CN108330182A - A kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe - Google Patents

Abstract

The invention discloses the method for identifying molecules that a kind of mRNA molecules transmit between plant anvil fringe, are related to field of plant molecular biology.The present invention builds the carrier progress transgenosis that gene carries fluorescent protein tag, transgenic rootstock and wild type scion are subjected to micrografting in sterile glass vials, because there is the characteristic for easily growing adventitious root in a humid environment in scion, the presence or absence of the fluorescence that scion adventitious root is sent out is detected by laser confocal microscope, to judge being transmitted at a distance between whether gene has occurred anvil fringe；Or judge transmitted at a distance between whether gene has occurred anvil fringe by the way that whether Chao Shi PCR verification anvil fringes specific band all occur, final realize can intuitively observe the purpose that gene is transmitted at a distance between stock and scion, the method of the present invention is with intuitive, quick, sensitive, accuracy is high, various plant genes be can be applied to without species limited, had broad application prospects in field of plant molecular biology.

Description

A kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe

Technical field

The present invention relates to field of plant molecular biology, and in particular to be that a kind of mRNA molecules transmit between plant anvil fringe Fluorescence Identification method.

Background technology

During garden crop production practices, it can improve plant resistance using graft technology, increase yield, improvement fruit The raising of real quality, the improvement to kind, economic value suffers from very important meaning, wherein being grafted more in production of fruit trees It is to have obtained extensive utilization.Found in production of fruit trees, different stocks to the physio-biochemical characteristics of scion, blossom and bear fruit, environment Adaptability and Growth status development etc. can generate different influences, and these influences are eventually to the formation of fruit And the economic value in later stage play the role of it is critical.

Up to the present, it has been found that many endogenous mRNA that can be at a distance transmitted by plant phloem points Son, such as arabidopsis Aux/IAA18 and Aux/IAA28 (Notaguchi et al., 2012), pumpkin CmNACP (Ruiz- Medrano et al.,1999)、CmPP16(Xoconostle-Cazares et al.,1999)、CmGAIP(Haywood et Al., 2005), tomato the PFP-LeT6fusion gene (Kim et al., 2001), potato StBEL5 (Banerjee et al.,2006；Hannapel,2010)、POTH1(a KNOTTED1-Like transcription factor) Herbaceous plant such as (Mahajan et al., 2012).Currently, what is identified on xylophyta fruit tree can be passed at a distance The endogenous mRNA molecules passed, the GAI for having apple endogenous (Xu et al., 2010), pears endogenous KN1 (Zhang Wenna, 2012), GAI (Zhang et al.,2012)、NACP(Zhang et al.,2013)、WoxT1(Duan et al.,2015).These mRNA Molecule can be transmitted to scion from stock, influence the physiology and developmental process of scion.Therefore, by being transmitted at a distance between anvil fringe MRNA molecules carry out identification and mechanism study, can be not only mRNA interaction machines between the remote transport mechanism of plant and anvil fringe Reason research provide fundamental basis, and can also be used discovery transmit mRNA develop it is a kind of can be with orderly improvement scion character Novel stock breeding method, and then regulate and control scion character by grafting means, harvest transgenic product is not only avoided, but also available Specific merit product, achievees the purpose that increase economic efficiency, and can also provide guidance for garden crop breeding from now on and production.

3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is that the of terpene substances are synthesized in mevalonate pathway One important rate-limiting enzyme, be cytoplasm terpenoid metabolism in important regulating and controlling site (Bach, 1986；Choi et al., 1992), play the role of in the growth and development process of plant very important.HMGR polypeptides chain part is all by N- in plant This four part of end region, bonding pad, transmembrane region and the end regions C- forms, and wherein C- terminal catalytics region is by nearly 3 ' end in gene Coding is formed, and highly conserved in most plants (Roberts, 2007).Currently, the birch-leaf pear PbHMGR1 of pear by gram Grand to obtain (citrine etc., 2015), transferabilities of the mRNA between anvil fringe has also been proved to.

