CN106591321A - Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method - Google Patents
Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method Download PDFInfo
- Publication number
- CN106591321A CN106591321A CN201611081459.4A CN201611081459A CN106591321A CN 106591321 A CN106591321 A CN 106591321A CN 201611081459 A CN201611081459 A CN 201611081459A CN 106591321 A CN106591321 A CN 106591321A
- Authority
- CN
- China
- Prior art keywords
- ccpils
- gene
- race
- auxin
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method. The method includes the steps of: material preparation, RNA extraction and purification, CcPILS gene RACE, bioinformatics analysis, and temporal and spatial expression analysis. The method provided by the invention clones Carya cathayensis PIL homologous gene CcPILS by RACE technique, the gene has a full-length sequence of 1541bp, wherein the open reading frame sequence is 1263bp, bioinformatics software analysis is applied to predict that the sequence encodes 420 amino acids, the CcPILS protein molecular weight is about 46.22KD, PI is 5.38, the protein is located at an endoplasmic reticulum membrane, is equipped with 5 transmembrane hydrophobic structural domains respectively at the N terminal and C terminal, and is separated by a middle hydrophilic loop. The gene has high homology with Arabidopsis thaliana AtPILS5 and AtPILS7, and belongs to Clade III subtribe of PILS gene family. Fluorescence quantitative RT-PCR analysis shows that before and after Carya cathayensis grafting, CcPILS has expression trend in scion consistent with that in rootstock, and provides a beneficial promoting effect on solution of the problems of difficult Carya cathayensis grafting, difficult variety breeding and difficult quality improvement, etc.
Description
【Technical field】
The present invention relates to the technical field of Semen Caryae Cathayensis, more particularly to a kind of Semen Caryae Cathayensis auxin output carrier protein CcPILS
Gene diffusion analysis method.
【Background technology】
Plant is adjusted by auxin metabolism and intercellular transport and cellulose content is grown between different tissues, and then produces signals-modulating
The growth of plant tropism, the generation of lateral organ and development etc..Polar translocation is the key character of auxin, and polar translocation is by growing
Plain input carrier AUX/LAX protein families (AUXIN RESISTANT1/LI-KE AUX1) and output carrier PIN-FORMED
(PIN) protein family collaboration is completed.Barbez et al. is found that another has the transmitter loss phase of the auxin of 7 members
Correlation gene family.Although the gene (10%～18%) very low with the sequence similarity of PIN, topological structure and PIN albumen
It is similar, therefore it is named as PILS (PIN-LIKES, PILS).PILS can be divided into Clade I, Clade II and Clade III
Three subfamilies, wherein CladeIII subtribes include PILS5 and PILS7, tracheophyte evolve in during have important work
With.
Semen Caryae Cathayensis (Carya cathayensis Sarg.) belong to Juglandaceae hickory plant, the different flower of hermaphroditism.Mountain
Semen Juglandiss are important traditional oil trees, are also the important nonwood forest trees in Zhejiang Province, and economic worth is big, to improving hill farmer
Income is significant.But hickory grafting is difficult, breed breeding is difficult, and quality is difficult to improve.To overcome an above difficult problem, around mountain
The Physiology and biochemistry mechanism that walnut grafting is survived, it is necessary to propose Semen Caryae Cathayensis auxin output carrier protein CcPILS gene cloning sides
Method, and CcPILS expression regulations are analyzed, it is auxin during Semen Caryae Cathayensis CcPILS gene functions and hickory grafting
The research of Regulation Mechanism provides favourable help, promotes, breed breeding difficulty difficult to hickory grafting, quality to be difficult to raising problem
Solution.
【The content of the invention】
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, there is provided a kind of Semen Caryae Cathayensis auxin exports carrier egg
White CcPILS gene diffusion analysis methods, it is aimed to solve the problem that, and hickory grafting is difficult, breed breeding is difficult in prior art,
Quality is difficult to the technical problem for improving.
