CN101942453A - Molecular identification method based on transfer of GAI mRNA molecules between pear rootstock and scion - Google Patents
Molecular identification method based on transfer of GAI mRNA molecules between pear rootstock and scion Download PDFInfo
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Abstract
The invention discloses amino acid sequences of GAI proteins of Pyrus betulaefolia and Pyrus bretschneideri, and nucleotide sequences for coding the GAI proteins, wherein the sequences are disclosed as SEQ ID NO1-4 in the sequence table. The invention also discloses a molecular identification method based on transfer of GAI mRNA between pear rootstock and scion, which comprises the following steps: (1) micrografting seedlings in test tubes by using Pyrus bretschneideri as scion and using Pyrus betulaefolia as rootstock; (2) looking for the respective specific enzyme-digestion sites of the GAI genes of the Pyrus bretschneideri and the Pyrus betulaefolia, and respectively designing a primer at the upstream and downstream of the enzyme-digestion site; (3) and carrying out enzyme-digestion on the Pyrus betulaefolia, the Pyrus bretschneideri, and the RT-PCR product of the Pyrus betulaefolia and the Pyrus bretschneideri after grafting, and comparing the enzyme-digestion spectra before and after the grafting to identify the transfer conditions. The method is quick, sensitive, accurate, simple and convenient, and is applicable to other pear plants.
Description
Technical field
The present invention relates to the molecular biology of plants field, what be specifically related to is the GAI gene mRNA molecule molecular assay method that long distance is transmitted between pear plant stock and scion.
Background technology
In the garden crop production process, the widespread use graft technology obtains proterties such as resistance, dwarfing, and to increase output and to improve product quality, wherein the application to grafting is more general in the cultivation of fruit tree.Find in the production process, same scion grafting The Characters difference to the different stocks, as variations such as fruit color and luster, soluble solid content, flowering period, the change of many proterties can have influence on formation, growth and the growth of fruit, and then influences the economic benefit in later stage.
A series of studies show that, some RNA molecules shift in phloem in iuntercellular and some the phloem albumen form with protein-RNA complex body in the plant materials, and can be passed to the apical meristem of scion by stock by the grafting mouth, influence growing of scion.Therefore, by evaluation and research to the transportation of the bast minister system of distance of RNA in the fruit-tree grafting process, can be for we get in the fruit tree pass through mechanism of RNA molecule between stock and scion clear from now on, and utilize and to transmit the autotelic improvement stock of RNA transgenosis, the raising that makes scion acquisition good character be beneficial to fruit production and economic benefit lays the foundation, and also provides positive directive function for production of fruit trees.
Plant hormones regulators,gibberellins (GA) is the plant hormone that plays an important role in plant growth and development process.Find in the research to GA signals-modulating aspect, the GA signal plays the negative regulation effect in the GAI Arabidopis thaliana in the GRAS family, and its aminoacid sequence N end comprises that a conservative zone is called as DELLA (Alvey etc., 2005), DELLA district disappearance plant can show half and downgrade (Olszewski etc., 2003).The proteic gene of encoding D ELLA has 5 in the Arabidopis thaliana at present, is respectively GAI, RGL, RGL1, RGL2 and RGL3 (Dill et al., 2001; Lee et al., 2002).The proteic gene of encoding D ELLA that apple ' loud, high-pitched sound is drawn ' kind is logined in NCBI has three groups of totally 6 MdRGL1a/b, MdRGL2a/b and MdRGL2a/b (Foster et al., 2007), 2 MhGAI1 and MhGAI2 are arranged in the Hubei Chinese flowering crabapple, 2 MxGAI1 and MxGAI2 are arranged in the malus xiaojinensis, apple ' Fuji ' has 2 MhGAI1 and MhGAI2 (Xu Haiyan etc., 2009), other plant such as paddy rice (Su et al., 2005), also be cloned into the proteic GRAS family gene of encoding D ELLA in the barley (Ikeda et al., 2001).
Up to now, discovering to grow apart from gene mRNA and the encoded protein thereof transmitted at plant phloem has a lot, as KN1, BEL5, NACP, FT albumen, SUT1 etc., the checking that whether can transmit about the RNA and the albumen of these genes also only is to use transgenosis to detect carrier and carries specificity label (Haywood et al., 2005), and nido RT-PCR (Harada et al., 2009), technology such as Northern blot, though the transitivity of these technology for detection genes is more accurate, but its process is loaded down with trivial details, require high, length consuming time, obviously exist workload big, weak point such as the work period is long, and efficient is low, particularly because fruit tree transgenosis and regeneration are difficult, we are in the process transmitted between the anvil fringe of research RNA, and the transmission of transgenic method checking gene RNA is very difficult, and so just the research of carrying out the endogenous mRNA grafting transmission of fruit tree for us has more increased difficulty.
