CN107033228A - Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application - Google Patents
Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application.The protein that the MsMYBPA1 albumen that the present invention is provided is made up of the amino acid sequence shown in sequence in sequence table 1.The present invention also protects the application of MsMYBPA1 albumen, is following (b1) and/or (b2) and/or (b3) and/or (b4)：(b1) the flavanols compounds content of plant is regulated and controled；(b2) the flavanols compounds content of plant is increased；(b3) Anthocyanin Content of plant is regulated and controled；(b4) Anthocyanin Content of plant is increased.Present invention finds MsMYBPA1 albumen and its function, available for the genetically modified plants that flavanols compounds and/or Anthocyanin Content change are cultivated, there is major application prospect in plant breeding.
Description
Technical field
The present invention relates to a kind of flavanols and anthocyanin modulin MsMYBPA1 and its coding obtained from functional form apple
Gene and application.
Background technology
" doctor's food homology " is developing direction.Apple storage property is good, and supply cycle is long, is worldwide fruit, and especially fruit contains
Have higher proportion, human body be easier absorb free polyphenol, with well it is anti-oxidant, antitumor, prevention cardiovascular and cerebrovascular
Disease and liver protection etc. are acted on, and healthy nutritive value is high, there is " one day apple, doctor is away from me " (An apple a day
keeps the doctor away！) good reputation, in the world considerable country be all classified as major consumers fruit and energetically
Recommend.But finding in recent years shows, on the one hand, more than 1000 apple variety being bred as both at home and abroad in the past few decades,
80% it is the hybridization of kinds such as ' Jin Shuai ', grows directly from seeds or bud selection offspring, this " inbreeding " often brings the hereditary basis of kind
The problems such as narrow and resistance declines；On the other hand, characteristic, many anti-and diversity fruits turn into the important directions of industry development.
Malus sieversii and its red meat modification (Malus sieversii f.neidzwetzkyana) are world's cultivation apples
Ancestors' kind of fruit, not only genetic diversity extremely enrich, and rich in function, the health-care components such as flavonoids, be carry out it is degeneration-resistant with
The precious gene pool of quality breeding.But because of reasons such as the farmland reclamation of wastelands, the genetic diversity of Malus sieversii is just seriously damaged,
It is endangered；China is maximum in the world apple production and country of consumption, wherein 39,500,000 tons of apple of production in 2012, main to use
In eating raw, and nearly 70% is the relatively low Fuji's kind of Flavonoid Content.
Therefore, around " scientific conservation of Malus sieversii resource is opened up with Sustainable and highly-efficient use, cultivar hereditary basis
Exhibition, Apple Industry transition and upgrade are together to side structure reform and Practices Sustainable Increasing Income of Farmers and level of human health lifting ", joined
Tackling key problem and integration and demonstration are closed, the present inventor and the professor of Cornell Univ USA cooperate, to Malus sieversii and Europe
Worldwide 97 parts of apple resources such as forest apple have carried out genome and have resurveyed sequence and bioinformatic analysis, construct new
Boundary red meat apple specify that Malus sieversii colony loses with one, two generation segregating populations of the apple variety first generation of hybrid and backcrossing, research
Pass the hereditary variation feature of the characters such as technical parameter, Flavonoid Content that structure is built with genetic diversity feature, Core Germplasms
And development mechanism, it is proposed that the concept of " functional form apple " and " wide row high level cadre, inter-row green covering, to grass fertilising, fertilize the soil support root "
Modern orchard management philosophy, creates the apple high-efficient breeding technique system that conventional hybridization is organically combined with biotechnology, initiative
A collection of new varieties and excellent germplasm, have developed Variety of Apple highly effective matched cultivation technical system.At present, authorized and declared
The remainder of patent of invention 10, is colonized the strain of hybrid seedling more than 40,000, is bred as new varieties (being) 16；Deliver correlative study paper 120
A piece, the wherein piece of SCI papers more than 20, these achievements in research are totally in the top standard of international similar research.
The content of the invention
It is an object of the invention to provide a kind of flavanols obtained from functional form apple and anthocyanin modulin MsMYBPA1
And its encoding gene and application.
The protein that the present invention is provided, obtained from apple, is named as MsMYBPA1 albumen, be following (a1) or (a2) or
(a3)：
(a1) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(a2) by the amino acid sequence of sequence 1 is by the substitution of one or several amino acid residues and/or missing and/or adds
Plus and the protein as derived from sequence 1 related to plant flavanols compounds content；
(a3) by the amino acid sequence of sequence 1 is by the substitution of one or several amino acid residues and/or missing and/or adds
Plus and the protein as derived from sequence 1 related to plant Anthocyanin Content.
