CN107033228B - Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application - Google Patents
Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application Download PDFInfo
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Abstract
The invention discloses a kind of flavanols obtained from functional form apple and anthocyanin modulin MsMYBPA1 and its encoding gene and applications.The MsMYBPA1 albumen protein that amino acid sequence forms shown in sequence in sequence table 1 provided by the invention.The present invention also protects the application of MsMYBPA1 albumen, for following (b1) and/or (b2) and/or (b3) and/or (b4)：(b1) regulate and control the flavanols compounds content of plant；(b2) increase the flavanols compounds content of plant；(b3) regulate and control the Anthocyanin Content of plant；(b4) increase the Anthocyanin Content of plant.Present invention finds MsMYBPA1 albumen and its function, the genetically modified plants that can be used for cultivating flavanols compounds and/or Anthocyanin Content changes have major application foreground in plant breeding.
Description
Technical field
The present invention relates to a kind of flavanols obtained from functional form apple and anthocyanin modulin MsMYBPA1 and its coding
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, have well it is anti-oxidant, antitumor, prevent cardiovascular and cerebrovascular
The effects that disease and liver protection, healthy nutritive value is high, there is " one day apple, doctor is far 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, a apple variety more than 1000 be 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 bring the hereditary basis of kind
The problems such as narrow and resistance declines；On the other hand, characteristic, more anti-and diversity fruits become 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 is extremely abundant, but also rich in functions, health-care components such as flavonoids, be carry out it is degeneration-resistant with
The precious gene pool of quality breeding.But because reasons, the genetic diversities of Malus sieversii such as the farmland reclamation of wasteland are just seriously damaged,
It is endangered；China is that maximum apple production and country of consumption mainly use wherein producing within 2012 39500000 tons of apple in the world
In fresh food, and nearly 70% is the lower 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 promoted to side structure reform and Practices Sustainable Increasing Income of Farmers and level of human health together ", 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 specifies that Malus sieversii group 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, the 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.Currently, having authorized and having declared
10 remainder of patent of invention is colonized more than 40,000 strain of hybrid seedling, is bred as new varieties (being) 16；Deliver correlative study paper 120
, wherein more than 20 piece of SCI papers, these achievements in research are totally in the top standard of international similar research.
Invention content
The object of the present invention is to provide a kind of flavanols obtained from functional form apple and anthocyanin modulin MsMYBPA1
And its encoding gene and application.
Protein provided by the invention is obtained from apple, is named as MsMYBPA1 albumen, be following (a1) or (a2) or
(a3)：
(a1) protein that amino acid sequence forms shown in sequence in sequence table 1；
(a2) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and with the relevant protein derived from sequence 1 of plant flavanols compounds content；
(a3) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and with the relevant protein derived from sequence 1 of plant Anthocyanin Content.
In order to make MsMYBPA1 albumen in (a1) convenient for purifying and detection, can in by sequence table ammonia 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 1 label of table
| 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 obtains.The encoding gene of above-mentioned (a2) or the MsMYBPA1 albumen in (a3) can be by by 2 institute of sequence in sequence table
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 hold the coded sequence for connecting label shown in table 1 to obtain at its 5 ' end and/or 3 '.
The gene (MsMYBPA1 genes) of coding MsMYBPA1 albumen also belongs to protection scope of the present invention.
