CN108611354A - A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe - Google Patents
A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe Download PDFInfo
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Abstract
The present invention relates to the method for identifying molecules that a kind of FT mRNA molecules transmit between congener anvil fringe, method provided by the invention can realize that can the gene that quick, sensitive, accuracy goes identification homology very high high, easy, economical and practically carry out long range transmission between stock and scion, have a vast market foreground.This method process is simple, it is of less demanding, it takes short, workload is smaller, work period is smaller, it is efficient, it spends cheap, on the one hand can be that scientific research person saves money, on the other hand, shorten experimental period, improve working efficiency, and with the popularity and generality of application, it can be applied on congener on a large scale, solve the problems, such as that common laboratory can not carry out RT PCR dCAPS identifications by polyacrylamide gel and the Vertial electrophorestic tank gene that homology is very high between congener stock and scion, eliminate the time spent by transgenosis, and RT PCR CAPS are also high to gene order requirement, general gene is difficult to meet.
Description
Technical field
The present invention relates to field of plant molecular biology, and in particular to be FT gene mRNAs molecule in Pyrus L stock
The method for identifying molecules transmitted over long distances between scion.
Background technology
The extensive utilization graft technology in production of fruit trees, stock can improve root system to nutrient with scion interaction
Absorption And Transportation improves degeneration-resistant scion, improvement fruit quality, increase fruit yield, so that scion is bloomed ahead of time, regulates and controls scion dwarfing
Deng, up to the present, between anvil fringe interaction relationship research it is special to be concentrated mainly on anatomy, nutrition transport, hormone and physiology
Property etc., related Study on Molecular Mechanism is less.It has been found that there are some RNA and protein signal molecule to lead between anvil fringe
It crosses plant phloem and carries out long range transmission, and then influence stock or the physiology and developmental process of scion.At present mainly in quasi- south
Some are identified in the herbaceous plant such as mustard, pumpkin, tomato, potato can carry out the endogenous mRNA and protein of long range transmission
Signaling molecule, and certain research has been carried out to molecular mechanism, and xylophyta fruit tree although identified it is several can be into
The endogenous mRNA molecules that row transmits over long distances, wherein finding that PbWoxT1mRNA can be compound with PbPTB3 formation RNP in companion cell
Body carries out movement over long distances, can regulate and control to tree-like development, tree vigo(u)r balance, in addition, transferable AtGAI mRNA are built
Expression vector obtains apple transgenic rootstock using transgenic technology, and after scion is grafted on transgenic rootstock, discovery can make
Scion is downgraded, therefore can carry out identification and transport mechanism research by transmitting mRNA molecules over long distances between fruit tree anvil fringe, is utilized
Transgenic technology, cultivating has exceptional function stock, regulates and controls scion (or stock) by grafting fixed point improvement stock (or scion)
Main Agronomic Characters are increased economic efficiency, and guidance is provided for fruit breeding and production.
FLOWERING LOCUS T (FT) mRNA using structure carry special signature carrier to Arabidopsis Mutants into
Row transgenosis, RT-PCR detections, can find stock transgenosis in scion after wild type scion is grafted on transgenic rootstock
Specific band, and it was found that so that scion is bloomed ahead of time, it was demonstrated that FT mRNA can carry out long range biography between plant anvil fringe
It passs.At present for identify xylophyta gene mRNA whether the method that can carry out long range transmission, only by structure take
Carrier with special signature carries out RT-PCR detections and digestion amplification polymorphism (Cleaved after transgenosis grafting
Amplified polymorphic sequences, CAPS) technology.Although these technologies detect the transitivity of gene than calibrated
Really, but wherein have the drawback that process is cumbersome, it is desirable that it is high, time-consuming, and workload is larger, and the work period is long, and efficiency is low etc.,
And during research perennial woody plant mRNA is transmitted between anvil fringe, the gene order of stock and scion need to be cloned, is led to
Sequence only exists the difference of several bases between normal congener anvil fringe, and method relatively simple at present is that derivative digestion amplification is polymorphic
Property (derived cleaved amplified polymorphic sequence, dCAPS) technology, introduces mispairing in primer
Base builds restriction enzyme enzyme recognition site, you can those SNPs for causing restriction enzyme site to change are detected by digestion,
Achieve the purpose that distinguish the different genes sequence transmitted between anvil fringe, but this method still has its shortcoming, because belonging to
The digestion products size that plant anvil fringe gene difference generates only differs 20bp or so, needs to use polypropylene so implementing this method
The Vertial electrophorestic tank instrument of acrylamide gel and costliness, therefore be unfavorable for common laboratory identification mRNA and passed between plant anvil fringe
Pass, the quantity that can transmit mRNA can be identified by thus greatly limiting, cause the research that fruit tree endogenous mRNA grafting is transmitted into
The endogenous mRNA that slow, new long range is transmitted is postponed to be difficult to find.