In order that the MsMYBPA1 albumen in (a1) is easy to purify and detected, ammonia that can be in as sequence table shown in sequence 1
The amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of base acid sequence composition.
The sequence of the label of table 1
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
MsMYBPA1 albumen in above-mentioned (a2) or (a3) can be artificial synthesized, also can first synthesize its encoding gene, then carry out
Biological expression is obtained.The encoding gene of MsMYBPA1 albumen in above-mentioned (a2) or (a3) can be by by the institute of sequence in sequence table 2
The codon of one or several amino acid residues is lacked in the DNA sequence dna shown, and/or carries out the missense of one or several base-pairs
Mutation, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect the label shown in table 1.
The gene (MsMYBPA1 genes) of coding MsMYBPA1 albumen falls within protection scope of the present invention.
MsMYBPA1 genes are following (1) or (2) or (3) or (4) or (5)：
(1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(2) the DNA sequence dna hybridization limited under strict conditions with (1) and coding and plant flavanols compounds content phase
The protein DNA molecule of pass；
(3) DNA sequence dna limited with (1) has more than 90% homology and coding and plant flavanols compounds content
Related protein DNA molecule；
(4) the DNA sequence dna hybridization limited under strict conditions to (1) and the coding albumen related with plant Anthocyanin Content
The DNA molecular of matter；
(5) there is more than 90% homology and the coding egg related with plant Anthocyanin Content to the DNA sequence dna that (1) is limited
The DNA molecular of white matter.
Above-mentioned stringent condition can be that 0.1%SDS solution is miscellaneous in DNA or RNA with 0.1 × SSPE (or 0.1 × SSC)
Hand over and hybridize in experiment at 65 DEG C and wash film.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue containing MsMYBPA1 genes or again
Group bacterium belongs to protection scope of the present invention.
The recombinant expression carrier of MsMYBPA1 genes can be contained with existing plant expression vector construction.The plant expression
Carrier includes double base agrobacterium vector and the carrier available for plant micropellet bombardment etc..Use the gene constructed restructuring tables of MsMYBPA1
During up to carrier, any enhanced, composing type, organizing specific type or induction type can be added before its transcription initiation nucleotides and is opened
Mover, they can be used alone or are used in combination with other plant promoters；In addition, using the gene constructed restructuring of MsMYBPA1
During expression vector, enhancer, including translational enhancer or transcriptional enhancer are it is also possible to use, these enhancer regions can be ATG
Initiation codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure whole sequence
Correct translation.The source of the translation control signal and initiation codon is extensive, can be natural or conjunction
Into.Translation initiation region can come from transcription initiation region or structural gene.For the ease of to transgenic plant cells or plant
Thing is identified and screened, and plant expression vector used can be processed, and color change can be produced by such as adding the expression in plant
The enzyme of change or the gene of luminophor, resistant antibiotic marker or anti-chemical reagent marker gene etc..From turn
The security consideration of gene plant, can be not added with any selected marker, directly with phenotypic screen transformed plant.The restructuring
The carrier that sets out of expression vector can be pRI101 carriers.The recombinant expression carrier is concretely in the Nde I of pRI101 carriers
The recombinant plasmid pRI101- that double chain DNA molecule between BamH I restriction enzyme sites shown in the sequence 2 of insetion sequence table is obtained
MsMYBPA1。
The plant tissue concretely plant callus, more specifically can be plant embryo callus or plant spire
Callus.The plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.
The rosaceous plant concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin '
Apple.
The present invention also protects the application of MsMYBPA1 albumen, is following (b1) and/or (b2) and/or (b3) and/or (b4)：
(b1) the flavanols compounds content of plant is regulated and controled；
(b2) the flavanols compounds content of plant is increased；
(b3) Anthocyanin Content of plant is regulated and controled；
(b4) Anthocyanin Content of plant is increased.
The plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.
The rosaceous plant concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin '
Apple.
The present invention also protection MsMYBPA1 genes are cultivating increased turn of the content of flavanols compounds and/or anthocyanin
Application in gene plant.The plant can be monocotyledon or dicotyledon.The dicotyledon concretely rose
Common vetch section plant.The rosaceous plant concretely Malus.The Malus concretely apple, more specifically may be used
For ' Wang Lin ' apple.
