CN107090022B

CN107090022B - Obtained from the MsVGT1L albumen and its encoding gene of functional form apple and application

Info

Publication number: CN107090022B
Application number: CN201710249630.6A
Authority: CN
Inventors: 许海峰; 陈学森; 王楠; 姜生辉; 王意程; 毛志泉; 姜远茂
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2017-04-17
Filing date: 2017-04-17
Publication date: 2018-08-31
Anticipated expiration: 2037-04-17
Also published as: CN107090022A

Abstract

The invention discloses a kind of MsVGT1L albumen obtained from functional form apple and its encoding gene and applications.The present invention provides a kind of protein, are obtained from apple, are named as MsVGT1L albumen, are following (a1) or (a2)：(a1) protein that amino acid sequence forms shown in sequence in sequence table 1；(a2) by the amino acid sequence of sequence 1 by one or several amino acid residues substitution and/or lack and or add and with the relevant protein derived from sequence 1 of the total Flavonoid Content of plant.The gene of coding MsVGT1L albumen also belongs to protection scope of the present invention.The present invention also protects the application of MsVGT1L albumen, for following (b1) or (b2)：(b1) regulate and control total Flavonoid Content of plant；(b2) total Flavonoid Content of plant is reduced.Present invention finds MsVGT1L albumen and its function, it can be used for cultivating the genetically modified plants that total Flavonoid Content changes, there is major application foreground in plant breeding.

Description

Obtained from the MsVGT1L albumen and its encoding gene of functional form apple and application

Technical field

The present invention relates to a kind of MsVGT1L albumen obtained from functional form apple and its encoding gene and applications.

Background technology

" doctor's food homology " is developing direction, and " eating nutrition, eat health " has become the common recognition of people.Apple storage property is good, Supply cycle is long, is worldwide fruit, especially fruit contain higher proportion, human body be easier absorb free polyphenol, tool There is the effects that anti-oxidant, antitumor, prevention cardiovascular and cerebrovascular disease well and liver protection, healthy nutritive value is high, there is " one day apple Fruit, doctor is far from me " (An apple a day keeps the doctor away！) good reputation, considerable state in the world Family is all classified as major consumers fruit and is recommended energetically.Finding in recent years shows, on the one hand, in the past few decades state A apple variety more than the 1000 of inside and outside incubation 80% is the hybridization of kinds such as ' Jin Shuai ', is grown directly from seeds or bud selection offspring, this " close relative The problems such as breeding " often brings the hereditary basis of kind narrow and resistance decline；On the other hand, characteristic, more anti-and diversity Fruit becomes 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 MsVGT1L albumen obtained from functional form apple and its encoding gene and applications.

The present invention provides a kind of protein, are obtained from apple, are named as MsVGT1L albumen, are following (a1) or (a2)：

(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 the total Flavonoid Content of plant.

In order to make MsVGT1L albumen in (a1) convenient for purifying and detection, can in by sequence table amino shown in sequence 1 The amino terminal or the upper label as shown in Table 1 of carboxyl terminal connection of the protein of 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

MsVGT1L albumen in above-mentioned (a2) can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression It obtains.The encoding gene of MsVGT1L albumen in above-mentioned (b) can be by will lack in DNA sequence dna shown in sequence in sequence table 2 The codon of one or several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 ' The coded sequence that end and/or 3 ' ends connect label shown in table 1 obtains.

The gene (being named as MsVGT1L genes) of coding MsVGT1L albumen also belongs to protection scope of the present invention.

MsVGT1L genes are following (1) or (2) or (3)：

(1) DNA molecular shown in sequence 2 in coding region sequence table；

(2) hybridize and encode and the relevant egg of the total Flavonoid Content of plant with the DNA sequence dna that (1) limits under strict conditions The DNA molecular of white matter；

(3) DNA sequence dna limited with (1) has 90% or more homology and coding is relevant with the total Flavonoid Content of plant The DNA molecular of protein.

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 MsVGT1L genes or again Group bacterium all belongs to the scope of protection of the present invention.

The recombinant expression carrier of MsVGT1L 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 MsVGT1L 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 MsVGT1L 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 concretely in the SalI of pRI101 carriers and The recombinant plasmid that double chain DNA molecule obtains shown in the sequence 2 of insetion sequence table between KpnI restriction enzyme sites.