Invention content
In view of the deficiencies in the prior art, method provided by the invention solve common laboratory can not be by poly- third
Acrylamide gel and the Vertial electrophorestic tank gene that homology is very high between congener stock and scion carry out RT-PCR-
The problem of dCAPS is identified, while also solving that fruit tree transgenic technology is more difficult, and time-consuming and is inconvenient to identify, RT-PCR-CAPS
High, general gene also implacable problem is required gene order.The present invention exists between utilizing different cultivars in gene order
SNPs (single nucleotide polymorphism), pass through and design special primer, introduce base mismatch structure restriction enzyme and identify position
Point so that gene order there is no natural discrepant restriction enzyme site and then obtains digestion position originally between different cultivars anvil fringe
Point designs special a pair of of long primer and carries out nested PCR amplification to last round of pcr amplification product again, later finally using limit
Property endonuclease digestion processed is detached digestion products with Ago-Gel by conventional Horizontal electrophoresis tank, you can relatively more same
Source is grafted with whether the digestion spectrogram after heterologous grafting, identification mRNA transmit between plant anvil fringe, finally solves congener
Can stock and the very high gene of scion homology carry out transmitting the problem of identifying over long distances by simple instrument and routine operation,
With quick, sensitive, accuracy is high, easy, economical and practical, the characteristics of can be applied to other plant.
To achieve the above objectives, the technical solution adopted by the present invention is that：
The amino acid sequence of the nucleotide sequence of birch-leaf pear and pear FT provided by the invention and the albumen by the nucleotide coding
Row, as shown in SEQ 1~4.
A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe, includes the following steps：
(1) according to https:The gold pendant pears (GenBank announced in //www.ncbi.nlm.nih.gov/:
KF240775.1 coding FT gene orders carry out analysis design primer, separation and clone's pear (Pyrus in)
Bretschneideri FT full length genes), are encoded in birch-leaf pear (Pyrus betulaefolia Bunge), utilize bioinformatics
Method carries out sequence similarity analysis, it was demonstrated that gained sequence belongs to FT genes；
(2) the FT gene orders for analyzing pear and birch-leaf pear, find the site of the two gene difference, the upstream and downstream in this site
Specific primer is separately designed, to constitute restriction enzyme site；
(3) pear is scion, and birch-leaf pear is that stock carries out test tube seedling micrografting and the self micrografting of pear, birch-leaf pear are micro- self
Grafting；
(4) respectively after extraction pear autoplastic graft, birch-leaf pear autoplastic graft and grafting stock and scion total serum IgE, reversion
CDNA is recorded into, respective FT genetic fragments are obtained with specific primer first round PCR amplification；
(5) genetic fragment for recycling first round pcr amplification product FT reuses a pair of of long primer and carries out as template
Second wheel PCR amplification, obtains respective FT genetic fragments again；
(6) PCR recovery products are taken turns with restriction enzyme pair second and carries out digestion, after comparing isogenetic graft and heterologous grafting
Digestion spectrogram, if there are the bi-directional of mRNA in grafting system, the meeting after the amplified production digestion of scion pear
The band for stock birch-leaf pear specificity occur equally also will appear scion pear specificity after the amplified production digestion of stock birch-leaf pear
Band；If there is the unidirectional delivery of mRNA, then it is special to will appear stock birch-leaf pear after the amplified production digestion of scion pear
The band of property or the band that will appear scion pear specificity after the amplified production digestion of stock birch-leaf pear；If mRNA is not sent out
It is raw to transmit, then the band of respective respective sample will be shown in the restriction enzyme digestion and electrophoresis figure of amplified production and without other miscellaneous bands；
It is described for expanding birch-leaf pear and the overall length primer of pear FT homologous genes is：
Upstream 5 '-ATGCCTCGGGACAGGGACC-3 '
Downstream 5 '-TTATCTTCTCCTCCCACCGGAG-3 '；
It is described for expanding birch-leaf pear and the nido first round primer of pear FT homologous gene fragments is：Upstream 5 '-
GTTGTCCAGCAACCTAGAGTTGGTAC-3’
Downstream 5 '-TTATCTTCTCCTCCCACCGGAG-3 '；
The nido second for expanding birch-leaf pear and pear FT homologous gene fragments takes turns primer：Upstream
5’-CAATGGTTGTGAGCTCAAACCTTCTCAAGTTGTCCAGCAACCTAGAG-3’
The downstream and
5’-AATCCAAGGTTATAAAGCTCGGCGAAGTCTCTGGTATTGAAGTTTTGG-3’；
The restriction enzyme is KpnI.
On the basis of said program, the sample for extracting total serum IgE selects stock birch-leaf pear stem section bast after micrografting 30d
Portion and graft union and scion pear stem section bast.
