CN108503699A - Matrimony vine gene and its coding protein, recombinant vector, and application thereof - Google Patents
Matrimony vine gene and its coding protein, recombinant vector, and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of matrimony vine gene and its coding protein, recombinant vector, and application thereof.The amino acid sequence of the matrimony vine gene is as shown in SEQ ID NO.1 or SEQ ID NO.2.Nucleotide sequence is as shown in SEQ ID NO.3 or SEQ ID NO.4.The invention also discloses the recombinant vector containing the gene, expression cassette, transgenic cell line, recombinant bacterium or host cells.The invention also discloses the gene regulation and control anthocyanidin synthesis in application and a kind of regulation and control plant in anthocyanidin synthetic method.The present invention selects lycium ruthenicum LMH1 to be sequenced with red matrimony vine Ningqi7 fruit transcript profiles, and analysis predicts the major gene resistance of control lycium ruthenicum black fruit properties, provides the foundation to research matrimony vine anthocyanidin synthesis mechanism.Also direction and target spot are provided to improve the content of anthocyanidin for genetic engineering means plant modification.
Description
Technical field
The invention belongs to plant genetic engineering field, it is related to regulating and controlling the Gene A N2 of anthocyanidin synthesis and metabolism, including the base
Recombinant plasmid, host and its application of cause.
Background technology
Lycium ruthenicum is a kind of distinctive wild plant resource in northwest China Desert Area.In Tibetan medicine classical works《Brilliant pearl sheet
Grass》Middle lycium ruthenicum is a kind of traditional Chinese herbal medicine, has used thousands of years of history in China, it can be used for treating heart disease, menstruation
Uncomfortable and climacteric etc. (J.Zheng etc., 2011).Lycium ruthenicum plant has the characteristics that the special physiological of drought resistance and salt tolerance,
It can prevent soil desertification from alleviating soil salinization degree, this is all extremely important to the ecosystem and agricultural of border district
(H.Zhang etc., 2007).Anthocyanidin content can reach 3.1% in the fruit of lycium ruthenicum.Petunia pigment accounts for total flower
The 95% of green element, delphinidin and malvidin account for remaining 5%.For human health, anthocyanidin be considered to have it is anti-inflammatory,
Anti- mutation, anticancer and it is anti-oxidant and other effects (C.S.Bowen-Forbes etc., 2010；L.S.Wang etc., 2008；G.Mazza.,
2007).Pharmacological evaluation proves, lycium ruthenicum have it is antifatigue, hypoglycemic and anti-oxidant and other effects (W.Feng etc., 2010；
J.H.Wang etc., 2007).Although research has been carried out to its chemical composition and pharmacological properties, spent in lycium ruthenicum fruit
The high molecular genetic mechanism of green cellulose content is unclear.
The biosynthesis pathway of anthocyanidin has the research of decades in model plant.Due to being easy to the characteristic of observation,
Anthocyanidin anabolism regulatory pathway is to study to obtain more clear secondary metabolism access (forests and streams, 1998).The synthesis of anthocyanidin
First coumaric acyl CoA, the coumaric acyl CoA of 1 molecule and 3 molecule malonyl CoA are generated by three steps of enzymatic reactions by phenylalanine
Chalcone is generated under chalcone synthase (CHS) catalysis, chalcone seldom accumulates, in the catalysis of enzyme, namely chalcone isomerase (CHI)
Under, isomerization quickly forms naringenin (flavanones).Flavanones is under flavonoids -3- hydroxylases (F3H) catalysis, 3 hydroxyl of C ring positions
Change forms flavanonol.Flavanonol is the substrate of other two kinds of enzymes B rings hydroxylase i.e. F3'H and F3'5'H.F3H, class are yellow
Ketone -3'- hydroxylases (F3'H), flavonoids -3', 5'- hydroxylase (F3'5'H) belong to P450 superfamilies.By F3H, F3'H, F3'5'
The product of tri- kinds of be catalyzed reactions of hydroxylase of H is the direct precursor of the synthesis of anthocyanins.The hydroxylated degree of flavanonol and
Position is different, the type of the anthocyanin finally synthesized will be determined, to determine the color of flower, seed, fruit.From flavanone
The reaction that alcohol is transformed into anthocyanin is extremely complex, needs the effect (Johnson etc., 2001) of several different enzymes.ANS is double adds
Oxygenase is catalyzed the transformation from leucoanthocyanidin to anthocyanidin.After unstable anthocyanin formation, further glycosylation formation is stablized
Anthocyanin (Mato etc., 2001).In addition tri- transcription factors of MYB, bHLH and WD40 have anthocyanidin synthetic gene and adjust
Relationship (Koes etc., 2005) between control effect.Some myb genes can regulate and control the expression of bHLH transcription factors, and then be formed
One complex for including WD40 albumen.WD40 whether appear in this start DFR complexs in there is also a little queries.MYB
It can be bonded directly on DNA, and bHLH is combined possibly via an imaginary binding protein with DNA.Small R3-MYB
Be likely to can with bHLH protein bindings, to prevent its enter transcripting starting complex, start DFR transcription (Zhang etc.,
2014)。
By real-time polymerase chain reaction, comparative analysis, but this have been carried out to the accumulation of anthocyanidin in lycium ruthenicum
It is to be obtained from blade transcript profile database (S.Zeng etc., 2014) to synthesize relevant gene with anthocyanidin a bit, it means that certain
Specifically expressed gene should be missed in fruit a bit.In addition, lycium barbarum and lycium ruthenicum belong in Solanaceae family
In variety classes.It is necessary to evaluate the architectural difference and its regulatory factor of structural gene in the fruit of lycium barbarum and lycium ruthenicum,
And compare their transcriptional level.
High-flux sequence of new generation has proved to be the gene order-checking tool of a low-cost high-efficiency, it can be used for base
Because a group sequencing, genome resurvey sequence, miRNA expression pattern analysis and DNA methylation analysis.Recently, new transcript profile sequencing technologies
It is widely used in the non-mode plant for lacking reference gene group information.The hair of current two generations high throughput transcript profile sequencing technologies
Exhibition so that detach and identify that related gene becomes very convenient and warp from the species of unknown gene group using transcript profile data
Ji.
Invention content
In view of this, the invention discloses the major gene resistance for regulating and controlling anthocyanidin synthesis and metabolism in matrimony vine and including being somebody's turn to do
Recombinant plasmid, host and its application of gene.
According to an aspect of the present invention, the present invention relates to the protein of regulation and control lycium ruthenicum black fruit properties, amino
Acid sequence is sequence shown in SEQ ID NO.2.