And for the method that the mRNA of plant identification gene can be transmitted at a distance, at present only by building purpose The carrier that gene carries special signature carries out RT-PCR detections (Haywood et al., 2005) after transgenosis grafting, nido The technologies such as RT-PCR (Kanehira et al., 2010), RT-PCR-CAPS (Xu et al., 2010), although these technologies are examined The transitivity of cls gene is more accurate, but shortcoming present in it is to carry out pcr amplification reaction to be easily contaminated And false positive is led to the problem of, visual inspection is unable to the signal transmitted, cannot specify transmission position and direction of signal etc., and The method of current more visual inspection is that molecular probe special on mRNA labels with fluorescence on probe, is carried out micro- note Fluorescence spread condition (Xoconostle-Cazares et al., 1999) is detected after penetrating, but this method still has its deficiency Place, the signal that observation mRNA is transmitted are simply present in one or two of iuntercellular, belong to the identification method of intercellular short distance transmission, and And required instrument is very expensive, operating technology requires high, spends also higher, common laboratory is extremely difficult to, therefore The process transmitted at a distance between anvil fringe for how to study mRNA, harsh requirement are greatly limited and can be identified The quantity of mRNA thus further increases difficulty so that plant is in mRNA to carry out the research that plant endogenous mRNA graftings are transmitted Research in terms of remote transmission is difficult to promote, and the new endogenous mRNA transmitted at a distance is difficult to find.

Invention content

It is an object of the invention to overcome the prior art to carry out turning base by building the carrier of gene carrying special signature Because can not intuitively observe the problem of transmission has occurred in mRNA after grafting, a kind of mRNA molecules are provided and are transmitted between plant anvil fringe Fluorescence Identification method.

The Fluorescence Identification method that a kind of mRNA molecules provided by the invention transmit between plant anvil fringe is by building gene Carry the carrier of fluorescent protein tag, prepare transgenosis plant；Using genetically modified plants as stock, by it with wild type scion in nothing Carry out micrografting under the conditions of bacterium, there is situation in the fluorescence for observing stock and scion, if stock and scion adventitious root observe it is glimmering Light then illustrates that the mRNA molecules of the gene transmit between plant anvil fringe.

On the other hand, the present invention provides a kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe, passes through structure Build the carrier that gene carries fluorescent protein tag, prepare transgenosis plant；Using genetically modified plants as stock, it is connect with wild type Fringe aseptically carries out micrografting, is detected using Chao Shi PCR, if there is specific band in anvil fringe Chao Shi PCR products, Illustrate that the mRNA molecules of the gene transmit between plant anvil fringe.

The fluorescin is green fluorescent protein, red fluorescent protein, yellow fluorescence protein, blue fluorescent protein, orange Color fluorescin or bluish-green fluorescin.

In an embodiment of the present invention, the gene is birch-leaf pear HMGR1.

The carrier is to be overexpressed GFP-PbHMGR1 carriers, is prepared by the following method to obtain：Extract birch-leaf pear genome RNA, reverse transcription cDNA carry out PCR amplification, recycle target fragment and purify, and connect carrier T, transformed competence colibacillus cell, selection Correct positive colony is sequenced to obtain the final product, the sequence of the primer pair used in the PCR amplification is as shown in SEQ ID NO.2-3.

When whether the method for the present invention progress Chao Shi PCR detection anvil fringes specific band occur, the reaction of first round PCR uses Primer pair sequence is as shown in SEQ ID NO.4-5, the primer pair sequence such as SEQ ID NO.6-7 institutes of the second wheel PCR reaction uses Show.

Further, the present invention provides a kind of Fluorescence Identification method that HMGR1mRNA molecules transmit between plant anvil fringe, packet Include following steps：

(1) birch-leaf pear total serum IgE is extracted respectively, and reverse transcription is at cDNA, the overall length of PCR amplification HMGR1 genes；

(2) structure birch-leaf pear HMGR1 genes carry the carrier of fluorescent protein tag, prepare transgenosis plant；

(3) genetically modified plants are stock, itself and wild type scion are aseptically carried out micrografting, observation stock and There is situation in the fluorescence of scion, if stock and scion adventitious root observe corresponding fluorescence, illustrates birch-leaf pear HMGR1 genes MRNA molecules transmit between plant anvil fringe.