Description of the drawings
The present invention has following attached drawing：
Fig. 1 show pear and birch-leaf pear FT full length gene PCR amplification electrophoretograms.In figure：M：DNA molecular amount standard；1：Duck
Pears；2：Birch-leaf pear.
Fig. 2 show FT amino acid sequence homologies and compares figure.In figure：DL-FT is birch-leaf pear；YL-FT is pear；JZ-FT
Pears are fallen for gold.
Fig. 3 show FT gene homologies and compares figure.In figure：DLFT is birch-leaf pear；YLFT is pear.
Fig. 4 show pear and birch-leaf pear FT genetic fragment first round PCR amplification electrophoretograms.In figure：M：DNA molecular amount mark
It is accurate；1：Pear；2：Birch-leaf pear.
Fig. 5 show pear and birch-leaf pear FT genetic fragments second take turns PCR amplification electrophoretogram.In figure：M：DNA molecular amount mark
It is accurate；1：Pear；2：Birch-leaf pear.
Agarose is solidifying after Fig. 6 show pear and the wheel PCR recovery product digestion with restriction enzyme of birch-leaf pear FT genes second
Gel electrophoresis testing result figure.In figure：M：DNA molecular amount standard；Y’：Pear second takes turns PCR recovery products；D’：Birch-leaf pear second is taken turns
PCR recovery products；Y：Pear second takes turns PCR recovery product digestion results；D：Birch-leaf pear second takes turns PCR recovery product digestion results；
M’：Pear and birch-leaf pear equal amount of mixture second take turns PCR recovery product digestion results.
Fig. 7 show stock and the wheel RT-PCR result figures of scion the one or two after micrografting.In figure：1：Birch-leaf pear stem section bast
Portion；2：Graft union；3：Pear stem section bast；CK：Control group；Y：Pear grafts pear；D：Birch-leaf pear grafts birch-leaf pear；M’：Pear
It grafts pear and birch-leaf pear grafts birch-leaf pear equal amount of mixture.
Fig. 8 show the improved dCAPS Molecular Identifications schematic diagram of the invention.
Fig. 9 show micrografting scion and takes turns PCR recovery product digestion with restriction enzyme agar with stock FT genes second
Sugared detected through gel electrophoresis result figure.In figure：M：DNA molecular amount standard；1：Birch-leaf pear stem section bast；2：Graft union；3：Pear stem
Section bast；CK：Control group；Y：Pear grafts pear；D：Birch-leaf pear grafts birch-leaf pear；M’：Pear grafts pear and birch-leaf pear grafting Du
Pears equal amount of mixture.
Specific implementation mode
Below in conjunction with attached drawing 1-9, invention is further described in detail.
1 test tube micrografting of embodiment
Choose stock birch-leaf pear (Pyrus betulaefolia Bunge) and scion pear (Pyrus
Bretschneideri) tissue-cultured seedling seedling, constant light source, light intensity 1500lx, illumination 14h/10h day-night cycles, room temperature 23~25
DEG C, relative humidity 85%, 30~40d subcultures are primary.It is MS+0.5mgL that pear and birch-leaf pear, which use culture medium prescription,–1 6-BA+
0.1mg·L–1IBA。
Aseptically, the birch-leaf pear tissue-cultured seedling decaptitating after numerous 30d will be expanded, stays about 1.5cm long stem sections as stock, along top
The longitudinal sectional about 0.5cm opennings in portion；The pear tissue-cultured seedling of the high 1cm of seedling taking, top stay 2-3 piece leaves, base portion to be whittled into " V " type, are inserted into stock
Notch is wrapped with masking foil, merging culture medium MS+0.5mgL–1 6-BA+0.1mg·L–1It is grown in IBA.Take stock
Birch-leaf pear tissue culture seeding stem segment bast and scion pear tissue culture seeding stem segment bast are put into -80 DEG C of refrigerators after liquid nitrogen is handled and preserve
For gene cloning.
2 gene RNA of embodiment extracts
The extraction of vegetable material total serum IgE uses CTAB methods (Zhang Yugang etc., 2005), from stock birch-leaf pear stem section bast and connects
Total serum IgE is extracted in scion bast after stock bast, graft union, grafting after fringe pear stem section bast, and grafting, with 30
μ L DEPC water dissolutions, -80 DEG C of refrigerators of electrophoresis detection postposition save backup.