According to one embodiment, the present invention also provides a kind of genes, encode the amino acid sequence.Preferably,
The nucleotide sequence is as shown in SEQ ID NO.4.
According to an aspect of the present invention, the present invention relates to the genes of above-mentioned regulation and control black fruit fructus lycii black fruit properties red
Fruit matrimony vine allelic, amino acid sequence are sequence shown in SEQ ID NO.1.
According to one embodiment, the present invention also provides a kind of genes, encode the amino acid sequence.Preferably,
The nucleotide sequence is as shown in SEQ ID NO.3.
According to one embodiment, the present invention also provides the recombinant vector containing the DNA fragmentation, expression cassette, turn base
Because of cell line, recombinant bacterium or host cell.Preferably, the host cell is not that the reproduction cell of human or animal or embryo do carefully
Born of the same parents.
According to an aspect of the present invention, the present invention provides the protein or the gene in regulation and control anthocyanidin synthesis
In application.
According to an aspect of the present invention, the present invention provides anthocyanidin synthetic methods in regulation and control plant, including by institute
The gene stated is transfected into plant, and the gene is made to be expressed in the plant.Preferably, the method includes structures to contain
The plant expression vector of the gene converts plant cell with the expression vector of the structure, and by the plant of the conversion
Cell culture is at transfer-gen plant.
Preferably, the primer in the method for screening is:
The sequence of forward primer AN2cdsF is TGTTCTTAATGCTACTGATGG,
Reverse primer AN2cdsR sequences are ATGATGAATACTAGTGTTACTAT.
The present invention selects lycium ruthenicum LMH1 to be sequenced with red matrimony vine Ningqi7 fruit transcript profiles, and analysis predicts control
The major gene resistance of lycium ruthenicum black fruit properties provides the foundation to research matrimony vine anthocyanidin synthesis mechanism.Also it is genetic engineering
Means plant modification provides direction and target spot to improve the content of anthocyanidin.
Other features and advantages of the present invention will illustrate in the following description, also, partly become from specification
It obtains it is clear that understand through the implementation of the invention.The purpose of the present invention and other advantages are in specification, claims
And specifically noted structure is realized and is obtained in attached drawing.
To enable the above objects, features and advantages of the present invention to be clearer and more comprehensible, preferred embodiment cited below particularly, and coordinate
Appended attached drawing, is described in detail below.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is the fruit that sample lycium ruthenicum LMH1 and red matrimony vine No. Ningqi7 is sequenced in transcript profile；
Fig. 2 is the species distribution for predicting albumen homology albumen.
Fig. 3 is the Vean diagram for predicting albumen.Wherein Black1 and Black2 belongs to two repetitions of LMH1 fruits, Red1 and
Red2 belongs to two repetitions of Ningqi7 fruits.
Fig. 4 difference expression gene distribution maps between No. LMH1 and No. Ningqi7.These genes are divided into 3 classes, and red indicates
Gene is raised, as gene expression amount is higher than No. Ningqi7 in No. LMH1；Blue indicates down-regulated gene, as gene expression
Amount is higher than No. LMH1 in No. Ningqi7；Black indicates between the two the not gene of differential expression.
The GO that Fig. 5 is DEGs classifies.All genes are divided into three classes：Cell component, biological evolution and molecular function.
Fig. 6 is AN2 of the present invention and other control fruit anthocyanidin synthesize major gene resistance amino acid alignment schematic diagram.Respectively
Abbreviation represents plant (GenBank accession number)：Pimento CaAN2 (CAE75745)；Eggplant SmAN2 (AGK37072)；It is short
Lead a cow PhAN2 (ADW94951)；Tomato SlAN2 (ACT36603)；Potato StMTF2 (ABY40371).Black line region indicates
HTH_MYB structural domains；Orange line region indicates SANT structural domains；Blue line area indicates MYB-like DNA-binding structural domains.Side
Frame indicates main difference amino acid.
Fig. 7 is the system of the relevant myb transcription factor of anthocyanidin anabolism in AN2 genes of the present invention and other species
Development tree.
The accession number of these protein (or translation product) is as follows in GenBank databases：
Arabidopsis thaliana/AtPAP2：Q9XI60.1；Brassica oleracea/BoMYB2：
ADP76651.1；Arabidopsis thaliana/AtPAP1：O81439.1；Arabidopsis thaliana/
AtMYB113：Q9FNV9.1；Arabidopsis thaliana/AtMYB114：Q9FNV8.1；Nicotiana tabacum/
NtAN2：ACO52472.1；Ipomoea nil/InMYB2：BAE94709.1；Ipomoea purpurea/IpMYB1：
BAE94388.1；Petunia x hybrida/PhAN2：BAO51604.1；Solanum lycopersicum/SlAN2：NP_
001265992.1；Solanum tuberosum/StMTF2：ABY40371.1；Solanum melongena/SmAN2：
AGK37072.1；lanum tuberosum/StCAI：ABY40370.1；Solanum melongena/SmMYB1：
AMK01805.1；SoSolanum lycopersicum/SlANT1：NP_001265992.1；Solanum tuberosum/
StAN1：AFD31843.1；Gerbera hybrid cultivar/GhMYB10：CAD87010.1；Fragaria x
ananassa/FaMYB：ABX79947.1；Malus domestica/MdMYB110a：AFC88038.1；Pyrus
communis/PcMYB10：AGL81354.1；Malus domestica/MdMYB1：ABK58136.1；Malus domestica
MdMYB10a：ABB84753.1；Diplacus aurantiacus/MaMYB：ACA04006.1；Antirrhinum majus/
VENOSA：ABB83828.1；Antirrhinum majus/ROSEA1：AKB94073.1；Antirrhinum majus/
ROSEA2：ABB83827.1；Vitis vinifera/VvMYBA1：BAD18977.1；Medicago truncatula/
MtLAP2：ACN79539.1；Morella rubra/MrMYB1：ADG21957.1；Epimedium sagittatum/
EsMYBA1：AGT39060.1；Lilium hybrid division/LhMYB6：BAJ05399.1；Glycine max/
GmMYB112：ABH02852.1；Capsicum annuum/CaAN2：CAE75745；Malus domestica/MdMYB6：
ADE92933.1；
Fig. 8 is AN2 genes of the present invention overexpression in model plant Samsun tobaccos.A is that lycium ruthenicum LrAN2 crosses scale
Up to Transgenic Tobacco system；B figures are red matrimony vine LbAN2 overexpression Transgenic Tobaccos system.Purple is helped in wherein LrAN2 evoking tobaccos change
Color, Lb tobacco evoking tobaccos generate local purple；C figures are positive transgenic strain detection, and centre is Samsun pairs of wild-type tobacco
According to；D figures are the corresponding plants anthocyanidin content detection of overexpression Transgenic Tobacco system, and LrAN2 genes induction flower is shown in table
The ability ratio LbAN2 genes of green element synthesis are stronger.