For achieving the above object, the present invention proposes a kind of Semen Caryae Cathayensis auxin output carrier protein CcPILS gene clonings
And expression analysis method, comprise the following steps：
A), material is prepared：Vegetable material is hickory grafting sample, and in test site sampling is started before and after grafting, is transferred
Stock and scion are taken before connecing respectively as control, then grafting is gathered in grafting part every 3d, 7d, 14d after grafting, often
30～50 graftings of secondary collection, take back laboratory and save backup in -70 DEG C；
B), RNA extraction and purifications：To step A) the middle stock for sampling, scion and grafting, using the CTAB methods of improvement
Total serum IgE is extracted, then purified mRNA is operated to specifications using Oligotex mRNA Spin-Column test kits；
C), CcPILS genes RACE：
C1) with reference to design of primers principle in RACE kit specifications, to what is obtained in Semen Caryae Cathayensis bud transcript profile data
Auxin output carrier protein Homologous gene sequences fragment designs 5 ' RACE specific primer symRev and newpilsR, and difference
Successively it is used for 5 ' RACE nest-type PRCs with GSP and NGSP1；
C2) according to sequential design 3 ' RACE the specific primer newpilsF and SymFor of known fragment, and respectively with
GSP2 and NGSP2 is successively used for 3 ' RACE nest-type PRCs；
C3) product obtained by 5 ' RACE and 3 ' RACE PCR amplifications is carried out Jing after 1.5% agarose gel electrophoresiies inspection
Glue reclaim is cut, recovery product is added by rTaq enzymes and be connected with pMD-18 carriers after A tails, then is transformed in DH5 α competence bacterial strains,
Bacterial strain after conversion is coated on LB flat boards in 37 DEG C of overnight incubations, picking monoclonal, after colony PCR amplification, electrophoresis detection
Positive colony is selected to send company to be sequenced；
C4) specific primer Pilsfor and Pilsrev are designed according to sequencing result, by masterplate of cDNA CcPILS is expanded
Open reading frame (ORF), and be connected to after pMD-18 and determine sequence；
D), bioinformatic analysis：The sequence for being determined is translated into into aminoacid sequence, using Mega6.0 by the amino
Acid sequence and AtPILS1, AtPILS2, AtPILS3, AtPILS4, AtPILS5, AtPILS6, AtPILS7, DcPILS3,
DcPILS6, DcPILS7, MdPILS3, MdPILS5, MdPILS6 phylogenetic tree construction, using instrument and program biology is carried out
Bioinformatics analysis；
E), spatial and temporal expression analysis：Select one plant of 20 years raw Semen Caryae Cathayensis, take young root, diameter 1cm stems, spire, leaf bud,
Staminate inflorescence and fruit, and different times stock and scion before and after hickory grafting is chosen, total serum IgE is extracted, and carry out cDNA's
Synthesis, using SYBR Premix ExTaqTM (the perfect real time) test kit of TAKARA quantitative RT-PCR point is carried out
Analysis, concrete grammar with reference to operating instruction, according to the auxin output carrier protein CcPILS protein gene for having obtained and interior
Ginseng actin gene orders separately design quantitative RT-PCR forward and reverse primer SymFor, symRev, actin For and actin
Rev, and enter performing PCR reaction respectively, derives data, carries out melting curve analysis, detect every part of sample aquaporin and
Actin gene C T value, every part of sample is repeated 3 times PCR, according to 2-△△CTMeansigma methodss and standard deviation are calculated, test the data obtained is used
Microsoft Excel 2010 map, and carry out spatial and temporal expression analysis.
Preferably, described step c1) in 5 ' RACE specific primer symRev sequences be 5 '-
TAGCTCCTCCCGAATCTGCTGAAG-3 ', newpilsR sequence is 5 '-TCCCGAATCTGCTGAAGAAATCCA-3 '.
Preferably, described step c1) in 5 ' RACE nest-type PRCs be divided into two-wheeled, first round PCR reaction system (50 μ
L)：The μ l of 5 × PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer symRev and downstream
The each 1 μ l of primer GSP1,5 '-RACE-Ready cDNA of masterplate 1 μ l, Prime STAR HS DNA Polymerase (2.5U/ μ
L) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extensions
1.5min, totally 30 circulations；Second wheel PCR reaction systems (50 μ L)：5 × PrimeSTAR Buffer 10 μ l, dNTP
The each 1 μ l of Mixture (2.5mM each) 4 μ l, forward primer newpilsR and downstream primer NGSP1, masterplate is first round PCR expansions
The volume increase μ l of thing 1, Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification
Program is：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations.
Preferably, described step c2) in 3 ' RACE specific primer newpilsF sequences be 5 '-
ATCTCCTCCTTATCATCGTCCCCG-3 ', SymFor sequence is 5 '-AGCACTTCAAGC AACTGAGGAGGT-3 '.
Preferably, described step c2) in 3 ' RACE nest-type PRCs be divided into two-wheeled, first round PCR reaction system (50 μ
L)：The μ l of 5 × PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer newpilsF are with
Trip primer GSP2 each 1 μ l, the μ l of 3 '-RACE-Ready cDNA of masterplate 1, Prime STAR HS DNA Polymerase (2.5U/
μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extensions
1.5min, totally 30 circulations；Second wheel PCR reaction systems (50 μ L)：5 × PrimeSTAR Buffer 10 μ l, dNTP
The each 1 μ l of Mixture (2.5mM each) 4 μ l, forward primer SymFor and downstream primer NGSP2, masterplate is first round PCR amplifications
The μ l of product 1, Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR expand journey
Sequence is：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations.
Preferably, described step c4) in specific primer Pilsfor sequence be 5 '-
The sequence of ATGGGTTTCTGGTCACTCTTTGAG-3 ', Pilsrev is 5 '-TCAAGACAAGATCCACATGAGACTG-3 '.