The present invention also protects a kind of method for cultivating genetically modified plants, comprises the following steps：By MsMYBPA1 channel genes
Set out plant, obtains genetically modified plants of the content higher than the plant that sets out of flavanols compounds and/or anthocyanin.It is described
The plant that sets out can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rose
Section plant concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.It is described
MsMYBPA1 genes can specifically pass through the plant that set out described in recombinant expression carrier importing described in any of the above.In methods described, tool
Body can set out the MsMYBPA1 channel genes callus of plant, and it is plant then to cultivate callus.It is described to be cured
Injured tissue concretely embryo callus or spire callus.Carry the recombinant expression carrier of the MsMYBPA1 genes
Can be routinely raw by Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated etc.
Thing method is transformed into plant cell or tissue.
The present invention also protects a kind of method for cultivating Transgenic plant tissue, comprises the following steps：By MsMYBPA1 genes
Importing is set out plant tissue, obtains content the turning higher than the plant tissue that sets out of flavanols compounds and/or anthocyanin
Gene plant tissue.The plant that sets out can be monocotyledon or dicotyledon.The dicotyledon concretely rose
Common vetch section plant.The rosaceous plant concretely Malus.The Malus concretely apple, more specifically may be used
For ' Wang Lin ' apple.The MsMYBPA1 genes can specifically pass through plant of being set out described in recombinant expression carrier importing described in any of the above
Thing tissue.The plant tissue concretely plant callus.The callus concretely embryo callus or children
Leaf callus.
The present invention also protects a kind of plant breeding method, comprises the following steps：Improve MsMYBPA1 albumen in purpose plant
Content and/or activity, so as to improve the content of flavanols compounds and/or anthocyanin in purpose plant.The purpose is planted
Thing can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant
Concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.
The present invention also protection any methods described of MsMYBPA1 albumen or MsMYBPA1 genes or more is in plant breeding
Application.The plant that the purpose of the plant breeding increases for the content of cultivation flavanols compounds and/or anthocyanin.It is described
Plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rose family is planted
Thing concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.
Flavanols compounds described in any of the above are the compound with female ring shown in formula (I)；
Flavanols compounds described in any of the above are compound shown in formula (II)；
R1=H, OH or OG；
R2=OH or H；
R3=H or OH.
R1=H, R2=OH, R3It is catechin during=H
R1=OH, R2=H, R3It is epicatechin during=H
R1=H, R2=OH, R3It is nutgall catechin during=OH
R1=OH, R2=H, R3It is epigallocatechin during=OH
R1=OG, R2=H, R3It is L-Epicatechin gallate during=H
R1=OG, R2=H, R3It is Epigallo-catechin gallate (EGCG) during=OH
Present invention finds MsMYBPA1 albumen and its function, available for cultivation flavanols compounds and/or anthocyanin
The genetically modified plants that content changes, have major application prospect in plant breeding.
Brief description of the drawings
Fig. 1 is the result that DMACA dyes identification.
Fig. 2 is the result of anthocyanin Qualitative Identification.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
PRI101 carriers (also known as " pRI101-AN DNA ")：Takala companies, Code No.3262.Agrobacterium
LBA4404：Tiangen companies, catalog number：CC2901.' Wang Lin ' apple：Bibliography：《The bHLH
transcription factor MdbHLH3promotes anthocyanin accumulation and fruit
colouration in response to low temperature in apples》.' purplish red No. 1 ' apple：Bibliography
《' purplish red No. 1 ' red meat apple pulp inoxidizability and anthocyanin analysis》.
1% methanol hydrochloride solution：97.2ml methanol is mixed with 2.8ml concentrated hydrochloric acids.Concentrated hydrochloric acid is commercially available 12mol/L hydrochloric acid.
KCl buffer solutions (pH=1,0.025M)：1.86g KCl 980ml distilled water dissolves, and adjusts pH to be 1.0 with concentrated hydrochloric acid,
It is transferred in 1L volumetric flasks, uses distilled water constant volume.
NaAC buffer solutions (pH=4.5,0.4M)：54.43g NaAC 960ml distilled water dissolves, and is with concentrated hydrochloric acid tune pH
4.5, it is transferred in 1L volumetric flasks, uses distilled water constant volume.
The method of spire callus is prepared referring specifically to document：Ji X H,Zhang R,Wang N,Yang L&Chen
X S.Transcriptome profiling reveals auxin suppressed anthocyanin biosynthesis
in red-fleshed apple callus(Malus sieversii f.niedzwetzkyana).Plant Cell Tiss
Organ Cult,2015,123:389–404.。
The discovery of embodiment 1, MsMYBPA1 albumen and its encoding gene
A new albumen is found that from " purplish red No. 1 " apple, as shown in the sequence 1 of sequence table, is named as
MsMYBPA1 albumen.It is MsMYBPA1 genes, such as sequence table of its ORFs by the unnamed gene for encoding MsMYBPA1 albumen
Sequence 2 shown in.