Fig. 9 each tissue site comparison charts between overexpression tobacco transgenic line.
Figure 10 is the express spectra in lycium ruthenicum and red matrimony vine different tissues organ of AN2 genes of the present invention.Wherein A figures are
In lycium ruthenicum AN2 in a organized way in transcriptional level be higher than red matrimony vine.AN2 transcriptional levels highest in black fruit, and
It can't detect in root, stem and blade；B figures are gradually forming with lycium ruthenicum black fruit, the transcriptional levels of AN2 genes also with
Raising.' Lr ' and ' Lb ' respectively represents lycium ruthenicum and red matrimony vine.
Figure 11 is the amino acid alignment figure of lycium ruthenicum and AN2 genes in red matrimony vine.There are different difference at two, first
Difference is histidine in lycium ruthenicum, is lysine in red matrimony vine；Another difference is glutamic acid in lycium ruthenicum, in red Chinese holly
It is serine in Qi.
Figure 12 is AN2 gene associations analysis chart of the present invention.A figures are the group PCR detections of AN2 Allelic Variation primers；B schemes
For 154 parts of red-black matrimony vine geographic distributions.' Lr ' and ' Lb ' respectively represents lycium ruthenicum and red matrimony vine.
Specific implementation mode
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with attached drawing to the present invention
Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Lower obtained every other embodiment, shall fall within the protection scope of the present invention.
Relevant key gene is synthesized for not finding and studying anthocyanidin in matrimony vine at present, the inventors discovered that mainly
Because currently synthesizing relevant gene with anthocyanidin is obtained from blade transcript profile database, specifically expressing in fruit is missed
Gene.For this purpose, inventor for the first time using high-flux sequence of new generation to anthocyanidin in matrimony vine synthesize relevant key gene into
Research is gone.
Compared with the prior art, the present invention has the following advantages：
1, the present invention is sequenced for No. LMH1 by lycium ruthenicum with red matrimony vine Ningqi7 fruit transcript profiles, and analysis predicts control
The major gene resistance of lycium ruthenicum black fruit properties processed：
(1) 192,869 single-genes, average length 1064bp is obtained in transcript profile sequencing.
(2) NR, Nt, Swissprot, KEGG, COG, Interpro, GO and other Protein Data Banks is utilized to analyze,
152,209 kinds of specific proteins of gained gene code.
(3) lycium ruthenicum belongs to Solanaceae family with red matrimony vine, belongs to same section, protein with potato, tobacco and tomato
Analysis shows the protein predicted in No. LMH1 and No. Ningqi7 has very high homology with these species.
(4) Vean diagram is analysis shows the specifically expressing of portion gene will lead to the formation of different fruits.
(5) by the Differential expression analysis of gene, compared with No. Ningqi7, there is 733,070 gene in LMH1 fruits
Up-regulated expression, 25,779 down regulation of gene expression.
(6) all sequencing genes can be divided into three classes：Cell component, biological evolution and molecular function.
(7) in anthocyanidin route of synthesis, in addition to two structural genes of F3 ' H and 3GT are not expressed, other structure bases
Because No. LMH1 all higher than No. Ningqi7 in expression.Wherein myb transcription factor is particularly special, thus filters out the black Chinese holly of regulation and control
Fructus lycii reality anthocyanidin synthesizes the highest candidate gene of possibility, and is named as AN2.
The present invention is using matrimony vine kind lycium ruthenicum LMH1 and red matrimony vine No. Ningqi7 for studying.Lycium ruthenicum has black
Fruit, and red matrimony vine has erythrocarpus.To solve the above problems, the present inventor is by turning No. LMH1 and Ningqi7 fruits
Group analysis is recorded, 192,869 single-genes, average length 1064bp is obtained.Using NR, Nt, Swissprot, KEGG, COG,
Interpro, GO and the analysis of other Protein Data Banks, these gene codes 152,209 kinds of specific proteins.With Ningqi7
It number compares, there is 733,070 gene expression up-regulation, 25,779 down regulation of gene expression in LMH1 fruits.It is synthesized in anthocyanidin
In approach, in addition to two structural genes of F3 ' H and 3GT are not expressed, No. LMH1 is all compared in other expression of structural gene levels
Ningqi7 high.Synthesizing relevant MYB and bHLH genes with anthocyanidin in No. LMH1 also has higher transcription, wherein MYB to turn
It is particularly special to record the factor.In lycium ruthenicum anthocyanin biosynthetic pathway, a kind of R2R3-MYB with higher transcriptional level turns
Factors A N2 is recorded, it can be as the candidate gene of myb transcription factor.AN2 has homology with other myb transcription factors.AN2 has
The structural domain for having myb transcription factor finds that AN2 belongs to point for adjusting anthocyanidin biosynthesis by phylogenetic tree construction
Branch.AN2 is expressed in model plant, can induce the synthesis of anthocyanidin.AN2 transcripts are only in the fruit from lycium ruthenicum
Expression designs specificity AN2cdsF AN2cdsR primers, can be used as selection markers of the AN2 in matrimony vine.
One, transcript profile sequencing analysis
It is lycium ruthenicum LMH1 black fruit and red matrimony vine Ningqi7 erythrocarpus (Fig. 1) that sample, which is sequenced,.
1, sequencing and sequence assembling
All transcripts of the acquisition biological tissue of efficient quick are capable of in transcript profile sequencing.It removes total after low-quality reading
60.57GB initial data and the reading of 403.94Mb are obtained altogether.192,869 genes are connected using software, are always obtained
The sequence fragment of 205,220,696bp.Average gene length is 1064bp, and wherein N50 numerical value length is 1831bp (table 1).
The sequencing of table 1, filtering and assembling statistical form
2, result is annotated
It is compared using NR, Nt, Swissprot, KEGG, COG, Interpro, GO and other protein database sequences
Analysis carries out albumen annotation to 192,869 genes.101,079,133,750,63,305,72,719,40 are predicted respectively,
270,62,303 and 18,541 kind of protein.152,209 albumen (table 2) are obtained in total after removal repetitive proteins.