The primer pair sequence that step (1) PCR amplification uses is as shown in SEQ ID NO.2-3.

The fluorescin that the embodiment of the present invention is selected is GFP, after micrografting, the adventitious root and anvil that take scion to grow out The wooden root is positioned over observation GFP fluorescence under laser confocal microscope, compares the GFP fluorescing matters of scion and stock, if transferring There are the transmission of mRNA in junctor system, then stock root will appear the GFP fluorescence that GFP-PbHMGR1 gives expression to, equally in scion cigarette The GFP fluorescence that careless adventitious root is given expression to due to also will appear remote transmit of between anvil fringe occurs mRNA by GFP-PbHMGR1；Such as Fruit is not transmitted, then only can will appear the GFP fluorescence that GFP-PbHMGR1 gives expression in stock root, in scion tobacco adventitious root Be not in then GFP fluorescence.

Wherein, for expanding birch-leaf pear HMGR1 genophore restriction enzyme site segments, the sample selection birch-leaf pear tissue culture of total serum IgE is extracted The bast of seedling；GFP-PbHMGR1 genes are overexpressed for identifying, the sample selection for extracting total serum IgE is regenerated by leaf disk method callus The out leaf of resistant buds；For identifying GFP-PbHMGR1 genes after micrografting, the sample selection micrografting 14d of total serum IgE is extracted The stem section and stock tobacco stem section of scion tobacco.

On the other hand, the present invention provides a kind of Fluorescence Identification method that HMGR1mRNA molecules transmit between plant anvil fringe, packet Include following steps：

(1) birch-leaf pear total serum IgE is extracted respectively, and reverse transcription is at cDNA, the overall length of PCR amplification HMGR1 genes；

(2) structure birch-leaf pear HMGR1 genes carry the carrier of fluorescent protein tag, prepare transgenosis plant；

(3) genetically modified plants are stock, it are aseptically carried out micrografting with wild type scion, using Chao Shi PCR is detected, if the specific band that length is 1084bp occur in anvil fringe Chao Shi PCR products, illustrates HMGR1 gene mRNAs point Son transmits between plant anvil fringe.

The primer pair sequence that the reaction of Chao Shi PCR first round PCR uses in step (3) is as shown in SEQ ID NO.4-5, and second The primer pair sequence of PCR reaction uses is taken turns as shown in SEQ ID NO.6-7.

The present invention provides application of the above-mentioned Fluorescence Identification method in plant germplasm resource improvement.

The present invention provides above-mentioned Fluorescence Identification methods can transmit mRNA molecules at a distance in identification between plant anvil fringe Application in gene.

Method provided by the invention solves the carrier progress transgenosis grafting that special signature is carried by building gene After can not intuitively observe the problem of transmission has occurred in mRNA, while also solving and marking special molecular probe on mRNA The problem of microinjection requires high and height, common laboratory is spent to be difficult to complete, and RT-PCR identification gene deliveries are vulnerable to pollution. The present invention carries out transgenosis using the carrier of structure gene Carrying Green Fluorescent Protein (GFP) label, by transgenic rootstock and open country Raw type scion carries out micrografting in sterile glass vials, since scion has the spy for easily growing adventitious root in a humid environment Property, the presence or absence of the GFP fluorescence that laser confocal microscope detection scion adventitious root is sent out is then passed through, to judge whether gene is sent out It has given birth to and has transmitted at a distance between anvil fringe, can finally solve gene can intuitively observe between stock and scion and be passed at a distance The problem of passing, have it is intuitive, quick, sensitive, accuracy is high, can be applied to various plant genes without spy species limited Point.