In GENBANK, with the homology highest albumen of sequence 1 as shown in the sequence 3 of sequence table.By the sequence 3 of sequence table
Shown protein is named as reference protein, and its encoding gene is named as crt gene (as shown in the sequence 4 of sequence table).
The Function Identification of embodiment 2, MsMYBPA1 albumen
First, construction recombination plasmid
Double chain DNA molecule shown in the sequence 2 of artificial synthesized sequence table is inserted to the Nde I and BamH of pRI101 carriers
Between I restriction enzyme sites, recombinant plasmid pRI101-MsMYBPA1 is obtained.
Double chain DNA molecule shown in the sequence 4 of artificial synthesized sequence table is inserted to the Nde I and BamH of pRI101 carriers
Between I restriction enzyme sites, control plasmid is obtained.
2nd, the acquisition of genetically modified organism
1st, recombinant plasmid pRI101-MsMYBPA1 is imported into Agrobacterium LBA4404, obtains recombinational agrobacterium.
2nd, the recombinational agrobacterium thalline that step 1 is obtained is taken, is suspended with 30ml liquid MS mediums, obtains OD600nm=0.8
Bacteria suspension, adds 30 μ L 100mM acetosyringones solution (solvent is DMSO), obtains infecting liquid.
3rd, the spire callus of ' Wang Lin ' apple is taken, is seeded on solidified MS media, 25 DEG C of dark culturings 15 days.
4th, complete after step 3, take callus, be immersed in that step 2 obtains infects in liquid, 160rpm shaken at room temperature 15-
20min, then blots surface with sterilizing filter paper.
5th, complete after step 4, take callus, be placed in co-cultivation culture medium and (contain 1mg/L 2,4-D and 0.5mg/L6-BA
Solidified MS media) on, 25 DEG C of dark culturings 36 hours.
6th, complete after step 5, take callus, be placed in screening and culturing medium (kanamycins containing 50mg/L, 250mg/L carboxylic Bians
Penicillin, 1mg/L 2,4-D and 0.5mg/L 6-BA solidified MS media) on, 25 DEG C of dark culturings 20-30 days.
7th, complete after step 6, take the resistant calli that can be grown on screening and culturing medium, enter performing PCR identification.
PCR identifications primer pair used is as follows：
35s-F：5’-GACGCACAATCCCACTATCC-3’；
MYBPA1-R：5’-GATTAACAGTGACTCGGC-3’.
The section that 35s-F corresponds in the 35S promoter on carrier framework, MYBPA1-R corresponds in MsMYBPA1 genes
Section, target sequence length is about 950bp.
The positive callus of PCR identifications is to turn MsMYBPA1 gene callus.
8th, the MsMYBPA1 gene callus that turns that step 7 is obtained is seeded to screening and culturing medium (that is mould for card containing 50mg/L
Element, 250mg/L carboxylic Bians penicillin, 1mg/L 2,4-D and 0.5mg/L 6-BA solidified MS media) on carry out squamous subculture.
3rd, the acquisition of control tissue
Replace recombinant plasmid pRI101-MsMYBPA1 to carry out step 2 control plasmid, obtain turning crt gene callus group
Knit.
Replace recombinant plasmid pRI101-MsMYBPA1 to carry out step 2 in pRI101 carriers, obtain turning empty carrier callus group
Knit.
4th, the identification of tissue
The culture of callus using screening and culturing medium (kanamycins containing 50mg/L, 250mg/L carboxylic Bians penicillin,
1mg/L 2,4-D and 0.5mg/L 6-BA solidified MS media).
1st, DMACA dyeing identification
DMACA decoration method principles：In acid condition, can be with flavanol compound to dimethylaminocinnamaldehyde (DMACA)
(flavanols compounds include three classes to compound：The first kind is natural flavan-3-alcohol class；Equations of The Second Kind is flavane -3,4- glycols,
That is leucoanthocyanidin；3rd class is OPC) colour developing generation blueness, its color is in positive relationship with concentration.Bibliography：
《The optimization of tea root OPC extraction process and detection method》.
Callus to be measured is respectively：Turn MsMYBPA1 genes callus, turn empty carrier callus, ' Wang Lin ' apple
Spire callus.The incubation time of each callus to be measured is identical.