2 annotation category of table
| Numerical value | Sum | Nr | Nt | Swissprot | KEGG | COG | Interpro | GO | It amounts to |
| Number | 192,869 | 101,079 | 133,750 | 63,305 | 72,719 | 40,270 | 62,303 | 18,541 | 152,209 |
| Percentage | 100% | 52.41% | 69.35% | 32.82% | 37.70% | 20.88% | 32.30% | 9.61% | 78.92% |
This sequencing material lycium ruthenicum and red matrimony vine belong to Solanaceae family with potato, tobacco and tomato, what data were predicted
Protein and these species all have very high homology.Wherein, 27.31% prediction albumen and potato in all prediction albumen
Albumen homology highest；There is 18.83% prediction albumen to spend albumen homology highest with camphor tree；16.4% prediction albumen and fine hair cigarette
Vegetable protein homology highest；12.12% prediction albumen and tomato albumen homology highest；Remaining 25.35% prediction albumen and its
He has close genetic affinity by species albumen.(Fig. 2)
It is sequenced in sample often in Black1 (LMH1), Black2 (LMH1), Red1 (Ningqi7) and Red2 (Ningqi7)
The albumen seen has 52,143 kinds, accounts for about the 1/3 of all albumen.There are 25,891 specific genes in lycium ruthenicum, in red matrimony vine
Specifically expressed gene has 16,636 (Fig. 3).The formation of different fruits is caused just because of the specifically expressing of these genes.
3, difference expression gene is analyzed
It calculates FPKM values according to being mapped to reference to the reading on transcription and determines possible differential expression single-gene, use
In the differential expression for identifying gene between No. LMH1 and No. Ningqi7.Pass through the Hes of FDR≤0.001 | log2Ratio | >=1 expression
Horizontal comparison, one shares 58,849 difference expression genes in No. LMH1 with No. Ningqi7.(Fig. 4) makees No. Ningqi7
For reference, there are 33,070 up-regulation gene, as these genes expression quantity higher in No. LMH1；There are 25,779 downward bases
Cause, as these genes the expression quantity higher in No. Ningqi7.
4, the GO function classifications of difference expression gene
58,849 differential genes are divided into 53 subclass by GO analyses.Wherein there are 20,520 DEGs to participate in biological mistake
Journey；19,734 DEGs are related with cell component；10,358 genes are grouped by molecular function.In bioprocess scope, absolutely
Most of genes and metabolic process (5,125DEGs), cell component (4,952DEGs) and single organization's process (3,310DEGs)
It is related.In cell component classification, most of difference expression genes are enriched in subtype of cells process (4,353DEGs), groups of cells
Divide (4,316DEGs) and organelle (3,198DEGs).In molecular function scope, the maximum ratio of difference expression gene is located at
' bookbinding ' (4,374DEGs) and ' catalytic activity ' (4182DEGs) (Fig. 5).
5, the expression of anthocyanidin biosynthesis related genes
At No. LMH1 with Ningqi7 transcript profile data, there are 31 genes related to the biosynthesis of anthocyanidin.Pass through
BlastX is analyzed, and it is related to anthocyanidin synthesis to filter out 13 genes.' 5 ' only PAL, C4H, C4L, CHS, CHI, F3H, F3 H,
DFR, LDOX, myb transcription factor and bHLH transcription factors have homologous gene (table 3).But it is not found in transcript profile database
The homologous gene with F3 ' H and 3GT.It being found after analysis result, No. LMH1 all expression of structural gene amounts are all higher than No. Ningqi7,
Illustrate that the anthocyanidin biosynthesis in No. LMH1 is activated.In No. LMH1, the Intensity of Transcription of Endothelial of all myb transcription factors all compares
Log2 rate value highests No. Ningqi7 strong, that wherein gene number is CL10341.Contig.In bHLH transcription factors, two
Expression of a gene in No. LMH1 is higher than No. Ningqi7, and expression of two genes in No. Ningqi7 is higher than
No. LMH1.
The differential expression of structural gene and controlling gene in 3 anthocyanin biosynthetic pathway of table
Remarks：Black represents lycium ruthenicum LMH1, and Red represents red matrimony vine No. Ningqi7
The major gene resistance LrAN2 of black fruit properties is controlled in lycium ruthenicum fruit, the gene source is in the black Chinese holly of matrimony vine kind
Myb transcription factor in Qi LMH1 fruits, it has nucleotide shown in SEQ ID NO.1 the 1st to 774 in sequence table.
LrAN2 genes include HTH_MYB, SANT and class MYB structural domains and function.
154 come from system of domestic different regions different colours matrimony vine group for association analysis black fruit properties and
AN2 allelic variations, for determining distribution of the AN2 allele in Natural Population, including：72 lycium ruthenicums and 72 red Chinese hollys
Qi.
Two, the molecular characterization of AN2
1, the preparation of total DNA, total serum IgE and cDNA
According to the method for Yan et al. (Yan etc., 2002), DNA is detached from the fruit of 1 34 -day-old of Post flowering.According to
The method (Ahmed etc., 2003) of Ahmed etc. is collected and prepared for root, leaf and stem sample.Use Tiangen RNAprep pure plants
Kit (Tiangen companies, BeiJing, China) extracts total serum IgE from about 0.5g fruits.Use Thermo RevertAid First
Strand cDNA kits (Thermo-Fisher Scientific, Shanghai, China) obtain cDNA from total serum IgE.
2, design primer detaches AN2 transcripts
Use Primer5 softwares (Premier Biosoft, the U.S. states Jia Lifoniya Palo Alto) design primer.
All primers used in this research are listed in Table 4 below.