Method provided by the invention can realize that quick, sensitive, accuracy is high, intuitively go to identify any one gene in anvil Can wood and scion be transmitted at a distance, and tobacco also has very wide generality, before having a vast market as model plant Scape.This method process is simple, it is desirable that not high, transgene efficiency is high, and cost is relatively inexpensive, on the one hand provides identification base for scientific research person Because on the other hand the method for transmission can be directly observed gene and be transmitted, positive evidence is provided for experiment, is eliminated The energy that many experiments are spent with the problem of remarking additionally is done, shortens experimental period indirectly, also saves time cost, is improved The efficiency of work.It can be applied on various plants on a large scale, solve gene and transmitted at a distance between stock and scion The problem of can not intuitively observing, and there is requirement height in molecular probe microinjection special on mRNA labels and spend height, generally The problem of laboratory is difficult to complete, and RT-PCR identification gene deliveries are vulnerable to pollution, if this method can be widelyd popularize, in this way Money and time can not only be saved, additionally it is possible to be applied to many plant genes, widen long distance between can identifying anvil fringe significantly From the quantity for transmitting mRNA, but also the research in terms of plant signal transmission is quickly propelled.

Description of the drawings

Fig. 1 show amplification birch-leaf pear HMGR1 genophore restriction enzyme site PCR amplification electrophoretograms.M in figure：DNA molecular amount mark It is accurate；1：Birch-leaf pear.

Fig. 2, which is shown, is overexpressed GFP-PbHMGR1 vector construction schematic diagrames.

Fig. 3 show a：Tobacco leaf disc method transgenosis；b：Transgenosis GFP-PbHMGR1 tobacco positive strains RT-PCR mirror It is fixed, wherein M：DNA molecular amount standard, swimming lane 1,2,3,4,5,6,7 are respectively different tobacco lines；c：Transgenosis GFP- PbHMGR1 tobaccos take root；d：Transgenosis GFP-PbHMGR1 Tobacco Flowerings are solid；e：Transgenosis GFP-PbHMGR1 tobaccos harvest kind Son.

Fig. 4 show a：Transgenosis GFP-PbHMGR1 tobacco seeds sow the latter moon；b：With transgenosis GFP- PbHMGR1 tobaccos are stock, and wild-type tobacco is scion micrografting；c：Transgenosis GFP-PbHMGR1 tobacco micrografting 14d scion Grow adventitious root.

Fig. 5 show tobacco scion adventitious root and stock root and observes GFP fluorescence, bar=50 μm.

Fig. 6 show scion stem section and stock stem section nest-type PRC as a result, positive control is transgene tobacco stock stem section, Negative control is wild-type tobacco stem section.

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention In the case of essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.

1 gene RNA of embodiment extracts

The extraction of birch-leaf pear bast total serum IgE uses CTAB methods (Zhang Yugang etc., 2005), with 30 μ L DEPC water dissolutions, electrophoresis Detection -80 DEG C of refrigerators of postposition save backup.

(1) DNA in RNA is removed：

(2) 30min is handled at 37 DEG C；550 μ L are added without RNase water (0.1%DEPC processing), add isometric 600 μ L 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.

The extraction of plant tobacco material total serum IgE uses Trizol methods, and total serum IgE is extracted from scion stem section, stock stem section, uses 30 μ L DEPC water dissolutions, the integrality of 1% agarose gel electrophoresis Detection and Extraction nucleic acid pass through survey with ultraviolet specrophotometer Absorbance at 260nm calculates the RNA concentration of extraction, and -80 DEG C of refrigerators of electrophoresis detection postposition save backup.

It is cDNA that embodiment 2, which is overexpressed GFP-PbHMGR1 vector constructions by RNA reverse transcriptions,

It is cDNA that the RNA that conventionally embodiment 1 is obtained, which carries out reverse transcription, and obtained cDNA is as following The template of PCR amplification.

According to birch-leaf pear (Pyrus betulaefolia Bunge) plant HMGR1 gene sequence informations, reference Restriction enzyme site on pCAMBIA1305 carriers, PbHMGR1CDS select Bgl II, Spe I as gene insertion site, underscore table Show that restriction enzyme site, design of primers are as follows：

Sense primer：5‘-AGATCTATGGACGTCCGAAGGC-3 ', (SEQ ID NO.2)

Downstream primer：5’-ACTAGTTTAAGCGGACGCAACAG-3’(SEQ ID NO.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 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min, 34 cycles；72℃ Finally extend 10min.

PCR product detects：According to big 1% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffers, 70- 110V electrophoresis about 15min, Ethidum Eremide dye, detection PCR product clip size under ultraviolet lamp (see Fig. 1).

