paper 120, wherein SCI paper more than 20 piece, these achievements in research are totally in the top standard of international similar research.
Summary of the invention
It is an object of the invention to provide a kind of anthocyanin modulin MsMYB111 available from functional type Fructus Mali pumilae and encoding gene thereof and application.
The protein that the present invention provides, available from Fructus Mali pumilae, named MsMYB111 albumen, is following (a1) or (a2):
(a1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(a2) by the aminoacid sequence of sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derives relevant to plant Anthocyanin Content.
In order to make the MsMYB111 albumen in (a) be easy to purification and detection, in by sequence table, label as shown in table 1 can be connected by amino terminal or the carboxyl terminal of the protein that the aminoacid sequence shown in sequence 1 forms.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
MsMYB111 albumen in above-mentioned (b) can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the MsMYB111 albumen in above-mentioned (b) can be by the codon by lacking one or several amino acid residue in the DNA sequence shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or connect the coded sequence of the label shown in table 1 at its 5 ' end and/or 3 ' ends and obtain.
The gene (MsMYB111 gene) encoding described MsMYB111 albumen falls within protection scope of the present invention.
Described gene is following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in coding region sequence table；
(2) protein DNA molecule relevant with plant Anthocyanin Content to the DNA sequence hybridization that (1) limits and coding under strict conditions；
(3) there is to the DNA sequence that (1) limits more than 90% homology and encode the protein DNA molecule relevant with plant Anthocyanin Content.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS, hybridizes and wash film in DNA or RNA hybrid experiment at 65 DEG C.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue or recombinant bacterium containing described MsMYB111 gene belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression carrier of MsMYB111 gene.Described plant expression vector includes double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.When using the gene constructed recombinant expression carrier of MsMYB111, can be plus any enhancement mode, composing type, organizing specific type or inducible promoter before its transcription initiation nucleotide, they can be used alone or be used in combination with other plant promoter；In addition, when using the gene constructed recombinant expression carrier of MsMYB111, it be also possible to use enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region start codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and start codon is widely, can be natural, it is also possible to be synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of transgenic plant cells or plant being identified and screening, plant expression vector used can be processed, can produce, as being added in plant to express, enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. that color changes.From the security consideration of transgenic plant, any selected marker can be not added with, directly with phenotypic screen transformed plant.The carrier that sets out of described recombinant expression carrier can be pRI101 carrier.The described recombinant expression carrier recombiant plasmid pRI101-MsMYB111 that concretely double chain DNA molecule shown in sequence 2 of insertion sequence table obtains between SalI and the BamHI restriction enzyme site of pRI101 carrier.
Described plant tissue concretely plant callus, can be more specifically plant spire callus.Described plant can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically Fructus Mali pumilae ' purplish red No. 1 '.
The present invention also protects the application of described MsMYB111 albumen, for as follows (b1) or (b2):
(b1) Anthocyanin Content of plant is regulated and controled；
(b2) Anthocyanin Content of plant is reduced.
Described plant can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically Fructus Mali pumilae ' purplish red No. 1 '.
The present invention also protects the application in cultivating the transgenic plant that Anthocyanin Content reduces of the described MsMYB111 gene.Described plant can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically Fructus Mali pumilae ' purplish red No. 1 '.
The present invention also protects a kind of method cultivating transgenic plant, and comprise the steps: to set out described MsMYB111 channel genes plant, obtain Anthocyanin Content be less than described in set out the transgenic plant of plant.The described plant that sets out can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically Fructus Mali pumilae ' purplish red No. 1 '.Described MsMYB111 gene specifically can be by the plant that sets out described in recombinant expression carrier importing described in any of the above.In described method, the callus of the plant that specifically described MsMYB111 channel genes can be set out, then callus is cultivated as plant.Described callus concretely spire callus.The recombinant expression carrier carrying described MsMYB111 gene can be transformed in plant cell or tissue by Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, the conventional biology methods such as agriculture bacillus mediated.
The present invention also protects a kind of method obtaining Transgenic plant tissue, and comprise the steps: to set out described MsMYB111 channel genes plant tissue, obtain Anthocyanin Content be less than described in set out the Transgenic plant tissue of plant tissue.The described plant that sets out can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically Fructus Mali pumilae ' purplish red No. 1 '.Described MsMYB111 gene specifically can be by the plant tissue that sets out described in recombinant expression carrier importing described in any of the above.Described plant tissue concretely plant callus.Described callus concretely spire callus.
The present invention also protects method application in plant breeding described in described MsMYB111 albumen or described MsMYB111 gene or any of the above.The purpose of described plant breeding is to cultivate the plant that Anthocyanin Content reduces.