(1) DNA in RNA is removed：
Ingredient is added and reaction system is as follows：
(2) 30min is handled at 37 DEG C；550 μ L are added without RNase water (0.1%DEPC processing), add isometric
600uL CI mixings；
(3) at 4 DEG C, 10000rpm centrifuges 10min, draws supernatant, adds isometric CI gently mixings；At 4 DEG C,
10000rpm centrifuges 10min；
(4) supernatant is drawn, the absolute ethyl alcohol of 2 times of volumes is added into pipe, at -20 DEG C, precipitates 1h；
(5) at 4 DEG C, 12000rpm centrifuges 20min；Supernatant is abandoned, 75% ethyl alcohol of 1mL is added and carries out rinsing precipitation,
12000rpm centrifuges 5min, and extra ethyl alcohol is sucked out with pipette tips after rinsing 2 times；
(6) it is put in superclean bench and is dried up, is dissolved in 30-50 μ L DEPC water, be put into -80 DEG C of refrigerators later and protect
It deposits with spare；
The integrality of (7) 1% agarose gel electrophoresis Detection and Extraction nucleic acid, with ultraviolet specrophotometer by surveying 260nm
The absorbance at place calculates the RNA concentration of extraction.
RNA reverse transcriptions are cDNA by embodiment 3
Reverse transcription system and program are as follows：
(1) following component is added into 0.2mL RNase-free PCR pipes on ice：
(2) brief centrifugation after mixing is put into 70 DEG C of 10min in PCR instrument, is placed in 5min on ice at once later；
(3) it continues up on ice in the PCR pipe in a step and following component is added：
(4) brief centrifugation after mixing is put into 42 DEG C of 60min in PCR instrument, 70 DEG C of 10min, after waiting for reaction to terminate, takes
Go out product and be placed in -20 DEG C to save backup.
Templates of the obtained cDNA as following PCR amplifications.
4 pear of embodiment and birch-leaf pear FT full length genes clone
According to https:The gold pendant pears (GenBank announced in //www.ncbi.nlm.nih.gov/:KF240775.1 in)
Design primer, separation and clone's pear (Pyrus bretschneideri), birch-leaf pear is compared in coding FT gene orders
FT full length genes are encoded in (Pyrus betulaefolia Bunge).
It is as follows using primer to expand FT full length genes：
Sense primer：5 '-ATGCCTCGGGACAGGGACC-3 ',
Downstream primer：5’-TTATCTTCTCCTCCCACCGGAG-3’.
Above-mentioned primer is synthesized by calm and peaceful Bioisystech Co., Ltd of Sino-U.S..
PCR reaction systems：2 × Es Taq MasterMix (Dye) are purchased from (Beijing health is the century limited public affairs of biotechnology
Department, CW0682S)
PCR response procedures are as follows：
94 DEG C of pre-degeneration 5min；
94℃30s；
60℃30s；
72℃30s；
34 cycles.
72 DEG C of last extension 10min.
PCR product detects：According to big 2% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffers, 70-
110V electrophoresis about 20min, Ethidum Eremide dye, and PCR product size is detected under ultraviolet lamp, can obtain the identical item of two sizes
Band.As shown in Figure 1.
5 target fragment Ago-Gel of embodiment recycles
Using AXYGEN companies Ago-Gel DNA QIAquick Gel Extraction Kits, method is with reference to its company's specification：
(1) agarose containing target DNA segment is cut, cuts off extra gel as possible；
(2) gel piece is put into 2mL centrifuge tubes, adds the gel melt agent Buffer DE-A of 3 times of volumes；
(3) it is positioned over water-bath 10min in 75 DEG C of water-baths, during which constantly leniently spins upside down centrifuge tube, to ensure glue
Block is dissolved completely in red sol solutions.After waiting for 10min, if also undissolved blob of viscose, it is molten can be supplemented some
Glue is placed a few minutes again, until being completely dissolved；
(4) the Buffer DE-B for drawing 0.5 Buffer DE-A volume are added in centrifuge tube, if the DNA pieces of recycling
When section is less than 400bp, the isopropanol of 1 gel volume should be also added；
(5) the yellow sol liquid that will have been dissolved, is added in recovery column, and at room temperature, 10000rpm centrifuges 1min, outwells receipts
Waste liquid in collector；
(6) operation repeated in (5) is primary；
(7) the rinsing liquid W1 of 500 μ L is added, at room temperature, 10000rpm centrifuges 1min, outwells the waste liquid in collecting pipe；
(8) the rinsing liquid W2 (plus absolute ethyl alcohol) of 700 μ L is added, at room temperature, 10000rpm centrifuges 1min, outwells receipts
Waste liquid in collector；
(9) recovery column is put back in collecting pipe, at room temperature, 10000rpm centrifuges 1min, removes extra rinsing liquid；
(10) recovery column is taken out, non-volatile ethyl alcohol is dried up on super-clean bench, is put into a new 1.5mL centrifuge tube,
The eluent that 35 μ L are pre-heated to 65 DEG C is added in recovery column film center, stands 2min at room temperature, PCR product is allowed to be completely dissolved
In eluent, 13000rpm centrifuges 1min to elute；
(11) 2-5 μ L recovery products are taken, electrophoresis detection recovering effect is carried out with a concentration of 1.0% Ago-Gel,
The PCR product that remaining recycling obtains is put into -20 DEG C and saves backup.