The plant tissue concretely plant callus, more specifically can be plant spire callus.The plant Can be monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant tool Body can be Malus.The Malus concretely apple, more specifically can be apple ' purplish red No. 1 '.

The present invention also protects the application of MsVGT1L albumen, for following (b1) or (b2)：

(b1) regulate and control total Flavonoid Content of plant；

(b2) total Flavonoid Content of plant is reduced.

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 that apple is ' purple Red No. 1 '.

The present invention also protects application of the MsVGT1L genes in cultivating the genetically modified plants that total Flavonoid Content reduces.Institute It can be monocotyledon or dicotyledon to state plant.The dicotyledon concretely rosaceous plant.The rose family Plant concretely Malus.The Malus concretely apple, more specifically can be apple ' purplish red No. 1 '.

The present invention also protects a kind of method for cultivating genetically modified plants, includes the following steps：MsVGT1L channel genes are gone out Plant is sent out, genetically modified plants of total Flavonoid Content less than the plant that sets out are obtained.The plant that sets out can be that unifacial leaf is planted Object or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant concretely Malus Plant.The Malus concretely apple, more specifically can be apple ' purplish red No. 1 '.The MsVGT1L genes specifically may be used Pass through the plant that sets out described in the importing of any description above recombinant expression carrier.It, specifically can be by the MsVGT1L bases in the method Because importing is set out the callus of plant, it is plant then to cultivate callus.The callus concretely be cured by spire Injured tissue.The recombinant expression carrier for carrying the MsVGT1L genes can be by Ti-plasmids, Ri plasmids, plant viral vector, straight The conventional biology methods such as DNA conversions, microinjection, conductance, agriculture bacillus mediated are connect to be transformed into plant cell or tissue.

The present invention also protects a kind of method for cultivating Transgenic plant tissue, includes the following steps：MsVGT1L genes are led Enter the plant tissue that sets out, obtains Transgenic plant tissue of total Flavonoid Content less than the plant tissue that sets out.It is described to set out 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 apple ' purplish red No. 1 '.It is described MsVGT1L genes can specifically pass through the plant tissue that sets out described in the importing of any description above recombinant expression carrier.The plant group Knit concretely plant callus.The callus concretely spire callus.

The present invention also protects a kind of plant breeding method, includes the following steps：Improve MsVGT1L albumen in purpose plant Content and/or activity, to reduce the content of total flavonoids in purpose plant.The purpose plant can be monocotyledon or double Cotyledon plant.The dicotyledon concretely rosaceous plant.The rosaceous plant concretely Malus.Institute Malus concretely apple is stated, more specifically can be apple ' purplish red No. 1 '.

The present invention also protects the MsVGT1L albumen or the MsVGT1L genes or any description above method to exist Application in plant breeding.The purpose of the plant breeding is the plant cultivated total Flavonoid Content and reduced.The plant can be Monocotyledon or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant specifically may be used For Malus.The Malus concretely apple, more specifically can be apple ' purplish red No. 1 '.

Present invention finds MsVGT1L albumen and its function, it can be used for cultivating the transgenosis that total Flavonoid Content changes and plant Object has major application foreground in plant breeding.

Description of the drawings

Fig. 1 is the photo of each callus in phenotypic evaluation.

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.Apple ' purplish red No. 1 ' (also known as ' purplish red No. 1 ' red meat apple)：Ginseng Examine document：《' purplish red No. 1 ' red meat apple pulp inoxidizability and anthocyanin analysis》.' red crisp No. 1 ' apple：Bibliography：《Newly 4 strain Flavonoid Contents of the boundary red meat apple first generation of hybrid and its synthesis related gene expression analysis》.

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 structural formula of rutin is as follows：

The discovery of embodiment 1, MsVGT1L albumen and its encoding gene

It is found that a new albumen is named as shown in the sequence 1 of sequence table from ' red crisp No. 1 ' apple MsVGT1L albumen.It is MsVGT1L genes, the sequence of open reading frame such as sequence table by the unnamed gene for encoding MsVGT1L albumen Shown in row 2.