MsMYBPA1 genes are following (1) or (2) or (3) or (4) or (5)：
(1) DNA molecular shown in sequence 2 in code area such as sequence table；
(2) hybridize and encode and plant flavanols compounds content phase with the DNA sequence dna that (1) limits under strict conditions
The DNA molecular of the protein of pass；
(3) DNA sequence dna limited with (1) has 90% or more homology and encodes and plant flavanols compounds content
The DNA molecular of relevant protein；
(4) hybridize and encode and the relevant albumen of plant Anthocyanin Content with the DNA sequence dna that (1) limits under strict conditions
The DNA molecular of matter；
(5) DNA sequence dna limited with (1) has 90% or more homology and encodes and the relevant egg of plant Anthocyanin Content
The DNA molecular of white matter.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
Recombinant expression carrier, expression cassette, transgenic cell line, Transgenic plant tissue containing MsMYBPA1 genes or again
Group bacterium all belongs to the scope of protection 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 can be used for the carrier etc. of plant micropellet bombardment.Use the gene constructed recombination tables of MsMYBPA1
When up to carrier, enhanced any type, composing type, organizing specific type or induction type can be added before its transcription initiation nucleotide and opened
Mover, they can be used alone or are used in combination with other plant promoters；In addition, using the gene constructed recombinations of MsMYBPA1
When expression vector, enhancer also can be used, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG
Initiation codon or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, to ensure entire sequence
Correct translation.The source of the translation control signal and initiation codon is extensive, can be natural, can also be to close
At.Translation initiation region can come from transcription initiation region or structural gene.For the ease of to transgenic plant cells or plant
Object is identified and is screened, and can be processed to plant expression vector used, and the expression in plant, which is such as added, can generate color change
The enzyme of the change or 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 recombination
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 obtains shown in the sequence 2 of insetion sequence table between BamH I restriction enzyme sites
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, for following (b1) and/or (b2) and/or (b3) and/or (b4)：
(b1) regulate and control the flavanols compounds content of plant；
(b2) increase the flavanols compounds content of plant；
(b3) regulate and control the Anthocyanin Content of plant；
(b4) increase the Anthocyanin Content of plant.
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 MsMYBPA1 genes in increased turn of content for cultivating 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, includes 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 sets out described in the importing of any description above recombinant expression carrier.In the method, 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
It can be routinely raw by Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated etc.
Object method is transformed into plant cell or tissue.
The present invention also protects a kind of method for cultivating Transgenic plant tissue, includes the following steps：By MsMYBPA1 genes
Importing is set out plant tissue, and the content for obtaining flavanols compounds and/or anthocyanin turns higher than the plant tissue that sets out
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 setting out described in the importing of any description above recombinant expression carrier
Object 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, includes the following steps：Improve MsMYBPA1 albumen in purpose plant
Content and/or activity, to improve the content of flavanols compounds and/or anthocyanin in purpose plant.The purpose is planted
Object 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 MsMYBPA1 albumen or MsMYBPA1 genes or any description above method in plant breeding
Application.The purpose of the plant breeding is the plant cultivated the content of flavanols compounds and/or anthocyanin and increased.It is described
Plant can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rose family is planted
Object concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.
Any description above flavanols compounds are the compound with female ring shown in formula (I)；
Any description above flavanols compounds 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 when=H
R1=OH, R2=H, R3It is epicatechin when=H
R1=H, R2=OH, R3It is nutgall catechin when=OH
R1=OH, R2=H, R3It is epigallocatechin when=OH
R1=OG, R2=H, R3It is L-Epicatechin gallate when=H
R1=OG, R2=H, R3It is Epigallo-catechin gallate (EGCG) when=OH
Present invention finds MsMYBPA1 albumen and its function, can be used for cultivating flavanols compounds and/or anthocyanin
The genetically modified plants that content changes have major application foreground in plant breeding.
Description of the drawings
Fig. 1 is the result of DMACA dyeing identifications.
Fig. 2 is the result of anthocyanin Qualitative Identification.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
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, that is, commercially available 12mol/L hydrochloric acid.
KCl buffer solutions (pH=1,0.025M)：1.86g KCl distill water dissolution with 980ml, are 1.0 with concentrated hydrochloric acid tune pH,
It is transferred in 1L volumetric flasks, with distilled water constant volume.
NaAC buffer solutions (pH=4.5,0.4M)：54.43g NaAC distill water dissolution with 960ml, are with concentrated hydrochloric acid tune pH
4.5, it is transferred in 1L volumetric flasks, with 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
It is found that a new albumen is named as shown in the sequence 1 of sequence table from " purplish red No. 1 " apple
MsMYBPA1 albumen.It is MsMYBPA1 genes, open reading frame such as sequence table by the unnamed gene for encoding MsMYBPA1 albumen
Sequence 2 shown in.
In GENBANK, with the highest albumen of 1 homology of sequence as shown in the sequence 3 of sequence table.By the sequence 3 of sequence table
Shown in protein be named as reference protein, encoding gene is named as crt gene (as shown in the sequence 4 of sequence table).
The Function Identification of embodiment 2, MsMYBPA1 albumen
One, construction recombination plasmid
Double chain DNA molecule shown in sequence 2 by artificial synthesized sequence table is inserted into 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 sequence 4 by artificial synthesized sequence table is inserted into the Nde I and BamH of pRI101 carriers
Between I restriction enzyme sites, control plasmid is obtained.
