CN106243207B - Obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application - Google Patents

Obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application Download PDF

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CN106243207B
CN106243207B CN201610789471.4A CN201610789471A CN106243207B CN 106243207 B CN106243207 B CN 106243207B CN 201610789471 A CN201610789471 A CN 201610789471A CN 106243207 B CN106243207 B CN 106243207B
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plant
mssut4
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albumen
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许海峰
陈学森
王楠
姜生辉
王意程
毛志泉
姜远茂
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Shandong Agricultural University
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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

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Abstract

The invention discloses a kind of obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application.The present invention provides a kind of protein, are named as MsSUT4 albumen, are following (a1) or (a2): the protein that (a1) amino acid sequence shown in sequence 1 in sequence table forms;(a2) by the amino acid sequence of sequence 1 by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to the total Flavonoid Content of plant.The gene for encoding the MsSUT4 albumen also belongs to protection scope of the present invention.The present invention also protects the application of the MsSUT4 albumen, regulates and controls total Flavonoid Content of plant for following (b1) or (b2): (b1);(b2) increase total Flavonoid Content of plant.Present invention finds MsSUT4 albumen and its function, it can be used for cultivating the genetically modified plants that total Flavonoid Content changes, there is major application prospect in plant breeding.

Description

Obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application
Technical field
The present invention relates to a kind of obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application.
Background technique
" 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 of 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 maximum apple production and country of consumption in the world, wherein 39,500,000 tons of apple of production in 2012, main to use 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 and the apple variety first generation of hybrid and one, two generation segregating populations of backcrossing, research specify that Malus sieversii group loses Pass the hereditary variation feature of the characters such as technical parameter, the Flavonoid Content that structure and genetic diversity feature, Core Germplasms construct And development mechanism, propose " functional form apple " concept 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 of conventional hybridization and biotechnology combination, 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, incubation new varieties (being) 16;Deliver correlative study paper 120 , wherein more than 20 piece of SCI paper, these research achievements are totally in the top standard of international similar research.
Summary of the invention
The object of the present invention is to provide a kind of obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application.
The present invention provides a kind of protein, are obtained from apple, are named as MsSUT4 albumen, are following (a1) or (a2):
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms;
(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 the protein as derived from sequence 1 relevant to the total Flavonoid Content of plant.
In order to make MsSUT4 albumen in (a) convenient for purifying and detection, can in as sequence table amino acid shown in sequence 1 The amino terminal or carboxyl terminal of the protein of sequence composition connect upper label as shown in Table 1.
The sequence of 1 label of table
Label Residue Sequence
Poly-Arg 5-6 (usually 5) RRRRR
Poly-His 2-10 (usually 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
MsSUT4 albumen in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain It arrives.The encoding gene of MsSUT4 albumen in above-mentioned (b) can be by will lack one in DNA sequence dna shown in sequence 2 in sequence table The codon of a or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.
The gene (MsSUT4 gene) for encoding the MsSUT4 albumen also belongs to protection scope of the present invention.
The gene is concretely following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in coding region sequence table;
(2) the DNA sequence dna hybridization limited under strict conditions to (1) and coding egg relevant with the total Flavonoid Content of plant The DNA molecular of white matter;
(3) there is 90% or more homology to the DNA sequence dna that (1) limits and coding is relevant with the total Flavonoid Content of plant The DNA molecular of protein.
Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be 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 the MsSUT4 gene or Recombinant bacterium all belongs to the scope of protection of the present invention.
The recombinant expression carrier of MsSUT4 gene can be contained with existing plant expression vector construction.The plant expression carries Body includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.It is carried using the gene constructed recombinant expression of MsSUT4 It, can be before its transcription initiation nucleotide plus any enhanced, composing type, organizing specific type or induction type starting when body Son, they can be used alone or are used in combination with other plant promoters;In addition, using the gene constructed recombinant expression of MsSUT4 When carrier, enhancer, including translational enhancer or transcriptional enhancer also can be used, these enhancer regions can be ATG starting Codon or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, to guarantee entire sequence just Really translation.The source of the translation control signal and initiation codon be it is extensive, can be natural, be also possible to synthesize 's.Translation initiation region can come from transcription initiation region or structural gene.For the ease of to transgenic plant cells or plant It is identified and is screened, plant expression vector used can be processed, the expression in plant, which is such as added, can produce color change Enzyme or gene, resistant antibiotic marker or the anti-chemical reagent marker gene of luminophor etc..From turning base Because of the security consideration of plant, any selected marker can be not added, directly with phenotypic screen transformed plant.The recombination table The carrier that sets out up to carrier can be pRI101 carrier.The recombinant expression carrier concretely in the BamHI of pRI101 carrier and The recombinant plasmid that double chain DNA molecule shown in the sequence 2 of insetion sequence table obtains between NotI restriction enzyme site.
The plant tissue concretely plant callus, more specifically can be plant spire callus.The plant It 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 ' Wang Lin ' apple.
The present invention also protects the application of the MsSUT4 albumen, for as follows (b1) or (b2):
(b1) regulate and control total Flavonoid Content of plant;
(b2) increase total Flavonoid 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 the MsSUT4 gene cultivating the application in the genetically modified plants that total Flavonoid Content increases.
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 a kind of method for cultivating genetically modified plants, includes the following steps: to lead the MsSUT4 gene Enter the plant that sets out, obtains the genetically modified plants that total Flavonoid Content is higher than the plant that sets out.