Preferably, described step D) in phylogenetic tree built using adjacent method (Neighbor-Joining), it is adjacent
The supporting rate for connecing tree is obtained with (boot-strap) method of bootstrapping, repeated sampling 1000 times.
Preferably, described step D) in utilize online tool ProtParam (http：//web.expasy.org/
Protparam/) the physicochemical property of analysing protein；Using ProtScale program (http：//web.expasy.org/
Protscale/) the hydrophobicity of analysing protein；Using TMpred program (http：//www.ch.embnet.org/
Software/TMPRED_form.html) prediction of transmembrane amino acid area is carried out；Using (the http of TargetP 1.1：//
Www.cbs.dtu.dk/services/TargetP/ the subcellular location of the gene) is predicted；By protein specialist system
Online tool (http：//www.expasy.org/) it is respectively completed prediction to Protein secondary, tertiary structure.
Preferably, described step E) in SymFor sequence be 5 '-AGCACTTCAAGCAACTGAGGAGGT-3 ',
The sequence of symRev is 5 '-TAGCTCCTCCCGAATCTGCTGAAG-3 ', the sequence of actin For is 5 '-
GTGAACGGGAAATTGTC-3 ', actin Rev sequences are 5 '-AGAGATGGCTGGAAGAGG-3 '.
Preferably, described step E) in PCR reaction carry out by following program：94 DEG C of denaturations 1min, next enter
94 DEG C of denaturations 5s of row, 60 DEG C of denaturations 34s, totally 35 circulations.
Beneficial effects of the present invention：Compared with prior art, a kind of Semen Caryae Cathayensis auxin output carrier that the present invention is provided
PROTEIN C cPILS gene diffusion analysis method, using RACE methods, clones Semen Caryae Cathayensis PIL homologous geness CcPILS,
Full length gene sequence 1541bp, wherein open reading frame sequence 1263bp.Analyze using bioinformatics software, predict the sequence
Row 420 aminoacid of coding, CcPILS molecular weight of albumen is about 46.22KD, and PI is 5.38, is positioned at endoplasmic reticulum, N, C two ends
Respectively there are 5 transmembrane hydrophobic domains, separated by middle hydrophilic loop.The gene has with arabidopsiss AtPILS5, AtPILS7
Compared with high homology, belong to the Clade III subtribes in PILS gene families.Fluorescence quantitative RT-RCR analysis shows, in Semen Caryae Cathayensis
Before and after grafting, it is consistent with the expression in stock that CcPILS expresses trend in scion, and the CcPILS genes participate in Semen Caryae Cathayensis and transfer
It is connected into during Auxin Signal Tranducation living and plays a part of gene expression regulation, breed breeding difficult to hickory grafting is stranded
Difficult, quality is difficult to the solution of the problems such as improving and provides favourable facilitation.
The feature and advantage of the present invention will combine accompanying drawing and be described in detail by embodiment.
【Description of the drawings】
Fig. 1 is the Semen Caryae Cathayensis CcPILS full length gene PCR amplification figures of the embodiment of the present invention；
Fig. 2 is the auxin output carrier protein aminoacid sequences of the Semen Caryae Cathayensis CcPILS with several plant of the embodiment of the present invention
Row sequence analysis dendrogram；
Fig. 3 is the homologous comparison diagrams of the Semen Caryae Cathayensis CcPILS with other plant PILS albumen of the embodiment of the present invention；
Fig. 4 is the transmembrane region analysis chart of the Semen Caryae Cathayensis CcPILS genes of the embodiment of the present invention；
Fig. 5 is the CcPILS Subcellular Localization and secondary structure schematic diagram of the embodiment of the present invention.
【Specific embodiment】
To make the object, technical solutions and advantages of the present invention of greater clarity, below by drawings and Examples, to this
Invention is further elaborated.However, it should be understood that specific embodiment described herein is only to explain the present invention,
It is not limited to the scope of the present invention.Additionally, in the following description, the description to known features and technology is eliminated, to keep away
Exempt from unnecessarily to obscure idea of the invention.
The embodiment of the present invention provides a kind of Semen Caryae Cathayensis auxin output carrier protein CcPILS gene diffusion analyses
Method, comprises the following steps：
A), material is prepared：Vegetable material is hickory grafting sample, and in test site sampling is started before and after grafting, is transferred
Stock and scion are taken before connecing respectively as control, then grafting is gathered in grafting part every 3d, 7d, 14d after grafting, often
30～50 graftings of secondary collection, take back laboratory and save backup in -70 DEG C.
In embodiments of the present invention, test site is located at Linan Zhejiang A ＆ F University Semen Caryae Cathayensis proving ground.