Two, the acquisition of genetically modified organism
1, recombinant plasmid pRI101-MsMYBPA1 is imported into Agrobacterium LBA4404, obtains recombinational agrobacterium.
2, the recombinational agrobacterium thalline that step 1 obtains is taken, is suspended with 30ml liquid MS mediums, obtains OD600nm=0.8
Bacteria suspension is added 30 μ L 100mM acetosyringone solution (solvent DMSO), obtains infecting liquid.
3, the spire callus for taking ' Wang Lin ' apple, is seeded on solidified MS media, 25 DEG C of dark culturings 15 days.
4, after completing step 3, callus is taken, be immersed in that step 2 obtains infects in liquid, 160rpm shaken at room temperature 15-
Then 20min blots surface with sterilizing filter paper.
5, after completing step 4, callus is taken, is placed in and co-cultures culture medium (containing 1mg/L 2,4-D and 0.5mg/L6-BA
Solidified MS media) on, 25 DEG C of dark culturings 36 hours.
6, after completing step 5, callus is taken, screening and culturing medium (kanamycins containing 50mg/L, 250mg/L carboxylic Bians are placed in
Penicillin, 1mg/L 2, the solidified MS media of 4-D and 0.5mg/L 6-BA) on, 25 DEG C of dark culturings 20-30 days.
7, after completing step 6, the resistant calli that can be grown on screening and culturing medium is taken, carries out PCR identifications.
PCR identifications primer pair used is as follows：
35s-F：5'-GACGCACAATCCCACTATCC-3'；
MYBPA1-R：5'-GATTAACAGTGACTCGGC-3'.
35s-F corresponds to the section in the 35S promoter on carrier framework, and 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.
8, the MsMYBPA1 gene callus that turns that step 7 obtains 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.
Three, the acquisition of control tissue
It replaces recombinant plasmid pRI101-MsMYBPA1 to carry out step 2 control plasmid, obtains turning crt gene callus group
It knits.
It replaces recombinant plasmid pRI101-MsMYBPA1 to carry out step 2 in pRI101 carriers, obtains turning empty carrier callus group
It knits.
Four, the identification organized
The culture of callus be all made of screening and culturing medium (kanamycins containing 50mg/L, 250mg/L carboxylic Bians penicillin,
The solidified MS media of 1mg/L 2,4-D and 0.5mg/L 6-BA).
1, DMACA dyeing identification
DMACA decoration method principles：It in acid condition, can be with flavanol compound to dimethylaminocinnamaldehyde (DMACA)
Closing object, (flavanols compounds include three classes：The first kind is natural flavan-3-alcohol class；Second class is flavane -3,4- glycols,
That is leucoanthocyanidin；Third class is procyanidine) colour developing generation blue, color is in positive relationship with concentration.Bibliography：
《The optimization of tea root procyanidine 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.
It is operated in accordance with the following steps：
(1) 1 parts by volume methanol and 1 parts by volume 6M HCl/water solution are mixed, obtains methanol-hydrochloric acid mixed solution.
(2) DMACA is dissolved in methanol-hydrochloric acid mixed solution, obtains the DMACA solution of 0.3g/100ml.
(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 is not dyed to navy blue.The callus group of ' Wang Lin ' apple
It knits and is not dyed to navy blue.The result shows that MsMYBPA1 albumen can regulate and control the synthesis of flavanols compounds.
It carries out five repetitions to test, as a result unanimously.
2, 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
The spire callus for turning empty carrier callus, 14 DEG C of Low- temperature cultures, 30 days ' Wang Lin ' apple of culture 30 days.
Callus photo is shown in Fig. 2.Turn MsMYBPA1 gene callus obviously 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.The result shows that MsMYBPA1 albumen can regulate and control cyanine glycoside chemical combination under cryogenic
The synthesis of object.
It carries out five repetitions to test, as a result unanimously.
3, flavanols Quantitative measurement
Callus to be measured is respectively：Turn MsMYBPA1 genes callus, turns crt gene callus, turns empty carrier
Callus.The incubation time of each callus to be measured is identical.