The plant that sets out can be monocotyledon or dicotyledon.The dicotyledon concretely plant by rosaceae Object.The rosaceous plant concretely Malus.The Malus concretely apple, more specifically can be ' king Woods ' apple.The MsSUT4 gene can specifically pass through the plant that sets out described in the importing of any description above recombinant expression carrier.It is described In method, then the callus for plant that specifically the MsSUT4 channel genes can set out is cultivated callus for plant. The callus concretely spire callus.The recombinant expression carrier for carrying the MsSUT4 gene can be by Ti matter The conventional biology methods such as grain, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus turn Change into plant cell or tissue.
The present invention also protects a kind of method for cultivating Transgenic plant tissue, includes the following steps: the MsSUT4 base Because importing is set out plant tissue, the Transgenic plant tissue that total Flavonoid Content is higher than the plant tissue that sets out is obtained.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 MsSUT4 gene can specifically pass through the plant tissue that sets out described in the importing of any description above recombinant expression carrier.The plant tissue Concretely plant callus.The callus concretely spire callus.
The present invention also protects a kind of plant breeding method, includes the following steps: to increase MsSUT4 albumen described in plant Content, so that total Flavonoid Content in plant be promoted to increase.
The present invention also protects the MsSUT4 albumen or the MsSUT4 gene or any description above method planting Application in object breeding.The purpose of the plant breeding is the plant cultivating total Flavonoid Content and increasing.The plant can be single Cotyledon plant or dicotyledon.The dicotyledon concretely rosaceous plant.The rosaceous plant is concretely Malus.The Malus concretely apple, more specifically can be ' Wang Lin ' apple.
Present invention finds MsSUT4 albumen and its function, it can be used for cultivating the transgenosis that total Flavonoid Content changes and plant Object has major application prospect in plant breeding.
Detailed description of the invention
Fig. 1 is the result of phenotypic evaluation.
Specific embodiment
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.
' Wang Lin ' apple (Wang Lin apple): bibliography: " The bHLH transcription factor MdbHLH3promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples".PRI101 carrier (also known as " pRI101-AN DNA "): Takala company, Code No.3262.Agrobacterium LBA4404: Tiangen company, catalog number: CC2901.
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, MsSUT4 albumen and its encoding gene
Have found that a new albumen is named as shown in the sequence 1 of sequence table from ' red crisp No. 1 ' apple MsSUT4 albumen.It is MsSUT4 gene, the sequence 2 of open reading frame such as sequence table by the unnamed gene for encoding MsSUT4 albumen It is shown.
Protein shown in sequence 3 by sequence table is named as reference protein (GENBANK ACCESSION NO.XP_ 017183866.1).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, MsSUT4 albumen
One, construction recombination plasmid
1, construction recombination plasmid pRI101-MsSUT4
(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 '-GGATCCATGCCAGCTCCAGAAGCA-3';
R1:5 '-GCGGCCGCTCATGTGACAGCTCTGGGCT-3’
(3) pcr amplification product obtained with restriction enzyme BamHI and NotI double digestion step (2), recycling digestion produce Object.
(4) restriction enzyme BamHI and NotI double digestion pRI101 carrier 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- MsSUT4.According to sequencing result, structure is carried out to recombinant plasmid pRI101-MsSUT4 and is described as follows: in pRI101 carrier Double chain DNA molecule shown in the sequence 2 of sequence table is inserted between BamHI and NotI restriction enzyme site.
2, control plasmid is constructed
The BamHI and NotI of the insertion pRI101 carrier of double chain DNA molecule shown in sequence 4 by artificial synthesized sequence table Between restriction enzyme site, control plasmid is obtained.
Two, turn the acquisition of MsSUT4 gene callus
1, recombinant plasmid pRI101-MsSUT4 is imported into Agrobacterium LBA4404, obtains recombinational agrobacterium.
2, the recombinational agrobacterium for obtaining step 1 is seeded to 30ml containing 50 μ g/ml kanamycins and 50 μ g/ml rifampins YEP fluid nutrient medium, 28 DEG C of shaken cultivations to OD600nmThalline were collected by centrifugation by=0.6,12000rpm, with 30ml ddH2O suspends, Acetosyringone is added and makes 100 μM of its concentration, obtains infected liquid.
3, Wang Lin apple spire callus is taken, is immersed into the infected liquid that step 2 obtains, shaken at room temperature 30min.
4, after completing step 3, callus is taken, is placed in containing 1mg/L 2, the MS solid culture of 4-D and 0.5mg/L 6-BA On base, 28 DEG C dark culture 2 days, be then transferred into containing 1mg/L 2,4-D, 0.5mg/L 6-BA, 50mg/L kanamycins and 50mg/ Continue dark culture 30 days or so on the MS solid medium of L rifampin.
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 MsSUT4 gene callus.
F2:5 '-GCTCCTACAAATGCCATCA-3 ';
R2:5 '-TCATGTGACAGCTCTGGG-3 '.
F2 corresponds to the 35S promoter partial sequence on carrier framework, and R2 corresponds to the partial sequence on MsSUT4 gene, target sequence Column length is about 1600bp.
6, after completing step 4, take and turn MsSUT4 gene callus, be placed in containing 1mg/L 2,4-D, 0.5mg/L 6-BA, Squamous subculture on the MS solid medium of 50mg/L kanamycins and 50mg/L rifampin.
Three, the acquisition of callus is compareed
It replaces recombinant plasmid pRI101-MsSUT4 to carry out step 2 control plasmid, obtains turning crt gene callus.
It replaces recombinant plasmid pRI101-MsSUT4 to carry out step 2 in pRI101 carrier, obtains turning empty carrier callus.
Four, phenotypic evaluation
The photo of each callus is shown in Fig. 1.In Fig. 1 be respectively step 26 in cultivate 30 days after turn MsSUT4 base 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 the Wang Lin apple spire callus in contemporaneity.
Wang Lin apple callus color is partially white, and total Flavonoid Content is lower.Turn MsSUT4 gene callus color compared with Huang, total Flavonoid Content improve.Turn empty carrier callus and the callus color of Wang Lin apple is almost the same.Turn control base Because callus is shown as the alternate color of white Huang, total Flavonoid Content, which is lower than, to be turned MsSUT4 gene callus and is higher than Wang Lin The callus of apple.
Five, total Flavonoid Content identification
Callus to be measured, which is respectively as follows: after cultivating 30 days in the 6 of step 2, turns MsSUT4 gene callus, step 3 6 in cultivate 30 days after turn crt gene callus, step 36 in cultivate 30 days after turn empty carrier callus and Wang Lin apple spire callus in contemporaneity.
Specific step 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 pre-cooling Amount) ethanol water, 4 DEG C are protected from light standing extraction 4h, and then 12000rpm is centrifuged 20min, collects supernatant.
2, the supernatant for taking 0.5mL step 1 to obtain sequentially adds 1mL 5g/100mL NaNO2Aqueous solution, 1mL10g/ 100mL Al(NO3)3Aqueous solution and 4mL 2molL-1NaOH aqueous solution mixes, is 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%) production 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 test every time and respectively take 10 parts, results are averaged.
It the results are shown in Table 2.
Table 2