Embodiment 6 prepares E. coli competent
(1) strain is taken out from -70 DEG C of refrigerators, standby after gently being tapped with sterilized toothpick before not thawing completely
It crosses on good 90mm culture mediums, 37 DEG C are incubated overnight 16h or so, and culture to sealing when visible single bacterium colony is protected in 4 DEG C of refrigerators
It deposits spare；
(2) LB liquid medium 300ml is prepared, the triangular flask (50ml/ bottles) of 6 200ml, autoclave sterilization are sub-packed in
20min is cooled to 55 DEG C or so, and 50 μ L of LB liquid medium is taken to be loaded on the same sterilized glass in each triangular flask
In test tube.Culture medium sealing is spare in 4 DEG C of refrigerators in triangular flask；
(3) LB liquid is equipped with pipette tips push-in in picking single bacterium colony on the cultured bacterium plates of 90mm with sterilized pipette tips
In the teat glass of culture medium, sealing, 228rpm is incubated overnight 16h or more in 37 DEG C of isothermal vibration beds；
(4) from the triangular flask for taking 50 μ L that fluid nutrient medium is housed in spare 6 in cultured liquid bacterium solution respectively,
With 1：100 ratios are inoculated with, and it is 0.6 that 225rpm, which is cultivated to OD600, about 3h or so；
(5) bacterium solution is dispensed into 12 50mL sterile polypropylene centrifuge tubes, places 10min on ice, then 4000rpm, 4
DEG C centrifugation 10min receive bacterium；
(6) supernatant is abandoned, is inverted centrifuge tube 1min thoroughly to remove supernatant.Often the 0.1M CaCl of 25ml precoolings are added in pipe2,
Gently mixing sets 30min on ice；
(7) 4000rpm, 4 DEG C of centrifugation 10min；
(8) supernatant is abandoned, is inverted centrifuge tube 1min thoroughly to remove supernatant.Often the 0.1M CaCl of 25ml precoolings are added in pipe2,
Gently mixing；
(9) competence can be used directly at this time, if needing to preserve for a long time, a concentration of 80% glycerine can be added, and make
Final glycerol concentration is 20%, in -80 DEG C of preservations.
7 target fragment T- carriers of embodiment connect and conversion
Take the PCR product of purifying that pMD19-T carriers and buffer solution (Takara companies, 3271, Japan) (reference is added
PMD19-T carrier connection descriptions book)
Linked system：
Mixing slightly centrifuges, 16 DEG C of water-bath 12h or more.The molar ratio 1 of carrier and genetic fragment:4.
(1) 50 μ L Escherichia coli trans5 α competent cells are taken, are placed on ice；
(2) etc. 10 μ L connection products are added until completely dissolved, by its gently mixing, are positioned over 30min on ice；
After carrying out heat shock 45s on (3) 42 DEG C of metal baths, it is positioned over 2-3min on ice at once；
(4) the LB culture mediums of 500 μ L are added, are put into 200rpm shaken cultivations 45min in 37 DEG C of shaking tables；
(5) 10000rpm centrifuges 1min at room temperature, the supernatant of 400 μ L is sopped up with pipette tips, then will be thin with remaining culture medium
Born of the same parents suspend；
(6) bacterium solution is equably coated on the solid LB media containing ammonia benzyl antibiotic；
(7) tablet is inverted to overnight incubation (16-18h) in 37 DEG C of incubators.With sterilized pipette tips picking positive colony
Contain in the LB liquid medium of ammonia benzyl antibiotic in 20 μ L, mixing is beaten in suction, is put into 37 DEG C of shaking tables and is cultivated 20min, and 1 μ L are drawn
Bacterium solution carries out the identification of PCR positive colonies as template.Positive control is set (using the product after PCR recovery purifyings as mould simultaneously
Plate) and negative control (using locus coeruleus bacterium solution as template) and single primer pair shine；Agarose gel electrophoresis detects Insert Fragment size.
Positive colony bacterium solution is selected, is sequenced by calm and peaceful Bioisystech Co., Ltd of Sino-U.S..Sequencing result shows two target fragments
It is 525bp, encodes 174 amino acid.
8 pear of embodiment and birch-leaf pear FT gene biological bioinformatics analysis
Gained gene order is translated as protein sequence and carries out Multiple Sequence Alignment point with DNAMAN (Lynnon BioSoft)
Analysis.Multiple Sequence Alignment result is shown：Pear and birch-leaf pear FT gene amino acid sequences and gold pendant pears amino acid sequence are completely the same, such as
Shown in Fig. 2, therefore illustrate that the gene order come is cloned in pear and birch-leaf pear belongs to FT genes, is then named as YL-FT,
It is corresponding by birch-leaf pear FT unnamed genes be DL-FT.