Present invention finds MsMYB111 albumen and function thereof, can be used for cultivating the transgenic plant that Anthocyanin Content changes, plant breeding has major application prospect.
Accompanying drawing explanation
Fig. 1 is the result of phenotypic evaluation.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
PRI101 carrier (also known as " pRI101-ANDNA "): Takala company, CodeNo.3262.Agrobacterium LBA4404: Tiangen company, catalog number: CC2901.Fructus Mali pumilae ' purplish red No. 1 ' (also known as ' purplish red No. 1 ' red meat Fructus Mali pumilae): list of references: " ' purplish red No. 1 ' red meat apple pulp non-oxidizability and anthocyanin are analyzed ".
Prepare the method for spire callus referring specifically to document: JiXH, ZhangR, WangN, YangL&ChenXS.Transcriptomeprofilingrevealsauxinsuppresse danthocyaninbiosynthesisinred-fleshedapplecallus (Malussieversiif.niedzwetzkyana) .PlantCellTissOrganCult, 2015,123:389 404..The preparation method of 1% methanol hydrochloride solution: add 2.8ml concentrated hydrochloric acid in 97.2ml methanol.The preparation method of 0.025MKCl buffer (pH=1.0): 1.86gKCl 980ml distilled water dissolves, and adjusts pH to 1.0 with concentrated hydrochloric acid, proceeds to, in 1L volumetric flask, use distilled water constant volume.The preparation method of 0.4MNaAc buffer (pH=4.5): claim 54.43gNaAC 960ml distilled water to dissolve, adjust pH to 4.5 with concentrated hydrochloric acid, transfer to 1L volumetric flask, use distilled water constant volume.
Embodiment 1, MsMYB111 albumen and the discovery of encoding gene thereof
It is found that a new albumen, as shown in the sequence 1 of sequence table, by its named MsMYB111 albumen from ' red crisp No. 1 ' Fructus Mali pumilae.Being MsMYB111 gene by the unnamed gene of coding MsMYB111 albumen, its open reading frame is as shown in the sequence 2 of sequence table.
In GENBANK, the albumen the highest with sequence 1 homology is as shown in the sequence 3 of sequence table.By the named reference protein of protein shown in the sequence 3 of sequence table, the named crt gene of its encoding gene, as shown in the sequence 4 of sequence table.
Embodiment 2, the Function Identification of MsMYB111 albumen
One, construction recombination plasmid
1, construction recombination plasmid pRI101-MsMYB111
(1) double chain DNA molecule shown in sequence 2 of artificial synthesized sequence table.
(2) DNA molecular obtained with step (1) is as template, uses the primer of F1 and R1 composition to carrying out PCR amplification, obtains pcr amplification product.
F1:5 '-GTCGACATGAGAAAACCTTGCTGTGAAA-3’；
R1:5 '-GGATCCTTATCTGAAAAGCACAAGGGTAG-3’。
(3) pcr amplification product obtained with restricted enzyme SalI and BamHI double digestion step (2), reclaims digestion products.
(4) with restricted enzyme SalI and BamHI double digestion pRI101 carrier, the carrier framework of about 10kb is reclaimed.
(5) digestion products of step (3) is connected with the carrier framework of step (4), obtains recombiant plasmid pRI101-MsMYB111.According to sequencing result, recombiant plasmid pRI101-MsMYB111 is carried out structure and is described as follows: between SalI and the BamHI restriction enzyme site of pRI101 carrier, insert the double chain DNA molecule shown in the sequence 2 of sequence table.
2, construction recombination plasmid pRI101-△ MsMYB111
(1) double chain DNA molecule shown in sequence 2 of artificial synthesized sequence table.
(2) DNA molecular obtained with step (1) is as template, uses the primer of F2 and R2 composition to carrying out PCR amplification, obtains pcr amplification product.
F2:5 '-GTCGACATGAGAAAACCTTGCTGTGAAA-3’；
R2:5 '-GGTTCAGGAAAAGATGATGGACCAGATGTTTC-3 '.
(3) DNA molecular obtained with step (1) is as template, uses the primer of F3 and R3 composition to carrying out PCR amplification, obtains pcr amplification product.
F3:5 '-TCCATCATCTTTTCCTGAACCTTCACTACAG-3 '
R3:5 '-GGATCCTTATCTGAAAAGCACAAGGGTAG-3’。
(4) to carrying out PCR amplification, pcr amplification product is obtained as template, the primer of employing F2 and R3 composition using after the pcr amplification product of step (2) and the pcr amplification product mixing of step (3).