It is operated in accordance with the following steps：
(1) 0.2g callus to be measured is taken, is transferred in centrifuge tube after liquid nitrogen grinding.
(2) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (1) to obtain
Aqueous acetone solution, collects supernatant by 4 DEG C, extraction 30min is stood under dark condition, remaining precipitation in centrifuge tube.
(3) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (2) to obtain
Aqueous acetone solution, collects supernatant by 4 DEG C, extraction 30min is stood under dark condition, remaining precipitation in centrifuge tube.
(4) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (3) to obtain
Aqueous acetone solution, collects supernatant by 4 DEG C, extraction 30min is stood under dark condition, remaining precipitation in centrifuge tube.
(5) 70% (volumn concentration) of 1ml ascorbic acid containing 1mg/ml is added in the centrifuge tube for taking step (4) to obtain
Aqueous acetone solution, collects supernatant by 4 DEG C, extraction 10 hours are stood under dark condition.
(6) supernatant and step obtained supernatant that supernatant that step (2) obtains, step (3) obtain, step (4)
Suddenly the supernatant mixing that (5) obtain, obtains mixed liquor.
(7) mixed liquor for taking step (6) to obtain, is added 3ml ether, -20 DEG C, stand that (split-phase, upper layer are under dark condition
Ether phase, lower layer are acetone phase, and flavanols compounds are mutually contained in lower layer), collect lower layer's phase.
(8) the DMACA solution prepared in 1ml methanol and 0.5ml steps 1 is added in the lower layer's phase for taking 2ml steps (7) to obtain,
It is stored at room temperature 20min, 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 mg/g).
It brings absorbance into calibration curve equation, contains to calculate the flavanols compounds in callus to be measured
Amount.
It carries out five repetitions to test, repeats the average value for taking 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.
4, 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 in 30 days.
It is operated in accordance with the following steps：
0.5g callus to be measured (0.5g callus grind into powder in liquid nitrogen to be measured) accurately is weighed,
1% methanol hydrochloride solution of 5ml precoolings is added.4 DEG C are protected from light extraction for 24 hours.2 parts of extracting solutions (every part of 1ml), 1 part of extracting solution are taken to add
Enter 4 DEG C of 4ml KCl buffer solutions (pH=1.0) mixing standing and be protected from light extraction 15min, NaAc buffer solutions (pH is added in another 1 part of extracting solution
=4.5) mixing stands 4 DEG C and is protected from light extraction 15min.8000r/min centrifuges 10min, measures 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).
It carries out five repetitions to test, repeats the average value for taking 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 functional form apple flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene and answer
With
<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 (9)
1. a kind of protein, the protein that amino acid sequence forms shown in sequence in sequence table 1.
It is DNA molecular shown in sequence 2 in code area such as sequence table 2. encoding the gene of protein described in claim 1.
3. the recombinant expression carrier, expression cassette containing gene described in claim 2 or recombinant bacterium.
4. the application of protein described in claim 1, for following (b2) and/or (b4)：
(b2) increase the flavanols compounds content of apple；
(b4) increase the Anthocyanin Content of apple.
5. gene described in claim 2 is in the increased transgenic apples of content for cultivating flavanols compounds and/or anthocyanin
In application.
6. a kind of method for cultivating genetically modified plants, includes the following steps：Channel genes described in claim 2 are set out plant,
Obtain genetically modified plants of the content higher than the plant that sets out of flavanols compounds and/or anthocyanin；The plant is apple
Fruit.
7. a kind of method for cultivating Transgenic plant tissue, includes the following steps：Channel genes described in claim 2 are set out plant
Object tissue obtains genetically modified plants group of the content higher than the plant tissue that sets out of flavanols compounds and/or anthocyanin
It knits；The plant is apple.
8. a kind of plant breeding method, includes the following steps：Improve the content of protein described in claim 1 in purpose plant
And/or activity, to improve the content of flavanols compounds and/or anthocyanin in purpose plant；The plant is apple.
9. protein described in claim 1, or, gene described in claim 2, or, claim 6 or 7 or 8 the methods,
Application in plant breeding；The plant is apple.
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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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| WO2019219200A1 (en) * | 2018-05-17 | 2019-11-21 | Enza Zaden Beheer B.V. | A solanaceae plant rich in anthocyanins concentration |
| 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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| 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 |
| 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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