9 pear of embodiment and birch-leaf pear FT segment first round PCR amplifications
YL-FT is up to 99.43% with DL-FT gene nucleotide series similitudes, and only there are three the difference of base, such as Fig. 3
It is shown, therefore specific primer is difficult design, is identified using the method for RT-PCR infeasible.And utilize DNAMAN analysis stock with
Restriction enzyme site difference between scion nucleotide sequence, the restriction enzyme site for selecting stock special to scion, this conventional CAPS technologies
Also infeasible, then inventor expects utilizing dCAPS technologies, and base mismatch structure restriction enzyme identification is introduced in primer
Site can detect those SNPs for causing restriction enzyme site to change by digestion, reach the different bases distinguished and transmitted between anvil fringe
Because of the purpose of sequence.
Primer is synthesized by calm and peaceful Bioisystech Co., Ltd of Sino-U.S..
PCR reaction systems：
PCR reaction conditions：
94 DEG C of pre-degeneration 5min；
94℃30s；
59℃30s；
72℃20s；
30 cycles.
72 DEG C of last extension 10min.
PCR product detects：According to big 2% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffers, 70-
110V electrophoresis about 20min, Ethidum Eremide dye, and PCR product size is detected under ultraviolet lamp, can obtain the identical item of two sizes
Band.As shown in Figure 4.
10 pear of embodiment and birch-leaf pear FT segments second take turns PCR amplification
By the recycling of first round PCR product, purifying, method is with embodiment 5, if to first round PCR recovery product using limitation
Property restriction endonuclease KpnI pear and birch-leaf pear can be cut into different size of segment, but digestion products size only differs 26bp, institute
It is needed using polyacrylamide gel and expensive Vertial electrophorestic tank instrument with implementing this method, therefore is unfavorable for generally testing
The mRNA transmitted between room identification anvil fringe, and detach digestion products and need to carry out the polyacrylamide of 4h or so under cryogenic
Gel electrophoresis is easily deformed band in laboratory is more demanding with scientific research personnel and finally runs out of running gel figure, is unfavorable for
The achievements exhibition of scientific research personnel.Then inventor expects special a pair of of the long primer of design again to last round of pcr amplification product
It carries out nido second and takes turns PCR amplification, finally reuse digestion with restriction enzyme, the digestion products size difference generated in this way can
Up to 50bp or so, digestion products are detached with Ago-Gel using conventional Horizontal electrophoresis tank, so that it may more homologous to transfer
Connect with the digestion spectrogram after heterologous grafting, analyze and identify whether mRNA transmits between plant anvil fringe.
Primer is synthesized by calm and peaceful Bioisystech Co., Ltd of Sino-U.S..
PCR reaction systems：
PCR reaction conditions：
94 DEG C of pre-degeneration 5min；
94℃30s；
57℃30s；
72℃20s；
34 cycles.
72 DEG C of last extension 10min.
PCR product detects：According to big 2% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffers, 70-
110V electrophoresis about 20min, Ethidum Eremide dye, and PCR product size is detected under ultraviolet lamp, can obtain the identical item of two sizes
Band.As shown in Figure 5.
Embodiment 11PCR product KpnI digestions
By the second wheel PCR product recycling, purifying, method then carries out digestion with embodiment 5, we use restricted interior
Enzyme cutting KpnI (Thermo companies, FD0524), you can pear is taken turns into PCR recovery products with birch-leaf pear FT segments second and is cut into Bu Tong greatly
Small segment.Digestion system is as follows：
12 agarose gel electrophoresis of embodiment
According to big 2% Ago-Gel of little makings of target fragment, with TAE electrophoretic buffers 70-110V electrophoresis about
30min, Ethidum Eremide dye, and PCR product clip size is detected under ultraviolet lamp.
It was found that the PCR product of birch-leaf pear cannot be digested, therefore generate band of the band of 351bp as birch-leaf pear specificity,
The PCR product of pear can be digested into two bands of 54bp, 297bp, as shown in fig. 6, since 54bp bands are brominated second pyridine
It dyes shallower and is easy to run out of outside glue, be not easy to observe in Jiao Tuzhong, so pear PCR recycling production in agarose gel electrophoresis figure
Object digestion can be used as the band of pear specificity at the band of 297bp, and final result is consistent with expected restriction enzyme mapping.Thus may be used
See, designed two pairs of primers and restriction enzyme KpnI can be used for the transmission for identifying FT gene mRNAs in pear birch-leaf pear.