B), RNA extraction and purifications：To step A) the middle stock for sampling, scion and grafting, using the CTAB methods of improvement
Total serum IgE is extracted, is then operated to specifications using Oligotex mRNA Spin-Column test kits (QIAGEN companies) pure
Change mRNA.
C), CcPILS genes RACE：
C1) with reference to primer in RACE test kits (BD SMARTTM RACE cDNA Amplification Kit) description
Design principle, the auxin output carrier protein Homologous gene sequences fragment design to obtaining in Semen Caryae Cathayensis bud transcript profile data
5 ' RACE specific primer symRev (5 '-TAGCTCCTCCCGAATCTGCTGAAG-3 ') and newpilsR (5 '-
TCCCGAATCTGCTGAAGAAATCCA-3 '), and respectively with GSP and NGSP1 successively for 5 ' RACE nest-type PRCs, 5 ' RACE nests
Formula PCR is divided for two-wheeled, first round PCR reaction system (50 μ L)：5 × PrimeSTAR Buffer 10 μ l, dNTP Mixture
The each 1 μ l of (2.5mM each) 4 μ l, forward primer symRev and downstream primer GSP1, the μ l of 5 '-RACE-Ready cDNA of masterplate 1,
Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98
DEG C degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations；Second wheel PCR reaction systems (50 μ L)：5×
The μ l of PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer newpilsR and downstream primer
The each 1 μ l of NGSP1, masterplate is the μ l of first round pcr amplification product 1, Prime STAR HS DNA Polymerase (2.5U/ μ l)
0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min,
Totally 30 circulations.
C2) according to the RACE specific primer newpilsF of sequential design 3 ' of known fragment (5 '-
ATCTCCTCCTTATCATCGTCCCCG-3 ') and SymFor (5 '-AGCACTTCAAGCAACTGAGGAGGT-3 '), and respectively with
GSP2 and NGSP2 is successively used for 3 ' RACE nest-type PRCs, and 3 ' RACE nest-type PRCs are divided into two-wheeled, first round PCR reaction system (50 μ
L)：The μ l of 5 × PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer newpilsF are with
Trip primer GSP2 each 1 μ l, the μ l of 3 '-RACE-Ready cDNA of masterplate 1, Prime STAR HS DNA Polymerase (2.5U/
μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extensions
1.5min, totally 30 circulations；Second wheel PCR reaction systems (50 μ L)：5 × PrimeSTAR Buffer 10 μ l, dNTP
The each 1 μ l of Mixture (2.5mM each) 4 μ l, forward primer SymFor and downstream primer NGSP2, masterplate is first round PCR amplifications
The μ l of product 1, Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR expand journey
Sequence is：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations.
C3) product obtained by 5 ' RACE and 3 ' RACE PCR amplifications is carried out Jing after 1.5% agarose gel electrophoresiies inspection
Glue reclaim is cut, recovery product is added by rTaq enzymes and be connected with pMD-18 carriers after A tails, then is transformed in DH5 α competence bacterial strains,
Bacterial strain after conversion is coated on LB flat boards in 37 DEG C of overnight incubations, picking monoclonal, after colony PCR amplification, electrophoresis detection
Positive colony is selected to send company to be sequenced；
C4) specific primer Pilsfor (5 '-ATGGGTTTCTGGTCACTCTTTGAG-3 ') is designed according to sequencing result
With Pilsrev (5 '-TCAAGACAAGATCCACATGAGACTG-3 '), by masterplate of cDNA CcPILS open reading frame is expanded
(ORF), and it is connected to after pMD-18 and determines sequence.
D), bioinformatic analysis：The sequence for being determined is translated into into aminoacid sequence, using Mega6.0 by the amino
Acid sequence and AtPILS1, AtPILS2, AtPILS3, AtPILS4, AtPILS5, AtPILS6, AtPILS7, DcPILS3,
DcPILS6, DcPILS7, MdPILS3, MdPILS5, MdPILS6 phylogenetic tree construction, phylogenetic tree adopts adjacent method
(Neighbor-Joining) build, the supporting rate of adjacent tree is obtained with (boot-strap) method of bootstrapping, repeated sampling 1000 times.
Using online tool ProtParam (http：//web.expasy.org/protparam/) analysing protein physicochemical property；
Using ProtScale program (http：//web.expasy.org/protscale/) analysing protein hydrophobicity；Utilize
TMpred program (http：//www.ch.embnet.org/software/TMPRED_form.html) carry out transmembrane amino acid
Predict in area；Using (the http of TargetP 1.1：//www.cbs.dtu.dk/services/TargetP/) predict the Asia of the gene
Cell position；By the online tool (http of protein specialist system：//www.expasy.org/) it is respectively completed to protein
Two grades, the prediction of tertiary structure.