(5) pcr amplification product obtained with restricted enzyme SalI and BamHI double digestion step (4), reclaims digestion products.
(6) with restricted enzyme SalI and BamHI double digestion pRI101 carrier, the carrier framework of about 10kb is reclaimed.
(7) digestion products of step (5) is connected with the carrier framework of step (6), obtains recombiant plasmid pRI101-△ MsMYB111.According to sequencing result, recombiant plasmid pRI101-△ MsMYB111 is carried out structure and is described as follows: between SalI and the BamHI restriction enzyme site of pRI101 carrier, insert the double chain DNA molecule shown in the sequence 5 of sequence table.
3, control plasmid is built
Double chain DNA molecule shown in the sequence 4 of the sequence table of synthetic is inserted between SalI and the BamHI restriction enzyme site of pRI101 carrier, obtain control plasmid.
Two, the acquisition of MsMYB111 gene callus is turned
1, recombiant plasmid pRI101-MsMYB111 is imported Agrobacterium LBA4404, obtain recombinational agrobacterium.
2, the recombinational agrobacterium that step 1 obtains is seeded to 30ml containing 50 μ g/ml kanamycin and the YEP fluid medium of 50 μ g/ml rifampicin, 28 DEG C of shaken cultivation to OD600nm=0.6,12000rpm are centrifugal collects thalline, uses 30mlddH2O suspends, and adds acetosyringone and to make its concentration be 100 μMs, obtain infecting liquid.
3, taking the spire callus of Fructus Mali pumilae ' purplish red No. 1 ', be immersed into that step 2 obtains infects in liquid, shaken at room temperature 30min.
4, after completing step 3, take callus, it is placed on the MS solid medium containing 1mg/L6-BA and 0.3mg/LNAA, 28 DEG C of light culture 2 days, are then transferred into alternation of light and darkness on the MS solid medium containing 1mg/L6-BA, 0.3mg/LNAA, 50mg/L kanamycin and 50mg/L rifampicin (16h illumination/8h is dark) and cultivate 30 days.
5, after completing step 4, taking callus, extract genomic DNA, use the primer of F4 and R4 composition to carrying out PCR qualification, PCR is accredited as being of the positive and turns MsMYB111 gene callus.
F4:5 '-GCTCCTACAAATGCCATCA-3 '；
R4:5 '-TTATCTGAAAAGCACAAGGGTAG-3 '.
35S promoter partial sequence on F4 correspondence carrier framework, the partial sequence on R4 correspondence MsMYB111 gene, target sequence length is about 750bp.
6, after completing step 4, take and turn MsMYB111 gene callus, be placed in successive transfer culture on the MS solid medium containing 1mg/L6-BA, 0.3mg/LNAA, 50mg/L kanamycin and 50mg/L rifampicin.
Three, the acquisition of control tissue
Recombiant plasmid pRI101-△ MsMYB111 replacement recombiant plasmid pRI101-MsMYB111 is carried out step 2, obtains turning △ MsMYB111 gene callus.
Control plasmid replaces recombiant plasmid pRI101-MsMYB111 carry out step 2, obtains turning crt gene callus.
PRI101 carrier replacement recombiant plasmid pRI101-MsMYB111 is carried out step 2, obtains turning empty carrier callus.
Four, phenotypic evaluation
Fig. 1 is shown in by the photo of each callus.6 of each callus respectively step 2 in Fig. 1 are cultivated after 30 days turn MsMYB111 gene callus, step 36 in cultivate 30 days after turn △ MsMYB111 gene callus, step 36 in cultivate 30 days after turn crt gene callus, step 36 in cultivate 30 days after turn empty carrier callus and be in Fructus Mali pumilae ' purplish red No. 1 ' the spire callus (representing with WT) of contemporaneity.The callus of Fructus Mali pumilae ' purplish red No. 1 ' is shown as darkviolet, shows that wherein Anthocyanin Content is higher.Turn MsMYB111 gene callus and be shown as light yellow, show that its Anthocyanin Content ' purplish red No. 1 ' relatively is substantially reduced.Turn △ MsMYB111 gene callus basically identical with the callus color of ' purplish red No. 1 '.Turn empty carrier callus basically identical with the callus color of ' purplish red No. 1 '.Turning crt gene callus and be shown as yellow purple alternate color, wherein Anthocyanin Content is higher than turning MsMYB111 gene callus and the callus less than ' purplish red No. 1 '.