Operated in accordance with the following steps：
(1) 1 parts by volume methanol and 1 parts by volume 6M HCl/waters solution are mixed, obtains methanol-hydrochloric acid mixed solution.
(2) DMACA is dissolved in methanol-hydrochloric acid mixed solution, obtains 0.3g/100ml DMACA solution.
(3) callus to be measured is taken, 15min is dyed in DMACA solution.
Photo after DMACA dyeing is shown in Fig. 1.Turn MsMYBPA1 gene callus and navy blue dyed by DMACA, it is meant that
There is the accumulation of a large amount of flavanols compounds.Turn empty carrier callus and be not dyed to navy blue.The callus group of ' Wang Lin ' apple
Knit and be not dyed to navy blue.As a result show, MsMYBPA1 albumen can regulate and control the synthesis of flavanols compounds.
Carry out five repetitions to test, as a result unanimously.
2nd, anthocyanin Qualitative Identification
Callus to be measured is respectively：14 DEG C of Low- temperature cultures 30 days turn MsMYBPA1 genes callus, 14 DEG C of low temperature
Culture turns empty carrier callus, the spire callus of 14 DEG C of Low- temperature cultures, 30 days ' Wang Lin ' apple in 30 days.
Callus photo is shown in Fig. 2.Turn MsMYBPA1 gene callus substantially has part to become red after Low- temperature culture
Color, it is meant that have the accumulation of cyanine glycosides compound.Turning empty carrier callus does not have any color change.' Wang Lin ' apple
Callus does not have any color change.As a result show, MsMYBPA1 albumen can regulate and control cyanine glycoside chemical combination under cryogenic
The synthesis of thing.
Carry out five repetitions to test, as a result unanimously.
3rd, flavanols Quantitative measurement
Callus to be measured is respectively：Turn MsMYBPA1 genes callus, turn crt gene callus, turn empty carrier
Callus.The incubation time of each callus to be measured is identical.
Operated in accordance with the following steps：
(1) it is transferred to after taking 0.2g callus to be measured, liquid nitrogen grinding in centrifuge tube.
(2) centrifuge tube for taking step (1) to obtain, adds 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml
Aqueous acetone solution, collects remaining precipitation in supernatant, centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(3) centrifuge tube for taking step (2) to obtain, adds 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml
Aqueous acetone solution, collects remaining precipitation in supernatant, centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(4) centrifuge tube for taking step (3) to obtain, adds 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml
Aqueous acetone solution, collects remaining precipitation in supernatant, centrifuge tube by 4 DEG C, stand under dark condition and extract 30min.
(5) centrifuge tube for taking step (4) to obtain, adds 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml
Aqueous acetone solution, collects supernatant by 4 DEG C, stand under dark condition and extract 10 hours.
(6) supernatant and step that supernatant that the supernatant that obtains step (2), step (3) are obtained, step (4) are obtained
Suddenly the supernatant mixing that (5) are obtained, obtains mixed liquor.
(7) mixed liquor for taking step (6) to obtain, adds 3ml ether, -20 DEG C, stand that (split-phase, upper strata is under dark condition
Ether phase, lower floor is acetone phase, and flavanols compounds are mutually contained in lower floor), collect lower floor's phase.
(8) the lower floor's phase for taking 2ml steps (7) to obtain, adds the DMACA solution prepared in 1ml methanol and 0.5ml steps 1,
20min is stored at room temperature, then 643nm measures absorbance.
Standard curve is made with (+)-Catechin (sigma products), calibration curve equation is as follows：Y=2.816x-
0.012, R2=0.996 (y：Light absorption value；x：Flavanols compounds concentration, unit are mg/g).
Absorbance is brought into calibration curve equation, so that the flavanols compounds calculated in callus to be measured contain
Amount.
Carry out five repetitions to test, repeat to take the average value of 10 parts of callus to be measured in testing every time.
The flavanols compounds content for turning MsMYBPA1 gene callus is 137.422mg/kg.Turn crt gene to be cured
The flavanols compounds content of injured tissue is 71.314mg/kg.Turn the flavanols compounds content of empty carrier callus
For 43.418mg/kg.What " kg " in this section referred to is callus fresh weight.
4th, anthocyanin Quantitative measurement
Callus to be measured is respectively：14 DEG C culture 30 days turn MsMYBPA1 genes callus, 14 DEG C cultivate 30 days
Turn crt gene callus, 14 DEG C of cultures turn empty carrier callus within 30 days.