Recovered target fragment simultaneously purifies, and connects T-simple carriers, conversion E. coli competent DH 5 α, bacterium colony PCR Detection selects positive colony bacterium solution, is sequenced by calm and peaceful Bioisystech Co., Ltd of Sino-U.S., will obtain be sequenced correct carrier and PCAMBIA1305 plasmids carry out double digestion with Bgl II, Spe I respectively.

Digestion system is as follows：10 × buffer 2 μ L, Bgl II 1 μ L, Spe I 1 μ L, 16 μ L of Plasmid DNA, 20 μ L of total volume.

To digestion products agarose gel electrophoresis, recycling, connection.

16 DEG C of connection 9-10h of metal bath are positioned over, 5 α of E. coli competent DH, bacterium colony PCR detections, selection sun are converted Property clone's bacterium solution, be sequenced by calm and peaceful Bioisystech Co., Ltd of Sino-U.S., will obtain and correct bacterium solution is sequenced extracts plasmid, obtained Recombinant plasmid (see Fig. 2).II-PbHMGR1-Spe of birch-leaf pear GFP-Bgl, I sequences are as shown in SEQ ID NO.1.

The preparation and conversion of 3 Agrobacterium tumefaciems competence of embodiment

1, the preparation of Agrobacterium tumefaciems competent cell

(1) picking single bacterium colony is added in the YEP fluid nutrient mediums that 20mL contains 20mg/L Rif, shaken cultivation (28 DEG C, 200rpm, under dark condition) 24-48h, Agrobacterium shaken cultivation to suitable 600 values of OD is 0.5.

(2) ice bath 30min；

(3) 4 DEG C, 5000rpm centrifuges 10min；

(4) it is operated in super-clean bench, abandons supernatant, the NaCl of 0.15M is added, be resuspended；

(5) 4 DEG C after ice bath 20min, 6000rpm centrifuges 5min；

(6) supernatant is abandoned, the CaCl of 20mM is added₂, it is resuspended, packing, often 200 μ L of pipe, isometric 50% glycerine is added ,- 80 DEG C of preservations.

2, the conversion of Agrobacterium tumefaciems competent cell

(1) 100 μ L competent cells are taken, are placed on ice, gently suspension cell after being completely dissolved.

(2) it is added the plant expression carrier plasmids that build of 5 μ L, mixing, 30min on ice.

(3) cold shock 60s in liquid nitrogen.

(4) 37 DEG C of heat shock 5min, place 2min on ice.

(5) 500 μ L YEP culture mediums are added, in 28 DEG C of 180rpm shaken cultivations 4h.

(6) room temperature 6000rpm collection bacterium discards 400 μ L supernatants, cell is suspended with remaining culture medium.

(7) bacterium is uniformly coated on the solid YEP culture mediums added with antibiotic 50mg/L Kan and 20mg/L Rif.

(8) tablet is inverted overnight incubation (24-48h) in 28 DEG C.

The preparation of embodiment 4 tobacco preculture and bacterium solution

1, tobacco preculture

Tobacco W38 (Nicotiana tabacum var.Winsconsin 38) conversions use leaf disk method (Murashige et al.,1962).Tobacco leaf master pulse both sides are cut into the slice of 0.5cm × 0.5cm sizes, are laid in MS+2.0mg/L 6- BA+0.5mg/L NAA culture mediums, cultivate 72h under light by 25 DEG C.

2, the preparation of bacterium solution

20mL contain 20mg/L Rif, 50mg/L Kan YEP fluid nutrient medium shaken cultivations thalline (28 DEG C, 200rpm, Under dark condition) 6-9h, Agrobacterium grows to logarithm plateau, and shaken cultivation to suitable 600 values of OD is 0.8-1.0.

5 Transformation of tobacco bud seedling of embodiment

Method is infected with reference to (1985) methods such as Hotsch, and is improved.Blade is placed in bacterium solution and is gently shaken 3min takes out, and extra bacterium solution is sucked with aseptic filter paper, puts back to former preculture solid medium, 25 DEG C of light culture 48h.It is transferred to de- In bacterium solid medium (precultivation medium+cephalosporin 250mg/L), continue to cultivate under light.Wait for that blade has callus after 7-10d Tissue is transferred to screening solid medium (de- bacterium solid medium+hygromycin 20mg/L) after generating, the formation of induction of resistance bud (see The a of Fig. 3 schemes).