Primer used in table 4 and sequence
| Number | Primer | Sequence (5 ' -3 ') |
| 1 | AN2cdsF | TGTTCTTAATGCTACTGATGG |
| 2 | AN2cdsR | ATGATGAATACTAGTGTTACTAT |
| 3 | AN2attb1 | AAAAAGCAGGCTTCATGATGAATACTAGTGTTAC |
| 4 | AN2attb2 | AGAAAGCTGGGTCCTAATTCAGTAGATTCCATA |
| 5 | Attb1 adapter | GGGGACAAGTTTGTACAAAAAAGCAGGCT |
| 6 | Attb2 adapter | GGGGACCACTTTGTACAAGAAAGCTGGGT |
| 7 | TublinF | CCATACCAGCATCACCATTCTTC |
| 8 | TublinR | GTCACACTTCCCACATTGCC |
| 9 | AN2-RT-F | ATGATGAATACTAGTGTTACTATTA |
| 10 | AN2-RT-R | AGTCTACAACTCTTCCTG |
| 11 | AN2spf | ACTAGTCATTATGCATAGAAAGTTG |
| 12 | AN2spr | CGTTTGCTGTTCTTCCCG |
Primer AN2cdsF and AN2cdsR are used for expanding cDNA to detach AN2 transcripts in red-black matrimony vine.It uses
GeneAmp PCRSystem 9700 (Thermo-Fisher Scientific) utilize high-fidelity Phushion archaeal dna polymerases
(Thermo-Fisher Scientific) carries out PCR amplification.PCR amplification program：98 DEG C are denaturalized 2 minutes；35 cycles：98℃
15 seconds, 64 DEG C 30 seconds, 72 DEG C 30 seconds；Then finally extend 10 minutes at 72 DEG C.It is purified using Tiangen TIANgel Midi
Kit (Tiangen companies) purified pcr product from 1.0% Ago-Gel.PCR product is cloned into pGEM-T Easy
In plasmid vector (Promega Corporation, the pungent state Madison of University of Wisconsin-Madison).Then by recombinant plasmid transformed to large intestine
In bacillus DH5 α cells.64 single bacterium colonies of random picking expand each thalline with primer AN2cdsF and AN2cdsR, obtain size one
The segment of cause.
5 positive colonies are sequenced by commercial company's (Hua Da gene, China Shenzhen), determine transcript sequence.
Transcriptome analysis based on Chinese wolfberry fruit, detached from lycium ruthenicum and red matrimony vine has high transcription water in black fruit
Flat myb gene AN2.Coding region sequences of the AN2 in lycium ruthenicum and red matrimony vine is 774bp.
3, the domain analysis of AN2 transcript structures and genealogical tree structure
In website (http://blast.ncbi.nlm.nih.gov/Blast.cgiPROGRAM=blastp＆PAGE_
TYPE=BlastSearch＆LINK_LOC=blasthome conservative functional domain is predicted on).The CDS of AN2 is 774bp, coding
257 amino acid (SEQ ID NO.1, SEQ ID NO.2).AN2 includes completely including HTH_MYB, SANT and class MYB structures
Domain.Compared with other Solanaceae species control fruit anthocyanidin synthesis major gene resistance, AN2 is complete in HTH_MYB and SANT structural domains
Unanimously；AN2 class MYB structural domains have differences section " TAPHQQERKYNNALKITENTILRPRPRTFTSSSAKNVSF " and
" HNNEILNICEKPTG " (Fig. 6).AN2 than other gene transcripts be more closely similar in pimento regulate and control anthocyanidin it is anabolic
Myb transcription factor CaAN2.HTH_MYB structural domains are the structural domains with myb transcription factor protein-protein interaction,
SANT structural domains are DNA binding domain, and class MYB structural domains interact with rna plymerase ii, startup transcription (Atchley etc.,
2000).It is particularly significant that three structural domains exercise its functional transcription for MYB protein.
The amino acid sequence system that MYB transcripts are built by adjacent method using MEGA 6.0 (Tamura etc., 2007) is sent out
Raw tree.In phylogenetic tree, AN2 obviously gathers with the anabolic protein of anthocyanidin is adjusted for a group (Fig. 7), shows AN2
It is likely to have the regulation and control anabolic function of anthocyanidin.
Three, the overexpression of AN2 can induce anthocyanidin to synthesize
Transgene expression vector is built using Gateway Cloning Kits (Thermo-Fisher Scientific)
PJAM1502：AN2, wherein carrier PJAM1502 carry 35S promoter.It is as follows：
Primer AN2attb1 and AN2attb2 of the design with ATTB connectors.
It is expanded from the pGEM-T Easy carriers for being connected with AN2 transcripts；Then it using the PCR product as template, uses
Attb1adapter and attb2adapter carries out PCR amplification；Amplified production carries out agarose electrophoresis, recycling, measured concentration.
BP reaction structure entry vectors pDONR207 is carried out according to Gateway Cloning Kit specifications：Wherein AN2 steps
It is as follows：
BP reacts：
(1) 200-400ng PCR recovery products；100ng pDONR207 carriers；1μl BP Clonase Ⅱ；Add water to 5 μ
l；25 DEG C of water-bath 12-16h.
(2) 0.5 μ l Proteinase K (2 μ g/ μ l), 37 DEG C of water-bath 15min are added into reaction system.
(3) by recombinant plasmid transformed into DH5 α, positive colony is screened and is sequenced, and then extracts the spare (TIANpure of plasmid
Mini PlasmidKit (Tiangen) kit).
(4) the structure of transgene expression vector：
LR reaction structure transient expression vectors PJAM1502 is carried out according to Gateway Cloning Kit specifications：AN2 steps
It is as follows：
LR reacts：(1) 300ngp DONR207 recombinant plasmids；100ng PJAM1502 transgene expression vectors；1μl LR
ClonaseⅡ；Add water to 5 μ l；25 DEG C of 12~16h of water-bath.(2) 0.5 μ l Proteinase K (2 μ g/ are added into reaction system
μ l), 37 DEG C of water-bath 15min.(3) recombinant plasmid transformed DH5 α, positive colony are screened and are sequenced, and it is spare then to extract plasmid.
(5) the acquisition of AN2 transgene tobaccos is overexpressed：
According to the method for (2006) Nature Protocols, recombinant vector is infected into tobacco by agriculture bacillus mediated
(Samsun) in, steps are as follows：
It is prepared by Agrobacterium：(1) over-express vector plasmid PJAM1502 is drawn：Agrobacterium kind LBA4404 is added in AN25 μ l,
200 μ l antibiotic-free LB culture solutions are added in ice bath 5min, liquid nitrogen 5min, 37 DEG C of water-bath 5min, ice bath 5min, at 28 DEG C,
Constant temperature cultivates 2h under the conditions of 200rpm；(2) draw 100 μ l culture solutions and be spread evenly across the LB containing kanamycins and rifampicin resistance
Solid culture ware surface is put into 28 DEG C of constant incubator 48h；(3) picking veneer carries out positive colony screening, and it is spare then to shake bacterium.
Transgenosis infects：(1) tobacco aseptic seedling young leaflet tablet is taken, 75% ethyl alcohol is put into and impregnates 1min, be transferred to 2% hypochlorous acid
Sodium 12min, sterile water wash 3~5 times, is uniformly cut into 1cm × 1cm or so sizes.(2) Agrobacterium shakes bacterium 48h, measures OD values and is
0.8, blade is impregnated into Agrobacterium 8min, filter paper exhaustion is put into and co-cultures base dark treatment 2d, is transferred to differential medium 7d, is transferred to
The 14d of differential medium containing kanamycins, seedling, which is cut, is transferred to root media until taking root.Using primer An2cdsF and
AN2cdsR is used for carrying out selective mechanisms (Fig. 8) to positive seedling.