Operated in accordance with the following steps：
0.5g callus to be measured (0.5g callus grind into powders in liquid nitrogen to be measured) accurately is weighed,
Add 1% methanol hydrochloride solution of 5ml precoolings.4 DEG C of lucifuges extract 24h.2 parts of extract solutions (every part of 1ml) are taken, 1 part of extract solution adds
Enter 4ml KCl buffer solutions (pH=1.0) and mix 4 DEG C of lucifuges extraction 15min of standing, another 1 part of extract solution adds NaAc buffer solutions (pH
=4.5) mix 4 DEG C of lucifuges extraction 15min of standing.8000r/min centrifuges 10min, determines 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).
Carry out five repetitions to test, repeat to take the average value of 10 parts of callus to be measured in testing every time.
The cyanine glycosides compounds content for turning MsMYBPA1 gene callus is 69.642mg/kg.Turn crt gene to be cured
The cyanine glycosides compounds content of injured tissue is 32.168mg/kg.Turn the cyanine glycosides compounds content of empty carrier callus
For 18.219mg/kg.What " kg " in this section referred to is callus fresh weight.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application
<130> GNCYX170829
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 295
<212> PRT
<213>Apple
<400> 1
Met Gly Arg Ala Pro Cys Cys Ser Lys Val Gly Leu His Arg Gly Pro
1 5 10 15
Trp Thr Pro Arg Glu Asp Thr Leu Leu Thr Lys Tyr Ile Glu Ala His
20 25 30
Gly Glu Gly His Trp Arg Ser Leu Pro Lys Lys Ala Gly Leu Leu Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Asn Ile Thr Pro Asp Glu Asp Asp Leu Ile Ile Arg
65 70 75 80
Leu His Ser Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu
100 105 110
Ser Lys Arg Leu Arg Asn Glu Gly Thr Asp Pro Asn Thr His Lys Lys
115 120 125
Leu Ser Glu Pro Ile Ala Arg Glu Asn Lys Arg Arg Lys Asn Gln Arg
130 135 140
Ser Lys Ser Asn Asn Asn Lys Lys Glu Met Val Met Thr Lys Asp Lys
145 150 155 160
Asn Asn Lys Thr Ala Gln His Val Glu Pro Gln Lys Pro Lys Val His
165 170 175
Leu Pro Lys Pro Thr Arg Phe Thr Ser Phe Leu Ser Leu Pro Arg Asn
180 185 190
Asp Ser Phe Thr Ser Ser Thr Thr Val Thr Thr Gly Ser Ser Ser Gln
195 200 205
Asp Leu Asn Gly Gly Gly Gly Arg Gly Gly Gly Gly Gly Gly Phe Gly
210 215 220
Val Asn Thr Trp Cys Asn Asn Gly Gly Leu Val Phe Cys Val Gly Asp
225 230 235 240
Glu Asp Gln Asp His Asp Pro Ile Asn Ser Ser Ala Asp Gly Gly Asp
245 250 255
Asp His Thr Leu Glu Asn Leu Tyr Glu Glu Tyr Leu Gln Ala Leu Leu
260 265 270
Lys Ile Asp His His His Asp His Gln Asn Gln Leu Glu Leu Glu Ser
275 280 285
Phe Ala Glu Ser Leu Leu Ile
290 295
<210> 2
<211> 888
<212> DNA
<213>Apple
<400> 2
atgggaaggg ctccttgttg ttccaaggtt ggtttgcata gaggtccatg gactcctaga 60
gaagacacat tactcaccaa gtatattgaa gctcatggtg aaggccattg gagatccttg 120
ccaaaaaaag ctggcctcct caggtgtggg aagagttgca ggctaaggtg gatgaactat 180
ctaagaccag acataaagag aggcaacata acccccgatg aagatgacct aattatcaga 240
ctacattcac ttcttggcaa ccgttggtct ctcatcgccg gtaggcttcc gggtcgaacc 300
gataatgaga tcaagaacta ctggaacacc catcttagca aaagactcag aaacgaaggc 360
accgacccaa acacccacaa aaaattatct gagccgatag ccagggaaaa taaaaggaga 420
aagaaccaaa gaagcaagag caacaacaat aagaaggaga tggtgatgac gaaagacaag 480
aacaataaaa ctgcccaaca tgtggagcca caaaagccta aggttcatct cccaaaacct 540
actaggttta cttccttttt atccctacca agaaatgaca gttttactag tagtactaca 600