Claims (10)

1. a kind of protein, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.
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 code area as shown in sequence 2 in sequence table DNA molecular.
4. recombinant expression carrier, expression cassette or recombinant bacterium containing gene described in Claims 2 or 3.
5. the application of protein described in claim 1, for the total Flavonoid Content for increasing plant.
6. gene described in Claims 2 or 3 is cultivating the application in the genetically modified plants that total Flavonoid Content increases.
7. a kind of method for cultivating genetically modified plants, includes the following steps: plant that channel genes described in Claims 2 or 3 set out Object obtains the genetically modified plants that total Flavonoid Content is higher than the plant that sets out.
8. a kind of method for cultivating Transgenic plant tissue, includes the following steps: channel genes described in Claims 2 or 3 Plant tissue is sent out, the Transgenic plant tissue that total Flavonoid Content is higher than the plant tissue that sets out is obtained.
9. a kind of plant breeding method includes the following steps: the content for increasing protein described in claim 1 in plant, thus Total Flavonoid Content in plant is promoted to increase.
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 plant breeding.
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CN101319209A (en) * 2008-06-05 2008-12-10 中国科学院植物研究所 Total flavone synthetic associated protein, encoding gene and application thereof

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Publication number Priority date Publication date Assignee Title
CN101319209A (en) * 2008-06-05 2008-12-10 中国科学院植物研究所 Total flavone synthetic associated protein, encoding gene and application thereof

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Title
Accession NO.: XP_017183866.1,PREDICTED: sucrose transport protein SUC4-like [Malus domestica];None;《Genbank Database》;20160622;DEFINITION、SOURCE、FEATURES及ORIGIN部分
液泡膜转运蛋白结构与功能的研究进展;胡军瑜等;《河南农业科学》;20091231(第6期);第5-10页

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