In application of the gene in regulation and control anthocyanidin synthesis, detached from matrimony vine kind lycium ruthenicum and red matrimony vine
To AN2 genes as the part in recombinant vector, the recombinant vector is thin as the part in host cell, wherein host
Born of the same parents refer to the cell in addition to the reproduction cell of human or animal or embryonic stem cell.In addition, from matrimony vine kind lycium ruthenicum and red Chinese holly
The transcript gene of isolated AN2 genes is as the part in host cell in Qi.
Application packet of the major gene resistance AN2 genes of black fruit properties in terms of cultivating transfection plant is controlled in structure fructus lycii reality
It includes：The plant expression vector of the AN2 genes and its transcript gene is built, and plant cell is converted using the expression vector, is carried
High target gene expression.
Four, in different tissues AN2 transcriptional level
1, in different tissues AN2 transcriptional level
Each tissue cDNA content is standardized with Tubulin, primer AN2-RT-F and AN2-RT-R are used for measuring AN2 in matrimony vine
Expression in different tissues.AN2 has apparent expression in lycium ruthenicum, but detects AN2 in red matrimony vine, root, stem and Ye Zhongneng
Trace expression.The transcriptional level of AN2 highest (Figure 10) in black fruit.In same tissue, the AN2 in lycium ruthenicum expresses water
It is flat to be apparently higher than red matrimony vine.
3, in lycium ruthenicum and red matrimony vine the reason of AN2 transcriptional level differences
In order to explain the difference of AN2 transcriptions in lycium ruthenicum and red matrimony vine, it converts AN2 coding sequences to amino acid
Sequence is compared.It was found that LrAN2 is with LbAN2, there are two amino acid of differences, are respectively histidine and paddy ammonia in lycium ruthenicum
Acid, and be respectively lysine and serine in red matrimony vine, single or multiple amino acid of differences may be to cause in lycium ruthenicum
The high reason of LrAN2 transcriptional levels.(Figure 11)
Five, in natural population AN2 allelic variations and black fruit properties distribution.
The special primer AN2spf and AN2spr across Insert Fragment is designed to distinguish the AN2 from lycium ruthenicum and red matrimony vine
Allelic variation (LrAN2 and LbAN2) (referring to table 3).AN2 genes are respectively in lycium ruthenicum and size in red matrimony vine genome
1405bp and 1417bp.Find that red matrimony vine is inserted into 15 bases at 861bp-876bp by sequence alignment
“ATATATATTTTTTTT”.It is used to distinguish the difference between lycium ruthenicum and red matrimony vine strain with this difference design specific primer.
(Figure 12) checks all 154 strains：72 lycium ruthenicum strains, 72 red matrimony vine strains.All strains containing LrAN2 have
Black fruit properties, all strains with erythrocarpus carry LbAN2.
Finally it should be noted that：Embodiment described above, only specific implementation mode of the invention, to illustrate the present invention
Technical solution, rather than its limitations, scope of protection of the present invention is not limited thereto, although with reference to the foregoing embodiments to this hair
It is bright to be described in detail, it will be understood by those of ordinary skill in the art that：Any one skilled in the art
In the technical scope disclosed by the present invention, it can still modify to the technical solution recorded in previous embodiment or can be light
It is readily conceivable that variation or equivalent replacement of some of the technical features；And these modifications, variation or replacement, do not make
The essence of corresponding technical solution is detached from the spirit and scope of technical solution of the embodiment of the present invention, should all cover the protection in the present invention
Within the scope of.Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence table
<110>Northwest Plateau-organisms Research Inst. of Chinese Academy of Sciences
<120>Matrimony vine gene and its coding protein, recombinant vector, and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 267
<212> PRT
<213>Red matrimony vine ()
<400> 1
Met Met Asn Thr Ser Val Thr Ile Thr Lys Ser Ser Gly Val Arg Lys
1 5 10 15
Gly Ala Trp Thr Glu Glu Glu Asp Leu Leu Leu Arg Lys Cys Ile Gln
20 25 30
Lys Tyr Gly Glu Gly Lys Trp His Gln Val Pro Ile Arg Ala Gly Leu
35 40 45
Asn Arg Cys Arg Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg
50 55 60
Pro His Ile Lys Arg Gly Asp Phe Ser Ser Glu Glu Val Asp Leu Ile
65 70 75 80
Leu Arg Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly
85 90 95
Arg Leu Pro Gly Arg Thr Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr
100 105 110
His Leu Gln Arg Lys Leu Thr Ala Pro His Arg Gln Glu Arg Lys Tyr
115 120 125
Asn Asn Ala Leu Lys Ile Thr Glu Asn Thr Ile Leu Arg Pro Arg Pro
130 135 140
Arg Thr Phe Thr Ser Ser Ser Ala Lys Asn Val Ser Phe Cys Ser Asn
145 150 155 160
Lys Ser Ile Thr Asn Thr Val Asp Lys Asn Ala His Asn Asn Glu Ile
165 170 175
Leu Asn Ile Cys Glu Lys Pro Thr Gly Glu Thr Thr Ser Val Asp Glu
180 185 190