Resistance seedling is cut, is connected on containing MS culture medium+IBA 1mg/L+ cephalosporin 250mg/L root inductions, about 20d Left and right obtains positive regenerated transfer-gen plant, extracts its DNA and RNA and carries out RT-PCR identifications (scheming see the b of Fig. 3), to obtaining Positive strain be enlarged breeding and culture of rootage (see the c figures of Fig. 3), in root media after root induction 2-3 weeks, set room Warm lower refining seedling one week, transplanting to compost (vegetation charcoal：Vermiculite=1：1) wait for harvest seed of blooming (see the d figures and e figures of Fig. 3).

The aseptic seeding and sterile glass vials tobacco seedling micrografting of 6 tobacco seed of embodiment

(1) about 100, the seed of harvest is taken to be put into 2mL EP pipes；

(2) 12.5% hypochlorite disinfectant 10min is added, blots solution later；

(3) seed is washed 5 times with sterile distilled water；

(4) seed is uniformly sowed on MS culture mediums.

Choose stock transgene tobacco and scion tobacco W38 (Nicotiana tabacum after sowing 30d Var.Winsconsin 38) seed seedling (scheming see a of Fig. 4), culture environment is constant light source, light intensity 1500lx, illumination 14h/ 10h day-night cycles, 23~25 DEG C of room temperature, relative humidity 85%.

Aseptically, by transgene tobacco seedling truncation decaptitating, stay about 1cm long stem sections as stock；Seedling taking is high by 3~ The tobacco seedling of 5cm, top stay 2-3 piece leaves, base portion truncation, the alignment of stock scion notch to be fixed with silica gel sleeve pipe, be placed in culture medium Growth (scheming see the b of Fig. 4) in MS.

The GFP that PbHMGR1mRNA is transmitted between transgene tobacco stock and wild-type tobacco scion after 7 micrografting of embodiment Fluorescence Identification

14d after micrografting, the adventitious root to grow out respectively from the sampling of scion wild-type tobacco (scheming see the c of Fig. 4) and anvil The wooden transgene tobacco root segment uses laser confocal microscope Olympus FluoView FV1000confocal laser The object lens of scanning microscope (Olympus, Tokyo, Japan) use × 20 and excitation wavelength 488nm observe root 1- GFP fluorescent protein expressions situation at 3mm (see Fig. 5).

The result shows that：GFP fluorescins are in transgenic tobacco plant root strong expression, after grafting, what scion was grown Also there are GFP fluorescins in adventitious root, and the wild-type tobacco scion on wild-type tobacco stock is grafted in control group, There is no the expression of GFP fluorescins for the adventitious root grown.

The nest that PbHMGR1mRNA is transmitted between transgene tobacco stock and wild-type tobacco scion after 8 micrografting of embodiment Formula PCR identifications

It extracts total serum IgE as described in Example 1, nest-type PRC is carried out after reverse transcription (see Fig. 6).To graft in wild type cigarette The cDNA of wild-type tobacco scion on careless stock is negative control, is positive right with the cDNA of transgene tobacco stock stem section According to.

First round PCR reaction system：2 × Es Taq MasterMix (Dye) 10 μ L, 10 μM of SEQ ID NO.4 and 5 institutes Primer each 1 μ L, cDNA1 μ L, the 7 μ L of distilled water shown amounts to 20 μ L.

PCR response procedures are as follows：94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 26 cycles；72℃ Finally extend 10min.

Second wheel PCR reaction systems：2 × Es Taq MasterMix (Dye) 10 μ L, 10 μM of SEQ ID NO.6 and 7 institutes Each 1 μ L of primer shown, 0.5 μ L of first round PCR reaction product, 7.5 μ L of distilled water amount to 20 μ L.

PCR response procedures are as follows：94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 34 cycles；72℃ Finally extend 10min.