Protein shown in sequence 3 by sequence table is named as reference protein (GENBANK ACCESSION NO.XP_ 008376911.1).The encoding gene of reference protein is named as crt gene, as shown in the sequence 4 of sequence table.

The Function Identification of embodiment 2, MsVGT1L albumen

One, construction recombination plasmid

1, construction recombination plasmid pRI101-MsVGT1L

(1) double chain DNA molecule shown in the sequence 2 of artificial synthesized sequence table.

(2) DNA molecular obtained using step (1) is carried out PCR amplification using the primer pair that F1 and R1 is formed, obtained as template To pcr amplification product.

F1：5’-GTCGACATGGCGTCCGATCCTGAG-3’；

R1：5’-GGTACCCTAAAGGCAATTGGCCTCAATT-3’。

(3) pcr amplification product obtained with restriction enzyme SalI and KpnI double digestion step (2), recycling digestion production Object.

(4) restriction enzyme SalI and KpnI double digestion pRI101 carriers are used, the carrier framework of about 10kb is recycled.

(5) digestion products of step (3) are connect with the carrier framework of step (4), obtains recombinant plasmid pRI101- MsVGT1L.According to sequencing result, structure is carried out to recombinant plasmid pRI101-MsVGT1L and is described as follows：In pRI101 carriers Double chain DNA molecule shown in the sequence 2 of sequence table is inserted between SalI and KpnI restriction enzyme sites.

2, control plasmid is built

Double chain DNA molecule shown in sequence 4 by artificial synthesized sequence table is inserted into the SalI and KpnI of pRI101 carriers Between restriction enzyme site, control plasmid is obtained.

Two, turn the acquisition of MsVGT1L gene callus

1, recombinant plasmid pRI101-MsVGT1L is imported into Agrobacterium LBA4404, obtains recombinational agrobacterium.

2, the recombinational agrobacterium that step 1 obtains is seeded to 30ml containing 50 μ g/ml kanamycins and 50 μ g/ml rifampins Liquid YEP medium, 28 DEG C of shaken cultivations to OD_600nmThalline were collected by centrifugation by=0.6,12000rpm, with 30ml ddH₂O suspends, Acetosyringone is added and makes its a concentration of 100 μM, obtains infecting liquid.

3, the spire callus for taking apple ' purplish red No. 1 ', be immersed into that step 2 obtains infects in liquid, shaken at room temperature 30min。

4, after completing step 3, callus is taken, the solidified MS media of 6-BA containing 1mg/L and 0.3mg/L NAA are placed in On, 28 DEG C of light cultures 2 days are then transferred into 6-BA containing 1mg/L, 0.3mg/L NAA, 50mg/L kanamycins and 50mg/L profits It is cultivated on the flat solidified MS media of good fortune 30 days or so (24 DEG C, 16h illumination/8h is dark).

5, after completing step 4, callus is taken, extracts genomic DNA, PCR is carried out using the primer pair that F2 and R2 is formed Identification, what PCR was accredited as the positive is to turn MsTMT1 gene callus.

F2：5’-GCTCCTACAAATGCCATCA-3’；

R2：5’-CTAAAGGCAATTGGCCTCAATT-3’.

F2 corresponds to the 35S promoter partial sequence on carrier framework, and R2 corresponds to the partial sequence on MsVGT1L genes, target Sequence length is about 1800bp.

6, complete step 5 after, take and turn MsVGT1L gene callus, be placed in 6-BA containing 1mg/L, 0.3mg/L NAA, Squamous subculture on the solidified MS media of 50mg/L kanamycins and 50mg/L rifampins.

Three, the acquisition of callus is compareed

It replaces recombinant plasmid pRI101-MsVGT1L to carry out step 2 control plasmid, obtains turning crt gene callus group It knits.

It replaces recombinant plasmid pRI101-MsVGT1L to carry out step 2 in pRI101 carriers, obtains turning empty carrier callus group It knits.