E), spatial and temporal expression analysis：Select one plant of 20 years raw Semen Caryae Cathayensis, take young root, diameter 1cm stems, spire, leaf bud,
Staminate inflorescence and fruit, and different times stock and scion before and after hickory grafting is chosen, total serum IgE is extracted, and carry out cDNA's
Synthesis, using SYBR Premix ExTaqTM (the perfect real time) test kit of TAKARA quantitative RT-PCR point is carried out
Analysis, concrete grammar with reference to operating instruction, according to the auxin output carrier protein CcPILS protein gene for having obtained and interior
Ginseng actin gene orders separately design the forward and reverse primer SymFor of quantitative RT-PCR (5 '-
AGCACTTCAAGCAACTGAGGAGGT-3’)、symRev(5’-TAGCTCCTCCCGAATCTGCTGAAG-3’)、actin For
(5 '-GTGAACGGGAAATTGTC-3 ') and actin Rev (5 '-AGAGATGGCTGGAAGAGG-3 '), and enter performing PCR respectively
Reaction, PCR reactions are carried out by following program：94 DEG C of denaturations 1min, followed by 94 DEG C of denaturations 5s, 60 DEG C of denaturations
34s, totally 35 circulations, derives data, carries out melting curve analysis, detects the aquaporin and actin genes of every part of sample
CT values, every part of sample is repeated 3 times PCR, according to 2-△△CTMeansigma methodss and standard deviation are calculated, the data obtained Microsoft is tested
Excel 2010 maps, and carries out spatial and temporal expression analysis.
As a result：
Refering to Fig. 1, the embodiment of the present invention extracts the total serum IgE of Semen Caryae Cathayensis spire using the CTAB methods of improvement, and Jing agaroses coagulate
Gel electrophoresis 3 counterband tapes of detection total serum IgE are bright, clear and complete.With cDNA as template, CcPILS amplified productions are 1541bp,
Its open reading frame sequence is 1263bp, identical with expected size.
Online tool ProtParam predicts that the molecular formula of the CcPILS albumen is C2141H3376N514O583S18, its coding 421
Aminoacid, predicted molecular weight is 46.22KD, and theoretical iso-electric point is 5.38, is made up of 20 kinds of aminoacid altogether, and wherein leucine contains
Amount highest (14.0%), histidine content minimum (0.5%).The instability index of CcPILS albumen is 41.99, belongs to unstable
Albumen, Aliphatic index reaches 120.64, and hydrophilic mean coefficient is 0.601.ProtScale programs predict the protein
Water repellent region, discovery respectively has a hydrophobic region, separated by middle hydrophilic area, the accounting that hydrophobic region accounts at N, C two ends
Larger, this is corresponding with higher Aliphatic index.The prediction according to more than can speculate that CcPILS is that hydrophobicity is unstable
Albumen.
Refering to Fig. 4, TMpred prediction CcPILS have 5 trans-membrane regions in N-terminal and C-terminal, and transmembrane region is corresponding with hydrophobic region.
Refering to Fig. 5, Predictprotein predicts that the albumen is positioned at endoplasmic reticulum, belongs to auxin output carrier
(Auxin efflux carrier)。
Refering to Fig. 2, the aminoacid sequence of other species PILS gene codes is searched in NCBI, and using MEGA6 by mountain
AtPILS1 (the GI of Semen Juglandiss CcPILS and arabidopsiss (Arabidopsis thaliana):1035997954)、AtPILS2(GI:
75169666)、AtPILS3(GI:75169730)、AtPILS4(GI:75169729)、AtPILS5(GI:75205686)、
AtPILS6(GI:75181312)、AtPILS7(GI:75171357), with the DcPILS3 (GI of Radix Dauci Sativae (Daucus carota):
1040866677)、DcPILS6(GI:1040913933)、DcPILS7(GI:1040905618) with Fructus Mali pumilae (Malus
Domestica MdPILS3 (GI):1039893723)、MdPILS5(GI:658038089)、MdPILS6(GI:
658061887) phylogenetic tree construction, it is found that the gene gathers at same point with AtPIL5, AtPIL7, DcPILS7 and MdPILS5
.
Refering to Fig. 3, CcPILS carries out homologous ratio with the protein sequence of AtPILS5, AtPILS7, DcPILS7 and MdPILS5
To finding, its consistency be respectively 58.9%, 61.9%, 61.3% and 64.1%, CcPILS and AtPILS5, AtPILS7 in egg
White N-terminal and C-terminal are that the homology of transmembrane region is higher, and then relatively low in centre, and have more 24 amino than AtPILS5, AtPILS7
Sour residue.
Space expression of the carrier protein CcPILS genes in Semen Caryae Cathayensis different tissues material is exported by Semen Caryae Cathayensis auxin
Analysis, as a result shows, Semen Caryae Cathayensis aquaporin CcPILS expressions in staminate inflorescence are minimum, next to that stem, fruit, spire,
Expression highest is root.