The identification that FT mRNA are transmitted between pear and birch-leaf pear after 13 micrografting of embodiment
30d after micrografting takes from stock birch-leaf pear stem section bast, graft union and scion pear stem section bast respectively
Sample extracts total serum IgE, carries out PCR by the method for embodiment 9,10 after reverse transcription, as shown in Figure 7.With respective pear and birch-leaf pear itself
The stem section bast cDNA of grafting is negative control, mixed with the stem section bast cDNA equivalent of pear and birch-leaf pear itself grafting
It is combined into positive control.Digestion is carried out with restriction enzyme KpnI to the second wheel PCR recovery products by embodiment 11, by embodiment
12 identify into row agarose gel electrophoresis, as shown in Figure 9.
The result shows that：After grafting 30 days, it can be seen that the not new digestion band of stock birch-leaf pear stem section bast occurs,
And scion pear stem section bast is other than containing autospecific digestion band, while also adding the special of stock birch-leaf pear
Property digestion band and pear and birch-leaf pear autoplastic graft seedling in control group and find no new enzyme-specific slitting band, explanation
FTmRNA can be passed uni-directionally to scion pear from stock birch-leaf pear, without being transmitted to stock birch-leaf pear from scion pear.
Advantages of the present invention：
Method provided by the invention can realize that quick, sensitive, accuracy goes identification homology high, easy, economical and practically
Can very high gene carry out long range transmission between stock and scion, have a vast market foreground.This method process is simple,
Of less demanding, time-consuming short, workload is smaller, and the work period is smaller, efficient, spends cheaply, on the one hand can be that scientific research person saves
On the other hand money shortens experimental period, improve working efficiency, and with the popularity and generality of application, can be a wide range of
It is applied on congener, solving common laboratory can not be planted by polyacrylamide gel and Vertial electrophorestic tank to belonging to
The gene that homology is very high between object stock and scion carries out the problem of RT-PCR-dCAPS identifications, eliminates spent by transgenosis
Time, and RT-PCR-CAPS is also high to gene order requirement, general gene is difficult to meet.If this method can be widelyd popularize, this
Sample can not only save money and time, additionally it is possible to be applied to other plant, widen significantly between can identifying anvil fringe over long distances
The quantity for transmitting mRNA, can also promote the research in terms of the xylophyta signal transmission of perennial genome height heterozygosis.
Sequence explanation
SEQ 1 and 2 is the amino acid sequence of the nucleotide sequence and FT of birch-leaf pear FT；SEQ 3 and 4 is the nucleotide of pear FT
The amino acid sequence of sequence and FT；SEQ 5 and 6 is the primer pair for expanding FT overall lengths；SEQ 7 and 8 is the first round to expand FT segments
Primer pair；SEQ 9 and 10 is the primer pair that the second wheel expands FT segments.
The content not being described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.
Sequence table
<110>China Agricultural University
<120>A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe
<141> 2018-05-11
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1683
<212> DNA
<213>A kind of method for identifying molecules (2 Ambystoma that FT mRNA molecules transmit between congener anvil fringe
laterale x Ambystoma jeffersonianum)
<400> 1
atntnvrsny rsbtaangat gcctcgggac agggaccccc ttgttgttgg acgagtggta 60
ggtgatgttt tagacccctt cacaaggtcc gtttctctga gggtgaccta cggcaataag 120
gaggttaaca atggttgtga gctcaaacct tctcaagttg tccagcaacc tagagttgat 180
actggtggtg acgatctcag gactttctac actctggtca tggtggatcc tgatgcaccc 240
agcccaagtg accccaacct aaaggaatat ttgcattggt tggttacgga tattccagca 300
acaacggcgg caagcttcgg gcaagagatc gtgtgttacg aaagtccacg gccaacagtt 360
gggattcatc gctttgtttt tgtgctgttt cgccaattgg gtaggcaaac agtgtatgct 420
cctggatggc gccaaaactt caataccaga gacttcgccg agctttataa tcttggatta 480
ccggtggctg ccgtctattt taactgccaa agggagagcg gctccggtgg gaggagaaga 540
taayrsbtaa ngtrrgsrgs raayrgaays asrhhrrgra rrgahryrys nysasnsnyy 600
sysrrnaann rrgashryys srghrhyrhr atasrsarrr rsrsnysyrs rahrsrahrh 660
raarhynays yrrrrgrhra ysrghahahr gnyrgnhray raryrrgnsn hsnhrrgsha 720
yrsnyraaaa yrhsnysnrg ryryyrgrgr gyrsbrtsch ndratgcctc gggacaggga 780
cccccttgtt gttggacgag tggtaggtga tgttttagac cccttcacaa ggtccgtttc 840
tctgagggtg acctacggca ataaggaggt taacaatggt tgtgagctca aaccttctca 900
agttgtccag caacctagag ttgataccgg tggtgacgat ctcaggactt tctacactct 960
ggtcatggtg gatcctgatg cacccagccc aagtgacccc aacctaaagg aatatttgca 1020
ttggttggtt acggatattc cagcaacaac cgcggcaagc ttcgggcaag agatcgtgtg 1080
ttacgaaagt ccacggccaa cagttgggat tcatcgcttt gtttttgtgc tgtttcgcca 1140
attgggtagg caaacagtgt atgctcctgg atggcgccaa aacttcaata ccagagactt 1200
cgccgagctt tataaccttg gattaccggt ggctgccgtc tattttaact gccaaaggga 1260
gagcggctcc ggtgggagga gaagataayr sbrtschndr trrgsrgsra ayrgaaysas 1320
rhhrrgrarr gahryrysny sasnsnyysy srrnaannrr gashryyssr ghrhyrhrat 1380
asrsarrrrs rsnysyrsra hrsrahrhra arhynaysyr rrrgrhrays rghahahrgn 1440
yrgnhrayra ryrrgnsnhs nhrrgshayr snyraaaayr hsnysnrgry ryyrgrgrga 1500
tgcctcggga cagggacctt atcttctcct cccaccggag gttgtccagc aacctagagt 1560
tggtacttat cttctcctcc caccggagca atggttgtga gctcaaacct tctcaagttg 1620
tccagcaacc tagagaatcc aaggttataa agctcggcga agtctctggt attgaagttt 1680
tgg 1683
Claims (6)
1. a kind of birch-leaf pear FT genes, nucleotide sequence is as shown in SEQ 1.