The result shows that：There are 1084bp specific bands in stock, and GFP-PbHMGR1 fusions are in transgenic rootstock cigarette Careless plant is largely transcribed, and the specific band of stock also occurs in scion after grafting, and the scion in control group does not go out Existing specific band.

Although above having used general explanation, specific implementation mode and experiment, the present invention is made to retouch in detail It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art 's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed Range.

SEQUENCE LISTING

<110>China Agricultural University

<120>A kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe

<130> KHP161119381.3

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 2556

<212> DNA

<213>II-PbHMGR1-Spe I of birch-leaf pear GFP-Bgl

<400> 1

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

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ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagaga 720

tctatggacg tccgaaggcg atctacgatg gatacacctg ccaccaaggc cagaaggggg 780

ccgatgaaag tgaaagtggt ggaccacgag aacgacgttg gtgtcgtcgg ggccaaggcc 840

tccgacgccc tgccgctgcc gttgtacctg actaacgccg tcttcttcac tctcttcttc 900

tccgtcgtct acttcctcct tactcgttgg cgcgagaaga tcaggacctc gacgccactc 960

cacgtcgtga acttctccga gatcgtcgcg atactcgcgt tcgtcgcctc cttcatctac 1020

ttgcttggat tcttcgggat cgatttcgtg cagtcgctca ttctccgccc cagcaatgac 1080

gtctgggccg ctgacgatga cgaggaggag cacgagcgct tgatattgaa agacgacgcc 1140

cggaaagtgc cgtgtggggc cggactcgac tgcagcccaa ttccccaaat tacccctgtt 1200

gctgctgccg cccccaaagc tgttgcacag aaggtgtttg ataaagaggt agtcctctcc 1260

actccctgcg atttcaccgc ccagccgttg acggaggaag atgaggaggt ggtcaagtcc 1320

gtggtggcgg gaaccatccc ttcctactct ctggagtcaa agctcggaga ttgcaggagg 1380

gcggcggcta tcaggcgcga ggcgcttcag aggattacag gaaagtctct gggtggtctg 1440

ccattggagg ggttcgatta cgagtcaatt ttgggtcagt gctgcgagat gccagttggg 1500

tatgttcaga ttccagttgg gattgctggg cctcttaggc tcgatggcag agagttttcc 1560

gtaccaatgg ccaccaccga aagttgcttg gttgccagca ccaaccgtgg cttcaaagct 1620

atcaacttgt ccggcggagc caccagtgtg ttgctgagag atgggatgac cagagcacct 1680

tgtgtgaggt tcaactctgc taagagagct gccgagttga agttctactt ggaagaaccc 1740

aacaattatg acaccttgtc cacggttttc aacaggtcaa gcagattcgg taggcttcag 1800

acaattaagt gtgccattgc tgggaagaac ttgtacatga gattcacctg cagcaccggt 1860

gatgctatgg ggatgaacat ggtctccaaa ggtgtgcaaa acgttttgga tttcctccag 1920

aacgacttcc ctgacatgga tgtgattgga atttccggca actactgctc tgacaagaag 1980

cccgctgcgg tgaactggat cgaaggtcgt ggaaaatcgg tggtctgtga ggctgtgatc 2040

aagggtgatg tggtgcagaa ggtgttgaaa accaatgtgg cgtccctgtg cgagcttaac 2100

atgctcaaga accttactgg gtctgcaatg gctggagccc tcggtggatt caacgcacat 2160

gccagcaaca tcgtctctgc catctacatc gctaccggcc aagacccagc tcagaatgtg 2220

gagagttctc actgcattac catgatggaa cccatcaatg atgggcagga ccttcacgtg 2280

tctgtcacca tgccttcaat tgaggttggt actgttggag gtgggaccca acttgcatct 2340

caatcagctt gtctgaacct tcttggagtg aagggtgcta acagggaggc accaggatct 2400

aatgcaagat tgttggccac tgttgtggct ggttctgttc ttgctggaga gctttctctc 2460

atgtctgcta tctcagctgg acagcttgtg aatagtcaca tgaaatacaa cagatcaagc 2520

aaagatgtct cagctgttgc gtccgcttaa actagt 2556

<210> 2

<211> 22

<212> DNA

<213>Artificial sequence

<400> 2

agatctatgg acgtccgaag gc 22

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence

<400> 3

actagtttaa gcggacgcaa cag 23

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence

<400> 4

agaagaacgg catcaaggtg 20

<210> 5

<211> 23

<212> DNA

<213>Artificial sequence

<400> 5

actagtttaa gcggacgcaa cag 23

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

ggtggtcaag tccgtcgtgg 20

<210> 7

<211> 21

<212> DNA

<213>Artificial sequence

<400> 7

tcctggtgcc tccctgttag c 21

Claims (10)