Gly Val Gln Trp Trp Thr Ser Leu Leu Glu Asn Cys Asn Glu Thr Glu
195 200 205
Glu Glu Ala Glu Ala Phe Gly Ser Phe Asp Glu Glu Asn Met Leu Gln
210 215 220
Ser Leu Leu His Glu Glu Ile Ser Pro Pro Met Gln Gln Gly Gln Ser
225 230 235 240
Gly Asn Trp Asp Asp Phe Ser Ala Asp Ile Asp Leu Trp Asn Leu Leu
245 250 255
Asn
<210> 2
<211> 257
<212> PRT
<213>Lycium ruthenicum ()
<400> 2
Met Met Asn Thr Ser Val Thr Ile Thr Lys Ser Ser Gly Val Arg Lys
1 5 10 15
Gly Ala Trp Thr Glu Glu Glu Asp His Leu Leu Arg Lys Cys Ile Gln
20 25 30
Lys Tyr Gly Glu Gly Lys Trp His Gln Val Pro Ile Arg Ala Gly Leu
35 40 45
Asn Arg Cys Arg Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg
50 55 60
Pro His Ile Lys Arg Gly Asp Phe Ser Ser Glu Glu Val Asp Leu Ile
65 70 75 80
Leu Arg Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly
85 90 95
Arg Leu Pro Gly Arg Thr Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr
100 105 110
His Leu Gln Arg Lys Leu Thr Ala Pro His Gln Gln Glu Arg Lys Tyr
115 120 125
Asn Asn Ala Leu Lys Ile Thr Glu Asn Thr Ile Leu Arg Pro Arg Pro
130 135 140
Arg Thr Phe Thr Ser Ser Ser Ala Lys Asn Val Ser Phe Cys Ser Asn
145 150 155 160
Lys Ser Ile Thr Asn Thr Val Asp Lys Asn Ala His Asn Asn Glu Ile
165 170 175
Leu Asn Ile Cys Glu Lys Pro Thr Gly Glu Thr Thr Ser Val Asp Glu
180 185 190
Gly Val Gln Trp Trp Thr Ser Leu Leu Glu Asn Cys Asn Glu Thr Glu
195 200 205
Glu Glu Ala Glu Ala Phe Gly Ser Phe Asp Glu Glu Asn Met Leu Gln
210 215 220
Ser Leu Leu His Glu Glu Ile Ser Pro Pro Met Gln Gln Gly Gln Ser
225 230 235 240
Gly Asn Trp Asp Asp Phe Ser Ala Asp Ile Asp Leu Trp Asn Leu Leu
245 250 255
Asn
<210> 3
<211> 774
<212> DNA
<213>Red matrimony vine ()
<400> 3
atgatgaata ctagtgttac tattactaaa tcatctggag tgaggaaagg tgcatggact 60
gaagaagaag atcttctttt gagaaaatgc attcaaaagt acggtgaagg aaaatggcat 120
caagttccca ttagagctgg tctaaataga tgcaggaaga gttgtagact gaggtggctg 180
aattatctaa ggccacatat aaagagaggt gacttctctt ctgaggaagt tgatcttatc 240
ttgaggcttc ataagctctt aggcaacaga tggtcactca tcgcgggtag acttccggga 300
agaacagcaa acgatgtcaa aaactactgg aacacacacc tacagaggaa gttaactgct 360
cctcatcgac aagagagaaa gtacaataat gctctcaaga tcacagaaaa caccatacta 420
agacctcgac ctcgaacctt cacatcaagt agtgcaaaaa atgtttcttt ttgcagcaac 480
aaaagtatca caaacacagt agataaaaac gcacacaaca atgaaatact aaatatttgt 540
gagaagccaa caggtgaaac gacgtcggta gacgagggag ttcaatggtg gacaagttta 600
ctggaaaatt gcaatgaaac tgaggaagaa gcagaagcat ttgggagctt tgatgaagaa 660
aatatgttac aaagtttgtt gcatgaggaa atttcaccac ccatgcaaca aggacaaagt 720
ggtaattggg atgacttttc cgctgatatt gacctatgga atctacttaa ttag 774
<210> 4
<211> 774
<212> DNA
<213>Lycium ruthenicum ()
<400> 4
atgatgaata ctagtgttac tattactaaa tcatctggag tgaggaaagg tgcatggact 60
gaagaagaag atcatctttt gagaaaatgc attcaaaagt acggtgaagg aaaatggcat 120
caagttccca ttagagctgg tctaaataga tgcaggaaga gttgtagact gaggtggctg 180
aattatctaa ggccacatat aaagagaggt gacttctctt ctgaggaagt tgaccttatc 240
ttgaggcttc ataagctctt aggcaacaga tggtcactca ttgcgggtag acttccggga 300
agaacagcaa acgatgtcaa aaactactgg aacacacacc tacagaggaa gttaactgct 360
cctcatcaac aagagagaaa gtacaataat gccctcaaga tcacagaaaa caccatacta 420
agacctcgac ctcgaacctt cacatcaagt agtgcaaaga atgtttcttt ttgcagcaac 480
aaaagtatca caaacactgt agataaaaac gcacacaaca atgaaatact aaatatttgt 540
gagaagccaa caggtgaaac gacgtcggta gacgagggag ttcaatggtg gacaagttta 600
ctggaaaatt gcaatgaaac tgaggaagaa gcagaagcat ttgggagctt tgatgaagaa 660
aatatgttac aaagtttgtt gcatgaggaa atttcaccac ccatgcaaca aggacaaagt 720
ggtaattggg atgacttttc cgctgatatt gacctatgga atctactgaa ttag 774
<210> 5
<211> 1417
<212> DNA
<213>Red matrimony vine ()
<400> 5
atgatgaata ctagtgttac tattactaaa tcatctggag tgaggaaagg tgcatggact 60
gaagaagaag atcttctttt gagaaaatgc attcaaaagt acggtgaagg aaaatggcat 120
caagttccca ttagagctgg taataataat tctgatacta tactctctag aagagaagta 180
cgattgagta taacttatat tattccacta gataagagta tgtgcatatg tctgatatat 240
gtgaatatgt gcaggtctaa atagatgcag gaagagttgt agactgaggt ggctgaatta 300
tctaaggcca catataaaga gaggtgactt ctcttctgag gaagttgatc ttatcttgag 360
gcttcataag ctcttaggca acaggcaagt tcattttcaa acactttact aatatagagg 420
tggatttaaa attactatgt tataggaagg catgatatct ttaacactac ttataagatt 480
taaagataac tacggttagg tctctttatg acaccatcct tatataacaa ctccttacta 540
taacaatcaa gtttttctcg gaatcgattt tttatgttat attttacctc tctataataa 600
ccttttacct ataacaacaa caaccatctt ataacggctc tttgtaaaat tacccctcta 660
ttaaaagtat cttcattttt ggtaatatat taattaacca tatttataga aataaaatct 720
ttaagattat taccaataat aagtctaaga gattttgaac agatatttta caaaccaaag 780
aaataccata tcatgtttaa aatactagtc attatgcata gaaagttgtc tttgatgtta 840
tgtattttta tatatatata tatatttttt tttttttttt gcttaattag atggtcactc 900
atcgcgggta gacttccggg aagaacagca aacgatgtca aaaactactg gaacacacac 960
ctacagagga agttaactgc tcctcatcga caagagagaa agtacaataa tgccctcaag 1020
atcacagaaa acaccatact aagacctcga cctcgaacct tcacatcaag tagtgcaaag 1080
aatgtttctt tttgcagcaa caaaagtatc acaaacacag tagataaaaa cgcacacaac 1140
aatgaaatac taaatatttg tgagaagcca acaggtgaaa cgacgtcggt agacgaggga 1200
gttcaatggt ggacaagttt actggaaaat tgcaatgaaa ctgaggaaga agcagaagca 1260
tttgggagct ttgatgaaga aaatatgtta caaagtttgt tgcatgagga aatttcacca 1320
cccatgcaac aaggacaaag tggtaattgg gatgactttt ccgctgatat tgacctatgg 1380