2. by the albumen of gene code described in claim 1, amino acid sequence is as shown in SEQ 2.
3. a kind of pear FT genes, nucleotide sequence is as shown in SEQ 3.
4. by the albumen of the gene code described in claim 3, amino acid sequence is as shown in SEQ 4.
5. the method for identifying molecules that a kind of FT mRNA molecules transmit between congener anvil fringe, which is characterized in that including following step
Suddenly：
(1) coding FT gene orders in pears are fallen according to gold and carries out analysis design primer, FT is encoded in separation and clone's pear, birch-leaf pear
Full length gene carries out sequence similarity analysis, it was demonstrated that gained sequence belongs to FT genes using bioinformatics method；
(2) the FT gene orders for analyzing pear and birch-leaf pear, find the site of the two gene difference, and the upstream and downstream in this site is distinguished
Specific primer is designed, to constitute restriction enzyme site；
(3) pear is scion, and birch-leaf pear is that stock carries out that test tube seedling micrografting and the self micrografting of pear, birch-leaf pear are self micro- to transfer
It connects；
(4) respectively after extraction pear autoplastic graft, birch-leaf pear autoplastic graft and grafting stock and scion total serum IgE, reverse transcription at
CDNA obtains respective FT genetic fragments with specific primer first round PCR amplification；
(5) genetic fragment for recycling first round pcr amplification product FT reuses a pair of of long primer and carries out second as template
PCR amplification is taken turns, obtains respective FT genetic fragments again；
(6) PCR recovery products are taken turns with restriction enzyme pair second and carries out digestion, compare the enzyme after isogenetic graft and heterologous grafting
Spectrogram is cut, if there are the bi-directionals of mRNA in grafting system, will appear after the amplified production digestion of scion pear
The band of stock birch-leaf pear specificity, equally also will appear the item of scion pear specificity after the amplified production digestion of stock birch-leaf pear
Band；If there is the unidirectional delivery of mRNA, then it will appear stock birch-leaf pear specificity after the amplified production digestion of scion pear
Band or the band that will appear scion pear specificity after the amplified production digestion of stock birch-leaf pear；If mRNA is not passed
It passs, then will show the band of respective respective sample in the restriction enzyme digestion and electrophoresis figure of amplified production and without other miscellaneous bands；
It is described for expanding birch-leaf pear and the overall length primer of pear FT homologous genes is：
Upstream 5 '-ATGCCTCGGGACAGGGACC-3 '
Downstream 5 '-TTATCTTCTCCTCCCACCGGAG-3 '；
It is described for expanding birch-leaf pear and the nido first round primer of pear FT homologous gene fragments is：Upstream 5 '-
GTTGTCCAGCAACCTAGAGTTGGTAC-3’
Downstream 5 '-TTATCTTCTCCTCCCACCGGAG-3 '；
The nido second for expanding birch-leaf pear and pear FT homologous gene fragments takes turns primer：Upstream
5’-CAATGGTTGTGAGCTCAAACCTTCTCAAGTTGTCCAGCAACCTAGAG-3’
The downstream and
5’-AATCCAAGGTTATAAAGCTCGGCGAAGTCTCTGGTATTGAAGTTTTGG-3’；
The restriction enzyme is KpnI.
6. the method for identifying molecules that FT mRNA molecules as claimed in claim 5 transmit between congener anvil fringe, feature exist
In the sample for extracting total serum IgE selects stock birch-leaf pear stem section bast and graft union and scion pear stem after micrografting 30d
Section bast.
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