PILS genes are shown in the relative expression quantity and standard deviation of different times according to Semen Caryae Cathayensis PILS genes RT-PCR
Change in the expression of hickory grafting different times.As a result it is as follows：Content of the PILS genes in stock and scion be all when 0 day
Than relatively low；And at 3 days after grafting, the expression in stock increased nearly 3 times than 0 day, peak value, at the same time, scion are reached
The expression of middle PILS genes has also reached peak value, but incrementss not as stock it is notable；Afterwards PILS gene expression amounts are in stock
All decline with scion, wherein stock rises and falls big to declining during after grafting 14 days in 7 days after grafting, and scion is relative in the meantime
The value of expression is basically stable at 0.5 or so.
Auxin is distributed widely in each organ of higher plant, with the unidirectional polar translocation mode of short distance.This
Invention with hickory grafting Seedling as material, using transcript profile sequencing technologies obtain Semen Caryae Cathayensis PILS albumen homology genes fragment
Design special primer, by RACE technologies, has cloned Semen Caryae Cathayensis CcPILS full length genes, to CcPILS protein amino acid sequences
Physicochemical property, domain, transmembrane region and subcellular location are predicted analysis shows PILS with two hydrophobic regions, and each is dredged
Aqua region has a 3-5 membrane spaning domain, and there is a hydrophilic loop in albumen central authorities, and hydrophilic loop is located in kytoplasm, subcellular location with
PIN5, PIN8 are similar, are both positioned at endoplasmic reticulum, are responsible for transport of the auxin from kytoplasm to endoplasmic, so as to participate in life
During long element polar translocation, embodiment of the present invention result not only shows CcPILS in various groups of Semen Caryae Cathayensis root, stem, leaf, flower etc.
Middle expression is knitted, and function is played during hickory grafting, be that hickory grafting is difficult, breed breeding is difficult, quality
The solution for being difficult to the problems such as improving provides favourable facilitation.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent or improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of Semen Caryae Cathayensis auxin exports carrier protein CcPILS gene diffusion analysis methods, it is characterised in that：Bag
Include following steps：
A), material is prepared：Vegetable material is hickory grafting sample, starts sampling before and after grafting in test site, before grafting
Stock and scion are taken respectively as control, then grafting is gathered in grafting part every 3d, 7d, 14d after grafting, are adopted every time
30～50 graftings of collection, take back laboratory and save backup in -70 DEG C；
B), RNA extraction and purifications：To step A) the middle stock for sampling, scion and grafting, extracted using the CTAB methods of improvement
Total serum IgE, then operates to specifications purified mRNA using Oligotex mRNA Spin-Column test kits；
C), CcPILS genes RACE：
C1) with reference to design of primers principle in RACE kit specifications, the growth to obtaining in Semen Caryae Cathayensis bud transcript profile data
Element output carrier protein Homologous gene sequences fragment designs 5 ' RACE specific primer symRev and newpilsR, and respectively with
GSP and NGSP1 is successively used for 5 ' RACE nest-type PRCs；
C2) according to sequential design 3 ' RACE the specific primer newpilsF and SymFor of known fragment, and respectively with GSP2 and
NGSP2 is successively used for 3 ' RACE nest-type PRCs；
C3) product obtained by 5 ' RACE and 3 ' RACE PCR amplifications is carried out cutting glue Jing after 1.5% agarose gel electrophoresiies inspection
Reclaim, recovery product is added by rTaq enzymes and be connected with pMD-18 carriers after A tails, then is transformed in DH5 α competence bacterial strains, will turn
Bacterial strain after change is coated on LB flat boards in 37 DEG C of overnight incubations, picking monoclonal, is selected after colony PCR amplification, electrophoresis detection
Positive colony send company to be sequenced；
C4) specific primer Pilsfor and Pilsrev are designed according to sequencing result, is opened by masterplate amplification CcPILS of cDNA
Reading frame (ORF), and be connected to after pMD-18 and determine sequence；
D), bioinformatic analysis：The sequence for being determined is translated into into aminoacid sequence, using Mega6.0 by the aminoacid sequence
Row with AtPILS1, AtPILS2, AtPILS3, AtPILS4, AtPILS5, AtPILS6, AtPILS7, DcPILS3, DcPILS6,
DcPILS7, MdPILS3, MdPILS5, MdPILS6 phylogenetic tree construction, using instrument and program bio information credit is carried out
Analysis；
E), spatial and temporal expression analysis：One plant of 20 years raw Semen Caryae Cathayensis is selected, young root, diameter 1cm stems, spire, leaf bud, male flower is taken
Sequence and fruit, and different times stock and scion before and after hickory grafting is chosen, extraction total serum IgE, and the synthesis of cDNA is carried out,
Quantitative RT PCR analysis are carried out using SYBR Premix ExTaqTM (the perfect real time) test kit of TAKARA, is had
Body method exports carrier protein CcPILS protein gene and internal reference with reference to operating instruction according to the auxin for having obtained
Actin gene orders separately design forward and reverse primer SymFor, symRev, actin For and actin Rev of quantitative RT-PCR,
And enter performing PCR reaction respectively, and data are derived, melting curve analysis are carried out, detect the aquaporin and actin bases of every part of sample
Because of CT values, every part of sample is repeated 3 times PCR, according to 2-△△CTMeansigma methodss and standard deviation are calculated, the data obtained Microsoft is tested
Excel 2010 maps, and carries out spatial and temporal expression analysis.
2. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step c1) in 5 ' RACE specific primer symRev sequences be 5 '-
TAGCTCCTCCCGAATCTGCTGAAG-3 ', newpilsR sequence is 5 '-TCCCGAATCTGCTGAAGAAATCCA-3 '.
3. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step c1) in 5 ' RACE nest-type PRCs be divided into two-wheeled, first round PCR reaction system (50 μ L)：5
The μ l of × PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer symRev and downstream primer
The each 1 μ l of GSP1,5 '-RACE-Ready cDNA of masterplate 1 μ l, Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5
μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30
Individual circulation；Second wheel PCR reaction systems (50 μ L)：5 × PrimeSTAR Buffer10 μ l, dNTP Mixture (2.5mM
Each each 1 μ l of) 4 μ l, forward primer newpilsR and downstream primer NGSP1, masterplate is the μ l of first round pcr amplification product 1,
Prime STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98
DEG C degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations.
4. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step c2) in 3 ' RACE specific primer newpilsF sequences be 5 '-
ATCTCCTCCTTATCATCGTCCCCG-3 ', SymFor sequence is 5 '-AGCACTTCAAGCAACTGAGGAGGT-3 '.
5. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step c2) in 3 ' RACE nest-type PRCs be divided into two-wheeled, first round PCR reaction system (50 μ L)：5
The μ l of × PrimeSTAR Buffer 10, dNTP Mixture (2.5mM each) 4 μ l, forward primer newpilsF and downstream are drawn
The each 1 μ l of thing GSP2,3 '-RACE-Ready cDNA of masterplate 1 μ l, Prime STAR HS DNA Polymerase (2.5U/ μ l)
0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration 10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min,
Totally 30 circulations；Second wheel PCR reaction systems (50 μ L)：5 × PrimeSTAR Buffer10 μ l, dNTP Mixture (2.5mM
Each each 1 μ l of) 4 μ l, forward primer SymFor and downstream primer NGSP2, masterplate is first round pcr amplification product 1 μ l, Prime
STAR HS DNA Polymerase (2.5U/ μ l) 0.5 μ l, the μ l of sterile purified water 32.5, PCR amplification programs are：98 DEG C of degeneration
10s, 55 DEG C of annealing 10s, 72 DEG C of extension 1.5min, totally 30 circulations.
6. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step c4) in specific primer Pilsfor sequence be 5 '-
The sequence of ATGGGTTTCTGGTCACTCTTTGAG-3 ', Pilsrev is 5 '-TCAAGACAAGATCCACATGTAGACTG-3 '.
7. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step D) in phylogenetic tree using adjacent method (Neighbor-Joining) build, adjoin tree
Supporting rate with bootstrap (boot-strap) method obtain, repeated sampling 1000 times.
8. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step D) in utilize online tool ProtParam (http：//web.expasy.org/
Protparam/) the physicochemical property of analysing protein；Using ProtScale program (http：//web.expasy.org/
Protscale/) the hydrophobicity of analysing protein；Using TMpred program (http：//www.ch.embnet.org/
Software/TMPRED_form.html) prediction of transmembrane amino acid area is carried out；Using (the http of TargetP 1.1：//
Www.cbs.dtu.dk/services/TargetP/ the subcellular location of the gene) is predicted；By protein specialist system
Online tool (http：//www.expasy.org/) it is respectively completed prediction to Protein secondary, tertiary structure.
9. a kind of Semen Caryae Cathayensis auxin as claimed in claim 1 exports carrier protein CcPILS gene diffusions analysis side
Method, it is characterised in that：Described step E) in SymFor sequence be 5 '-AGCACTTCAAGCAACTGAGGAGGT-3 ',
The sequence of symRev is 5 '-TAGCTCCTCCCGAATCTGCTGAAG-3 ', the sequence of actin For is 5 '-
GTGAACGGGAAATTGTC-3 ', actin Rev sequences are 5 '-AGAGATGGCTGGAAGAGG-3 '.