Four, phenotypic evaluation

The photo of each callus is shown in Fig. 1.Turn MsVGT1L bases after being cultivated 30 days in Fig. 1 be respectively step 26 Because callus, step 36 in cultivate 30 days after turn crt gene callus, step 36 in cultivate 30 days after Turn empty carrier callus and apple ' purplish red No. 1 ' spire callus (being indicated with WT) in contemporaneity.' purplish red No. 1 ' The callus of apple is shown as darkviolet, and total Flavonoid Content is higher.Turn MsVGT1L gene callus and is shown as light pink Color, total Flavonoid Content are relatively low.Turn empty carrier callus and the color of the callus of ' purplish red No. 1 ' apple is almost the same. Turn crt gene callus and be shown as the alternate color of purple-lightpink, total Flavonoid Content is cured higher than turning MsVGT1L genes Injured tissue and the callus for being less than ' purplish red No. 1 ' apple.

Five, total Flavonoid Content identification

Callus to be measured is respectively：Turn MsVGT1L genes callus, step after being cultivated 30 days in the 6 of step 2 After being cultivated 30 days in the 6 of three turn crt gene callus, step 36 in cultivate 30 days after turn empty carrier callus With ' purplish red No. 1 ' apple spire callus in contemporaneity.

It is as follows：

1,1g callus to be measured is weighed, with liquid nitrogen grinding at powder, then (volume basis contains the 65% of addition 5mL precoolings Amount) ethanol water, 4 DEG C are protected from light standing extraction 4h, and then 12000rpm centrifuges 20min, collects supernatant.

2, the supernatant for taking 0.5mL steps 1 to obtain sequentially adds 1mL 5g/100mL NaNO₂Aqueous solution, 1mL10g/ 100mL Al(NO₃)₃Aqueous solution and 4mL 2molL^-1NaOH aqueous solutions, mixing are then allowed to stand 15min.

3, after completing step 2, it is (water-soluble with the ethyl alcohol that volumn concentration is 80% to measure light absorption value at 510nm for sampling Liquid is as blank control).

With rutin standard items (rutin；Sigma chemical, St, Louis, USA, >=98%) make standard curve, mark Directrix curve equation is as follows：Y=0.9166x+0.0047 (R²=0.9997) (y：Light absorption value；x：Total flavonoids concentration, unit are Mg/g, " g " refer to the fresh weight of callus).

It carries out five repetitions to test, repeats each callus to be measured in testing every time and respectively take 10 parts, results are averaged.

It the results are shown in Table 2.

Table 2

SEQUENCE LISTING

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Gln Val Asp Glu Ile Leu Ala Glu Leu Ala Tyr Val Gly Glu Glu Lys