1. a kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe, which is characterized in that structure gene carries fluorescence The carrier of protein tag, prepare transgenosis plant；Using genetically modified plants as stock, aseptically with wild type scion by it Micrografting is carried out, situation occurs in the fluorescence for observing stock and scion, if stock and scion adventitious root observe fluorescence, illustrates The mRNA molecules of the gene transmit between plant anvil fringe.

2. a kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe, which is characterized in that structure gene carries fluorescence The carrier of protein tag, prepare transgenosis plant；Using genetically modified plants as stock, aseptically with wild type scion by it Micrografting is carried out, is detected using Chao Shi PCR, if specific band occur in anvil fringe Chao Shi PCR products, illustrates the gene MRNA molecules transmit between plant anvil fringe.

3. Fluorescence Identification method as claimed in claim 1 or 2, which is characterized in that the fluorescin be green fluorescent protein, Red fluorescent protein, yellow fluorescence protein, blue fluorescent protein, orange fluorescent protein or bluish-green fluorescin.

4. Fluorescence Identification method as claimed in claim 1 or 2, which is characterized in that the gene is birch-leaf pear HMGR1.

5. Fluorescence Identification method as claimed in claim 4, which is characterized in that the carrier is to be overexpressed GFP-PbHMGR1 to carry Body is prepared by the following method to obtain：Birch-leaf pear geneome RNA is extracted, reverse transcription cDNA carries out PCR amplification, recycles purpose Segment simultaneously purifies, and connects carrier T, transformed competence colibacillus cell, and correct positive colony is sequenced to obtain the final product in selection, the PCR amplification institute The sequence of primer pair is as shown in SEQ ID NO.2-3.

6. Fluorescence Identification method as claimed in claim 5, which is characterized in that the Chao Shi PCR, the reaction of first round PCR use Primer pair sequence as shown in SEQ ID NO.4-5, second wheel PCR reaction use primer pair sequence such as SEQ ID NO.6-7 It is shown.

7. a kind of Fluorescence Identification method that HMGR1mRNA molecules transmit between plant anvil fringe, includes the following steps：

(1) birch-leaf pear total serum IgE is extracted respectively, and reverse transcription is at cDNA, the overall length of PCR amplification HMGR1 genes；

(2) structure birch-leaf pear HMGR1 genes carry the carrier of fluorescent protein tag, prepare transgenosis plant；

(3) genetically modified plants are stock, itself and wild type scion are aseptically carried out micrografting, observe stock and scion Fluorescence there is situation, if stock and scion adventitious root observe fluorescence, illustrate that the mRNA molecules of birch-leaf pear HMGR1 genes exist It is transmitted between plant anvil fringe.

8. a kind of Fluorescence Identification method that HMGR1mRNA molecules transmit between plant anvil fringe, includes the following steps：

(1) birch-leaf pear total serum IgE is extracted respectively, and reverse transcription is at cDNA, the overall length of PCR amplification HMGR1 genes；

(2) structure birch-leaf pear HMGR1 genes carry the carrier of fluorescent protein tag, prepare transgenosis plant；

(3) genetically modified plants are stock, it is aseptically carried out micrografting with wild type scion, is examined using Chao Shi PCR It surveys, if the specific band that length is 1084bp occur in anvil fringe Chao Shi PCR products, illustrates that HMGR1 gene mRNA molecules exist It is transmitted between plant anvil fringe.

9. application of any Fluorescence Identification methods of claim 1-8 in plant germplasm resource improvement.

10. any Fluorescence Identification methods of claim 1-8 can transmit mRNA points at a distance in identification between plant anvil fringe Application in the gene of son.