10. a kind of Semen Caryae Cathayensis auxin output carrier protein CcPILS gene diffusion analyses as claimed in claim 1
Method, it is characterised in that：Described step E) in PCR reaction carry out by following program：94 DEG C of denaturations 1min, followed by
94 DEG C of denaturations 5s, 60 DEG C of denaturations 34s, totally 35 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611081459.4A CN106591321A (en) | 2016-11-30 | 2016-11-30 | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611081459.4A CN106591321A (en) | 2016-11-30 | 2016-11-30 | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106591321A true CN106591321A (en) | 2017-04-26 |
Family
ID=58593987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611081459.4A Pending CN106591321A (en) | 2016-11-30 | 2016-11-30 | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591321A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102050A (en) * | 2017-05-16 | 2017-08-29 | 肖玲君 | A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism |
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| CN110093358A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind OjCCoAOMT gene order relevant to Chinese celery lignin synthesis and its application |
| CN113005120A (en) * | 2021-03-19 | 2021-06-22 | 中国农业大学 | Method for effectively extracting dormant grape grafted DNA and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349527A (en) * | 2015-11-23 | 2016-02-24 | 浙江农林大学 | Carya cathayensis RCA gene cloning method |
| CN105543341A (en) * | 2015-11-23 | 2016-05-04 | 浙江农林大学 | Detection and analysis method of hickory nut RCA gene clone |
| CN105695576A (en) * | 2016-03-02 | 2016-06-22 | 浙江农林大学 | Analysis method for pecan Aux/IAA gene cloning |
-
2016
- 2016-11-30 CN CN201611081459.4A patent/CN106591321A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349527A (en) * | 2015-11-23 | 2016-02-24 | 浙江农林大学 | Carya cathayensis RCA gene cloning method |
| CN105543341A (en) * | 2015-11-23 | 2016-05-04 | 浙江农林大学 | Detection and analysis method of hickory nut RCA gene clone |
| CN105695576A (en) * | 2016-03-02 | 2016-06-22 | 浙江农林大学 | Analysis method for pecan Aux/IAA gene cloning |
Non-Patent Citations (2)
| Title |
|---|
| ELKE BARBEZ ET AL.: "A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants", 《NATURE》 * |
| 艾雪等: "山核桃水通道蛋白CcPIP同源基因克隆与表达分析", 《福建林学院学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102050A (en) * | 2017-05-16 | 2017-08-29 | 肖玲君 | A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism |
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| CN110093358A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind OjCCoAOMT gene order relevant to Chinese celery lignin synthesis and its application |
| CN113005120A (en) * | 2021-03-19 | 2021-06-22 | 中国农业大学 | Method for effectively extracting dormant grape grafted DNA and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Good et al. | Engineering nitrogen use efficiency with alanine aminotransferase | |
| CN107435047B (en) | Low-phosphorus-resistant key gene GmPHR25 in plant phosphorus signal network and application thereof | |
| CN106591321A (en) | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method | |
| Chen et al. | Identification of Rubisco rbcL and rbcS in Camellia oleifera and their potential as molecular markers for selection of high tea oil cultivars | |
| Zhang et al. | De novo transcriptome sequencing and comparative analysis of differentially expressed genes in kiwifruit under waterlogging stress | |
| CN110845590B (en) | Wild grape VyPPR gene and application of encoding protein thereof in drought stress | |
| CN101960012A (en) | Woody plants having improved growth charateristics and method for making the same using transcription factors | |
| CN112876551B (en) | Transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and application thereof | |
| CN110128514A (en) | Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and application | |
| Douglas | Populus as a model tree | |
| Zeng et al. | Differential expression patterns reveal the roles of cellulose synthase genes (CesAs) in primary and secondary cell wall biosynthesis in Miscanthus× giganteus | |
| Zhang et al. | Rice co-expression network analysis identifies gene modules associated with agronomic traits | |
| CN112601452A (en) | Benzophin bicarbonate transporter from green algae | |
| CN106399326A (en) | Soybean bHLH transcription factor gene GmFER and encoded protein and application thereof | |
| Zhang et al. | PacBio full-length sequencing integrated with RNA-seq reveals the molecular mechanism of waterlogging and its recovery in Paeonia ostii | |
| CN110964740B (en) | Preparation method and application of tobacco with high flavonol content | |
| CN114958906B (en) | Gene and promoter related to low potassium stress of tobacco and application of gene and promoter | |
| CN114621956B (en) | Drought-resistant lncRNA and application thereof | |
| Ohtsu et al. | Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana | |
| Gu et al. | Phosphoenolpyruvate carboxylase in the stem of the submersed species Egeria densa may be involved in an inducible C4-like mechanism | |
| CN105018519B (en) | Paddy gene ORYsa；LPR5 genetic engineering application | |
| CN107354162A (en) | Paddy gene ORYsa;SIZ2 genetic engineering application | |
| CN114292855A (en) | PagARR9 gene for regulating and controlling growth of xylem of poplar and application thereof | |
| CN109234289B (en) | Method for creating stress-resistant transgenic alfalfa | |
| CN109722441B (en) | Cucumber small heat shock protein Cu-sHSP gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170426 |