275 280 285

Glu Ala Thr Leu Gly Glu Met Phe His Gly Lys Cys Leu Lys Ala Leu

290 295 300

Val Ile Gly Val Gly Leu Val Leu Phe Gln Gln Ile Thr Gly Gln Pro

305 310 315 320

Ser Val Leu Tyr Tyr Ala Ala Ser Ile Phe Gln Ser Ala Gly Phe Ser

325 330 335

Glu Ala Ser Asp Ala Thr Arg Val Ser Ile Leu Leu Gly Val Phe Lys

340 345 350

Leu Ile Met Thr Gly Ala Ala Val Leu Val Val Asp Arg Leu Gly Arg

355 360 365

Arg Pro Leu Leu Leu Gly Gly Val Thr Gly Met Val Ile Ser Leu Phe

370 375 380

Leu Leu Gly Ser Tyr Tyr Leu Phe Phe Asp Asp Ala Pro Val Ala Ala

385 390 395 400

Val Val Ala Leu Leu Leu Tyr Val Gly Cys Tyr Gln Ile Ser Phe Gly

405 410 415

Pro Ile Gly Trp Leu Met Ile Ser Glu Val Phe Pro Leu Arg Leu Arg

420 425 430

Gly Arg Gly Leu Gly Ile Ala Val Leu Val Asn Phe Ala Ala Asn Ala

435 440 445

Leu Val Thr Phe Ala Phe Ser Pro Leu Lys Ala Leu Leu Gly Ala Gly

450 455 460

Ile Leu Phe Tyr Ala Phe Gly Val Ile Ala Val Ala Ser Leu Phe Phe

465 470 475 480

Ile Phe Phe Ile Val Pro Glu Thr Lys Gly Leu Thr Leu Glu Glu Ile

485 490 495

Glu Ala Asn Cys Leu

500

<210> 4

<211> 1506

<212> DNA

<213>Apple

<400> 4

atggcgtccg atcctgagca acacgcgccc tcttctctcg gaaaggtggg gaaatcttca 60

ggtgaaattg gcgacgcaca ggagccactt cttgaggggg ctcagaattc agaaaactat 120

tctcttattg ccgcaatcct accatttcta ttcccagctt ttggaggact actatatggc 180

tatgacattg gcgcaacatc atgcgctaca atttccatag agtcggccac attaagcgga 240

gtatcatggt acaacttgtc atctgtggaa attggtctca taactagtgg ctcattatat 300

ggggccttga ttggatcttt gctggccttt aacattgctg acttcttagg aagaaggagg 360

gagttgattt tggctgcttt attgtatctt ctcggaggcc tagtaacagc attagcaccc 420

gacttgcctg ttatggtgat tggacgcctc atatatggta taggaattgg actggcaatg 480

catgctgctc cgatgtatat tgccgagaca gctccaagtg cgatacgagg tcgcctaata 540

tcgctcaaag agttcttcat agtgcttggg atggttgcgg gttatggaat tggtagcctt 600

ttagttgaca cagtagctgg ttggcgctat atgtatggaa ttagtgcccc tttagcaata 660

attatgggta ttggaatgtg gtggctaccc gcatcaccta gatggatcct tctgcgtgcc 720

atacagggaa aaggaagtat ggatgagtta aaagtgactg caatcagttg cttgtgtcgg 780

cttaggggta gtgctattgg tgattcagct cctgcacaag tagatgagat actggctgag 840

cttgcttatg ttggtgaaga gaaagaagcc acattagggg agatgttcca tggaaaatgc 900

ttgaaagccc ttgtgattgg tgttggatta gtcttgttcc aacagatcac agggcaacca 960

agtgtgctgt actatgctgc atcgattttt cagagtgcag gattctctga agcatctgat 1020

gcaacacggg tatcaatttt acttggtgtt ttcaagttaa taatgactgg agcagctgtt 1080

cttgttgttg atagactcgg gaggagacct ctactacttg gaggtgttac ggggatggtc 1140

atctcattat tccttttggg atcatattac cttttctttg atgatgcacc agttgcagct 1200

gtagttgcac tgctgttgta tgttggatgt tatcagatat ctttcggtcc cattggttgg 1260

ctaatgatat cagaagtatt ccccttacgg ctaagggggc gaggactcgg tatagcagtg 1320

cttgtgaatt ttgctgcaaa tgcactggtg acatttgcat tttctcctct gaaggcattg 1380

ctgggagctg gaatattgtt ttatgcattt ggagtaatag cagtggcatc tctgtttttc 1440

attttcttca tcgtcccaga gaccaagggg cttacccttg aggaaattga ggccaattgc 1500

ctttag 1506

Claims

1. a kind of protein, the protein that amino acid sequence forms shown in sequence in sequence table 1.

2. encoding the gene of protein described in claim 1.

3. gene as claimed in claim 2, it is characterised in that：The gene is shown in sequence 2 in code area such as sequence table DNA molecular.

4. the recombinant expression carrier, expression cassette containing gene described in Claims 2 or 3 or recombinant bacterium.

5. the application of protein described in claim 1, to reduce total Flavonoid Content of Malus.

6. application of the gene described in Claims 2 or 3 in cultivating the genetically modified plants that total Flavonoid Content reduces；The plant For Malus.

7. a kind of method for cultivating genetically modified plants, includes the following steps：Channel genes described in Claims 2 or 3 are set out plant Object obtains genetically modified plants of total Flavonoid Content less than the plant that sets out；The plant is Malus.

8. a kind of method for cultivating Transgenic plant tissue, includes the following steps：Channel genes described in Claims 2 or 3 are gone out Plant tissue is sent out, Transgenic plant tissue of total Flavonoid Content less than the plant tissue that sets out is obtained；The plant is apple Fruit platymiscium.

9. 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 reduce the content of total flavonoids in purpose plant；The plant is Malus.

10. protein described in claim 1, or, gene described in Claims 2 or 3, or, claim 7 or 8 or 9 sides Method, the application in Malus breeding.