gttactactg ggtcttcaag ccaagactta aacggagggg gggggagagg aggaggagga 660
ggaggttttg gtgttaatac ttggtgtaat aatggtgggc ttgtgttttg tgttggtgat 720
gaagatcaag atcatgatcc tattaattct tcagctgatg gcggtgatga tcatacgctt 780
gaaaatctat atgaagaata tctacaggcg cttctgaaga tagaccatca tcatgatcat 840
caaaatcaac ttgaattaga gtcatttgcc gagtcactgt taatctga 888
<210> 3
<211> 293
<212> PRT
<213>Apple
<400> 3
Met Gly Arg Ala Pro Cys Cys Ser Lys Val Gly Leu His Arg Gly Pro
1 5 10 15
Trp Thr Pro Arg Glu Asp Thr Leu Leu Thr Lys Tyr Ile Glu Ala His
20 25 30
Gly Glu Gly His Trp Arg Ser Leu Pro Lys Lys Ala Gly Leu Leu Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Asn Ile Thr Pro Asp Glu Asp Asp Leu Ile Ile Arg
65 70 75 80
Leu His Ser Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu
100 105 110
Ser Lys Arg Leu Arg Asn Glu Gly Thr Asp Pro Asn Thr His Lys Lys
115 120 125
Leu Ser Glu Pro Ile Ala Arg Glu Asn Lys Arg Arg Lys Asn Gln Arg
130 135 140
Ser Lys Ser Asn Asn Asn Lys Lys Glu Met Val Met Thr Lys Asp Lys
145 150 155 160
Asn Asn Lys Thr Ala Gln His Val Glu Pro Gln Lys Pro Lys Val His
165 170 175
Leu Pro Lys Pro Thr Arg Phe Thr Ser Phe Leu Ser Leu Pro Arg Asn
180 185 190
Asp Ser Phe Thr Ser Ser Thr Thr Val Thr Thr Gly Ser Ser Ser Gln
195 200 205
Asp Leu Asn Gly Gly Gly Gly Arg Gly Gly Gly Gly Phe Gly Val Asn
210 215 220
Asn Trp Gly Asn Asn Gly Gly Leu Val Phe Cys Val Gly Asp Glu Asp
225 230 235 240
Gln Asp His Asp Pro Ile Asn Ser Ser Ala Asp Gly Gly Asp Asp His
245 250 255
Thr Leu Glu Asn Leu Tyr Glu Glu Tyr Leu Gln Ala Leu Leu Lys Ile
260 265 270
Asp His His His Asp His Gln Asn Gln Leu Glu Leu Glu Ser Phe Ala
275 280 285
Glu Ser Leu Leu Ile
290
<210> 4
<211> 882
<212> DNA
<213>Apple
<400> 4
atgggaaggg ctccttgttg ttccaaggtt ggtttgcata gaggtccatg gactcctaga 60
gaagacacat tactcaccaa gtatattgaa gctcatggtg aaggccattg gagatccttg 120
ccaaaaaaag ctggcctcct caggtgtggg aagagttgca ggctaaggtg gatgaactat 180
ctaagaccag acataaagag aggcaacata acccccgatg aagatgacct aattatcaga 240
ctacattcac ttcttggcaa ccgttggtct ctcatcgccg gtaggcttcc gggtcgaacc 300
gataatgaga tcaagaacta ctggaacacc catcttagca aaagactcag aaacgaaggc 360
accgacccaa acacccacaa aaaattatct gagccgatag ccagggaaaa taaaaggaga 420
aagaaccaaa gaagcaagag caacaacaat aagaaggaga tggtgatgac gaaagacaag 480
aacaataaaa ctgcccaaca tgtggagcca caaaagccta aggttcatct cccaaaacct 540
actaggttta cttccttttt atccctacca agaaatgaca gttttactag tagtactaca 600
gttactactg ggtcttcaag ccaagactta aacggagggg gagggagagg aggaggaggt 660
tttggtgtta ataattgggg taataatggt gggcttgtgt tttgtgttgg tgatgaagat 720
caagatcatg atcctattaa ttcttcagct gatggcggtg atgatcatac gcttgaaaat 780
ctatatgaag aatatctaca ggcgcttctg aagatagacc atcatcatga tcatcaaaat 840
caacttgaat tagagtcatt tgccgagtca ctgttaatct ga 882
Claims (10)
1. a kind of protein, is following (a1) or (a2) or (a3)：
(a1) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(a2) by the amino acid sequence of sequence 1 by the substitution and/or missing and/or addition of one or several amino acid residues and
The protein as derived from sequence 1 related to plant flavanols compounds content；
(a3) by the amino acid sequence of sequence 1 by the substitution and/or missing and/or addition of one or several amino acid residues and
The protein as derived from sequence 1 related to plant Anthocyanin Content.