Five, content is identified
Callus to be measured be respectively as follows: in the 6 of step 2 cultivate after 30 days turn MsMYB111 gene callus, step 36 in cultivate 30 days after turn △ MsMYB111 gene callus, step 36 in cultivate 30 days after turn crt gene callus, step 36 in cultivate 30 days after turn empty carrier callus and be in Fructus Mali pumilae ' purplish red No. 1 ' the spire callus (representing with WT) of contemporaneity.
Specifically comprise the following steps that
1, weighing 0.5g callus to be measured, add liquid nitrogen grind into powder, be subsequently adding 1% methanol hydrochloride solution of 5ml4 DEG C of pre-cooling, then 4 DEG C of lucifuges stand extraction 24h, and then 8000r/min is centrifuged 10min, collects supernatant.
2, taking the supernatant that 1ml step 1 obtains, add 4ml0.025MKCl buffer (pH=1.0) and mix, then 4 DEG C of lucifuges stand extraction 15min, and then 8000r/min is centrifuged 10min, collects supernatant.
3, taking the supernatant that 1ml step 1 obtains, add 4ml0.4MNaAc buffer (pH=4.5) and mix, then 4 DEG C of lucifuges stand extraction 15min, and then 8000r/min is centrifuged 10min, collects supernatant.
4, take supernatant that step 2 obtains and the supernatant that step 3 obtains respectively, measure the light absorption value under 510nm and 700nm.
Anthocyanin Content (mg/g)=△ A × 5 × 0.005 × 1000 × 449.2/ (26900 × 0.5)；
△ A=(A510nm-A700nm)(pH=1.0)-(A510nm-A700nm)(PH=4.5)。
" g " in Anthocyanin Content unit " mg/g " refers to the fresh weight of callus.
Carry out five times repeating test, repeat each callus to be measured in test every time and respectively take 10 parts, results averaged.
The results are shown in Table 2.Turn the Anthocyanin Content of MsMYB111 gene callus substantially less than to turn △ MsMYB111 gene callus, turn crt gene callus, turn empty carrier callus and be in Fructus Mali pumilae ' purplish red No. 1 ' the spire callus of contemporaneity.Result shows, MsMYB111 albumen can reduce the Anthocyanin Content of plant.
Anthocyanin Content in each callus to be measured of table 2
Claims (10)
1. a protein, is following (a1) or (a2):
(a1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(a2) by the aminoacid sequence of sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derives relevant to plant Anthocyanin Content.
2. the gene of protein described in coding claim 1.
3. gene as claimed in claim 2, it is characterised in that: described gene is following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in coding region sequence table；
(2) protein DNA molecule relevant with plant Anthocyanin Content to the DNA sequence hybridization that (1) limits and coding under strict conditions；
(3) there is to the DNA sequence that (1) limits more than 90% homology and encode the protein DNA molecule relevant with plant Anthocyanin Content.
4. contain the recombinant expression carrier of gene described in Claims 2 or 3, expression cassette, transgenic cell line, Transgenic plant tissue or recombinant bacterium.
5. the application of protein described in claim 1, for as follows (b1) or (b2):
(b1) Anthocyanin Content of plant is regulated and controled；
(b2) Anthocyanin Content of plant is reduced.
6. the application in cultivating the transgenic plant that Anthocyanin Content reduces of the gene described in Claims 2 or 3.
7. the method cultivating transgenic plant, comprise the steps: to set out channel genes described in Claims 2 or 3 plant, obtain Anthocyanin Content be less than described in set out the transgenic plant of plant.
8. the method cultivating Transgenic plant tissue, comprise the steps: to set out channel genes described in Claims 2 or 3 plant tissue, obtain Anthocyanin Content be less than described in set out the Transgenic plant tissue of plant tissue.
9. method as claimed in claim 8, it is characterised in that: described plant tissue is plant callus.