aatctactta attagcttca tccatcagta gcattaa 1417
<210> 6
<211> 1405
<212> DNA
<213>Lycium ruthenicum ()
<400> 6
atgatgaata ctagtgttac tattactaaa tcatctggag tgaggaaagg tgcatggact 60
gaagaagaag atcatctttt gagaaaatgc attcaaaagt acggtgaagg aaaatggcat 120
caagttccca ttagagctgg taataataat tctgatacta tactctctag aagagaagta 180
cgattgagta taacttatat tattccacta gataagagta tgtgcatgtg tctgatatat 240
gtgaatatgt gcaggtctaa atagatgcag gaagagttgt agactgaggt ggctgaatta 300
tctaaggcca catataaaga gaggtgactt ctcttctgag gaagttgacc ttatcttgag 360
gcttcataag ctcttaggca acaggcaagt tcattttcaa acactttact aatatagagg 420
tggatttaaa attactatgt tataggaagg catgatatct ttaacactac ttataagatt 480
taaagataac tacagttagg tctctctata acactatcct tatataacaa cttcttactc 540
taacaatcaa atttttctcg gaatcaattt tttatgttat attttacctc tctataataa 600
cattttacct ataacagcaa aaactatttt ctaacagctc tttgtaaaat taccactcta 660
ttaaaagtat cttcattttt tgtaatatat taattaacca tatttataga aatagaatcc 720
ctaagattat tatcaataat aagtctaaga gattttgaac agatattttt acaaaccaaa 780
gaaataccat atcatgttta aaatctacta gtcattatgc atagaaagtt gtctttgatg 840
ttatgtattt ttttatatat ttttttttgc ttaattagat ggtcactcat tgcgggtaga 900
cttccgggaa gaacagcaaa cgatgtcaaa aactactgga acacacacct acagaggaag 960
ttaactgctc ctcatcaaca agagagaaag tacaataatg ccctcaagat cacagaaaac 1020
accatactaa gacctcgacc tcgaaccttc acatcaagta gtgcaaagaa tgtttctttt 1080
tgcagcaaca aaagtatcac aaacactgtg gataaaaacg cacacaacaa tgaaatacta 1140
aatatttgtg agaagccaac aggtgaaacg acgtcggtag acgagggagt tcaatggtgg 1200
acaagtttac tggaaaattg caatgaaact gaggaagaag cagaagcatt tgggagcttt 1260
gatgaagaaa atatgttaca aagtttgttg catgaggaaa tttcaccacc catgcaacaa 1320
ggacaaagtg gtaattggga tgacttttcc gctgatattg acctatggaa tctactgaat 1380
tagcttcatc catcagtagc attaa 1405
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence ()
<400> 7
tgttcttaat gctactgatg g 21
<210> 8
<211> 23
<212> DNA
<213>Artificial sequence ()
<400> 8
atgatgaata ctagtgttac tat 23
Claims (8)
1. a kind of protein of matrimony vine, amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.
2. a kind of matrimony vine gene encodes protein described in claim 1.
3. a kind of matrimony vine gene, nucleotide sequence is as shown in SEQ ID NO.3 or SEQ ID NO.4.
4. the recombinant vector, expression cassette, transgenic cell line, recombinant bacterium containing DNA fragmentation described in Claims 2 or 3 or host
Cell.
5. the application of protein described in claim 1 or gene according to claim 2 or 3 in regulation and control anthocyanidin synthesis.
6. anthocyanidin synthetic method in a kind of regulation and control plant, including gene according to claim 2 or 3 is transfected into plant
In, so that the gene is expressed in the plant.
7. method as claimed in claim 6, wherein express load including building the plant containing the gene described in Claims 2 or 33
Body converts plant cell with the expression vector of the structure, and the plant cell of the conversion is cultivated into transfer-gen plant.
8. the method for claim 7, wherein be for screening the primer used in sun plant：
The sequence of forward primer AN2cdsF is TGTTCTTAATGCTACTGATGG,
Reverse primer AN2cdsR sequences are ATGATGAATACTAGTGTTACTAT.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440809A (en) * | 2020-04-08 | 2020-07-24 | 青海省农林科学院 | Application of structural gene in changing fruit color of medlar |
| CN112592925A (en) * | 2021-02-24 | 2021-04-02 | 沈阳农业大学 | Lycium ruthenicum stable genetic transformation system and application thereof |
| CN113444731A (en) * | 2021-06-18 | 2021-09-28 | 宁夏农林科学院枸杞科学研究所 | MYB transcription inhibitory factor LrETC1 related to synthesis of lycium ruthenicum anthocyanin and application thereof |
| CN116606880A (en) * | 2023-05-25 | 2023-08-18 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
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| CN114214336B (en) * | 2022-02-23 | 2022-05-03 | 中国科学院华南植物园 | Lycium ruthenicum LrNOR gene and application of protein thereof |
| US20230270073A1 (en) * | 2022-02-25 | 2023-08-31 | Benson Hill, Inc. | Compositions and methods comprising plants with modified anthocyanin content |
| CN116904506B (en) * | 2023-08-31 | 2023-12-12 | 中国科学院华南植物园 | Lycium ruthenicum LrANT1 gene and application of coded protein thereof |
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| CN111440809A (en) * | 2020-04-08 | 2020-07-24 | 青海省农林科学院 | Application of structural gene in changing fruit color of medlar |
| CN111440809B (en) * | 2020-04-08 | 2022-05-17 | 青海省农林科学院 | Application of structural gene in changing fruit color of medlar |
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| CN113444731A (en) * | 2021-06-18 | 2021-09-28 | 宁夏农林科学院枸杞科学研究所 | MYB transcription inhibitory factor LrETC1 related to synthesis of lycium ruthenicum anthocyanin and application thereof |
| CN116606880A (en) * | 2023-05-25 | 2023-08-18 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
| CN116606880B (en) * | 2023-05-25 | 2023-12-08 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
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| CN113337635B (en) | 2022-05-20 |
| CN113337635A (en) | 2021-09-03 |
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