2. encode the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that：The gene is following (1) or (2) or (3) or (4) or (5)：
(1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(2) hybridize and encode related with plant flavanols compounds content to the DNA sequence dna that (1) is limited under strict conditions
Protein DNA molecule；
(3) there is more than 90% homology to the DNA sequence dna that (1) is limited and coding is related with plant flavanols compounds content
Protein DNA molecule；
(4) hybridize under strict conditions to the DNA sequence dna that (1) is limited and encode the protein related with plant Anthocyanin Content
DNA molecular；
(5) there is more than 90% homology and the coding protein related with plant Anthocyanin Content to the DNA sequence dna that (1) is limited
DNA molecular.
4. recombinant expression carrier, expression cassette, transgenic cell line containing gene described in Claims 2 or 3, genetically modified plants group
Knit or recombinant bacterium.
5. the application of protein described in claim 1, is following (b1) and/or (b2) and/or (b3) and/or (b4)：
(b1) the flavanols compounds content of plant is regulated and controled；
(b2) the flavanols compounds content of plant is increased；
(b3) Anthocyanin Content of plant is regulated and controled；
(b4) Anthocyanin Content of plant is increased.
6. gene described in Claims 2 or 3 is planted in the increased transgenosis of content for cultivating flavanols compounds and/or anthocyanin
Application in thing.
7. a kind of method for cultivating genetically modified plants, comprises the following steps：Channel genes described in Claims 2 or 3 are set out plant
Thing, obtains genetically modified plants of the content higher than the plant that sets out of flavanols compounds and/or anthocyanin.
8. a kind of method for cultivating Transgenic plant tissue, comprises the following steps：Channel genes described in Claims 2 or 3 are gone out
Plant tissue is sent out, the content for obtaining flavanols compounds and/or anthocyanin is planted higher than the transgenosis of the plant tissue that sets out
Thing tissue.
9. a kind of plant breeding method, comprises the following steps：Improve the content of protein described in claim 1 in purpose plant
And/or activity, so as to improve the content of flavanols compounds and/or anthocyanin in purpose plant.
10. protein described in claim 1, or, gene described in Claims 2 or 3, or, side described in claim 7 or 8 or 9
Method, the application in plant breeding.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710249645.2A CN107033228B (en) | 2017-04-17 | 2017-04-17 | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application |
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| CN201710249645.2A CN107033228B (en) | 2017-04-17 | 2017-04-17 | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application |
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| CN112243345A (en) * | 2018-05-17 | 2021-01-19 | 安莎种子公司 | Solanaceae plant rich in anthocyanin |
| CN113683668A (en) * | 2020-11-06 | 2021-11-23 | 北京市农林科学院 | Application of AcAMS1 in regulation and control of synthesis of plant flavonoids |
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| CN105820224A (en) * | 2016-05-05 | 2016-08-03 | 山东农业大学 | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof |
| CN105820223A (en) * | 2016-05-05 | 2016-08-03 | 山东农业大学 | Anthocyanin-regulation protein MsMYB16 from functional apple and encoding gene and application thereof |
| CN105837669A (en) * | 2016-05-05 | 2016-08-10 | 山东农业大学 | Anthocyanin regulatory protein MsARF3 obtained from functional apples and encoding gene and application of protein |
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| CN105820224A (en) * | 2016-05-05 | 2016-08-03 | 山东农业大学 | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof |
| CN105820223A (en) * | 2016-05-05 | 2016-08-03 | 山东农业大学 | Anthocyanin-regulation protein MsMYB16 from functional apple and encoding gene and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112243345A (en) * | 2018-05-17 | 2021-01-19 | 安莎种子公司 | Solanaceae plant rich in anthocyanin |
| CN112243345B (en) * | 2018-05-17 | 2024-05-07 | 安莎种子公司 | Solanaceae plants enriched in anthocyanin concentration |
| CN113683668A (en) * | 2020-11-06 | 2021-11-23 | 北京市农林科学院 | Application of AcAMS1 in regulation and control of synthesis of plant flavonoids |
| CN113683668B (en) * | 2020-11-06 | 2023-05-30 | 北京市农林科学院 | Use of AcAMS1 in regulating and controlling plant flavonoid synthesis |
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| CN107033228B (en) | 2018-10-26 |
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