10. protein described in claim 1, or, gene described in Claims 2 or 3, or, method described in claim 7 or 8 or 9, the application in plant breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610293357.2A CN105820224A (en) | 2016-05-05 | 2016-05-05 | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610293357.2A CN105820224A (en) | 2016-05-05 | 2016-05-05 | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105820224A true CN105820224A (en) | 2016-08-03 |
Family
ID=56528440
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610293357.2A Pending CN105820224A (en) | 2016-05-05 | 2016-05-05 | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105820224A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106883292A (en) * | 2017-04-17 | 2017-06-23 | 山东农业大学 | Available from the MsLBD13 albumen and its encoding gene of functional form apple and application |
| CN107033228A (en) * | 2017-04-17 | 2017-08-11 | 山东农业大学 | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application |
| CN113024646A (en) * | 2021-02-22 | 2021-06-25 | 山东省果树研究所 | Anthocyanin regulatory protein JrMYB114 and coding gene and application thereof |
| CN113024647A (en) * | 2021-02-22 | 2021-06-25 | 山东省果树研究所 | Anthocyanin regulatory protein JrMYB10 and coding gene and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045642A (en) * | 2012-09-13 | 2013-04-17 | 浙江大学 | Method for cultivating tobacco rich in anthocyanin and application thereof |
-
2016
- 2016-05-05 CN CN201610293357.2A patent/CN105820224A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045642A (en) * | 2012-09-13 | 2013-04-17 | 浙江大学 | Method for cultivating tobacco rich in anthocyanin and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LIN-WANG, KUI等: "High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex", 《PLANT, CELL AND ENVIRONMENT》 * |
| NONE: "Accession ID: NM_001294077，Malus domestica myb-related protein 308-like (MYB111), mRNA.", 《GENBANK DATABASE》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106883292A (en) * | 2017-04-17 | 2017-06-23 | 山东农业大学 | Available from the MsLBD13 albumen and its encoding gene of functional form apple and application |
| CN107033228A (en) * | 2017-04-17 | 2017-08-11 | 山东农业大学 | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application |
| CN107033228B (en) * | 2017-04-17 | 2018-10-26 | 山东农业大学 | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application |
| CN113024646A (en) * | 2021-02-22 | 2021-06-25 | 山东省果树研究所 | Anthocyanin regulatory protein JrMYB114 and coding gene and application thereof |
| CN113024647A (en) * | 2021-02-22 | 2021-06-25 | 山东省果树研究所 | Anthocyanin regulatory protein JrMYB10 and coding gene and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016231586A1 (en) | Isolated polynucleotides and polypeptides and methods of using same for increasing plant yield, biomass, growth rate, vigor, oil content, abiotic stress tolerance of plants and nitrogen use efficiency | |
| CN105820224A (en) | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof | |
| CN105820223A (en) | Anthocyanin-regulation protein MsMYB16 from functional apple and encoding gene and application thereof | |
| CN105837671B (en) | Obtained from the flavonols modulin MsMYB22 and its encoding gene of functional form apple and application | |
| CN106554397B (en) | Protein OsGRF4-M and its relevant biological material from rice are regulating and controlling the application in plant organ size | |
| CN108218968A (en) | A kind of plant seed character-related protein and its encoding gene and application | |
| CN106883292B (en) | Obtained from the MsLBD13 albumen and its encoding gene of functional form apple and application | |
| CN106749580B (en) | Plant salt tolerance GAP-associated protein GAP TaPUB15-D and its encoding gene and application | |
| CN105837669B (en) | Obtained from the anthocyanin modulin MsARF3 and its encoding gene of functional form apple and application | |
| CN106243207B (en) | Obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application | |
| CN106591320A (en) | Betula platyphylla BplSPL1 gene for promoting precocious flowering and encoded protein thereof | |
| CN107033228B (en) | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application | |
| CN106432444B (en) | One plant glutelin transhipment storage GAP-associated protein GAP GPA4 and its encoding gene and application | |
| CN105949289B (en) | Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application | |
| CN107022014B (en) | Obtained from the MsTMT1L albumen and its encoding gene of functional form apple and application | |
| CN103694327B (en) | Drought tolerance in plants associated protein DSM1 and encoding gene thereof and application | |
| CN103012570B (en) | Plant adverse resistance related protein PpLEA3-21 and coding gene and application thereof | |
| CN105949288B (en) | Obtained from the flavanols modulin MsMYB6L and its encoding gene of functional form apple and application | |
| CN107022013B (en) | Obtained from the MsMYBL2 albumen and its encoding gene of functional form apple and application | |
| CN107446031A (en) | A kind of plant glutelin transhipment storage GAP-associated protein GAP OsVHA E1 and its encoding gene and application | |
| CN107090022B (en) | Obtained from the MsVGT1L albumen and its encoding gene of functional form apple and application | |
| Zhang et al. | Pepper (Capsicum annuum) xylogen-like arabinogalactan protein (XYLP) 1 and XYLP2 promote synthesis of lignin during stem development to cope with stresses | |
| CN106834343B (en) | Sucrose synthase is in the regulation developmental application of fruit | |
| CN103408647B (en) | Plant green-staying related protein as well as coding gene and application thereof | |
| CN102964438B (en) | Stress-resistance-related protein PpLEA3-23 of plant as well as coding gene and application of protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160803 |
|
| RJ01 | Rejection of invention patent application after publication |