Summary of the invention
The purpose of this invention is to provide the nucleotide sequence of birch-leaf pear and ' pear ' GAI and by the proteic aminoacid sequence of this nucleotide coding, shown in SEQ ID NO1～4.
Another object of the present invention provides the molecular assay method that a kind of GAI gene mRNA transports between pear anvil fringe, comprise the steps:
(1) according to plant GAI gene conserved regions design primers such as Arabidopis thaliana, pumpkin, apples, the proteic GAI full length gene of encoding D ELLA among separation and clone's birch-leaf pear (Pyrus betulaefolia), ' pear ' (Pyrus bretschneideri), utilize bioinformatics method to carry out sequential analysis, and the gained gene carried out comparison and analyze, confirm that the gained gene belongs to GRAS family;
(2) ' pear ' is scion, and birch-leaf pear is that stock carries out the test-tube plantlet micrografting;
(3) analyze the GAI gene order of ' pear ' and ' birch-leaf pear ', seek special separately restriction enzyme site, design primer respectively in the upstream and downstream of this restriction enzyme site;
(4) extract stock after ' pear ', birch-leaf pear and the grafting and total RNA of scion respectively, reverse transcription becomes cDNA, the gene fragment of pcr amplification GAI;
(5) with restriction enzyme the PCR product being carried out enzyme cuts, relatively the enzyme before and after the grafting is cut spectrogram, if in the grafting system, there is the transmission of RNA, the specific band of scion ' pear ' then after the amplified production enzyme of stock birch-leaf pear is cut, can occur, after the amplified production enzyme of scion ' pear ' is cut, also can occur the specific band of stock birch-leaf pear equally; If do not transmit, will demonstrate the band of respective sample separately so among the restriction enzyme digestion and electrophoresis figure of amplified production and do not have other assorted bands.
The described birch-leaf pear that is used to increase with the homogenic primer of ' pear ' GAI is: TTGATTTCCGAGCCCTACCC and AACTCGGTCATCGCTCACTGA.
Described restriction enzyme is the Hap II.
Wherein, be used to extract scion ' pear ' always blade, stem apex, stem section phloem or the grafting mouth of sample selection grafting 3～4d of RNA ' pear '.
Another object of the present invention provides the application in the GAI mRNA molecule transmission evaluation between pear other plant anvil fringe of described authentication method.
Method provided by the invention has solved the high gene of homology can't carry out the problem that RT-PCR identifies by the design Auele Specific Primer between stock and scion, it is difficult also to have solved the fruit tree transgenic technology simultaneously, length consuming time and the problem of being inconvenient to identify.Method provided by the invention is quick, sensitive, and the accuracy height is easy, can be applied to other pear plants.
Description of drawings
Figure 1 shows that ' pear ' and birch-leaf pear GAI gene PCR amplification electrophorogram.M:DNA molecular weight standard among the figure; 1: ' pear '; 2: birch-leaf pear.
Figure 2 shows that the GAI amino acid sequence homology relatively.YL-GAI among the figure: ' pear '; DL-GAI: birch-leaf pear; MfGAI: apple ' Fuji '; MxGAI: malus xiaojinensis; MdRGL1a: apple ' loud, high-pitched sound '.
Figure 3 shows that the analysis of GAI aminoacid sequence systematic evolution tree
Figure 4 shows that ' pear ' and birch-leaf pear GAI RT-PCR product restriction enzyme digestion electrophoresis detection result.M:DNA molecular weight standard among the figure; 1: ' pear ' RT-PCR product enzyme is cut the result; 2: birch-leaf pear RT-PCR product enzyme is cut the result; 3: ' pear ' and birch-leaf pear equal amount of mixture RT-PCR product enzyme are cut the result; 4: ' pear ' RT-PCR product; 5: birch-leaf pear RT-PCR product.
Figure 5 shows that stock and scion RT-PCR result after the grafting.Among the figure 1: ' pear ' stem apex; 2: ' pear ' blade; 3: ' pear ' stem section phloem; 4: the grafting mouth; 5: birch-leaf pear stem section phloem; 6: birch-leaf pear stem section xylem; CK: control group; Y: ' pear ' of grafting not; D: the birch-leaf pear of grafting not; M: not ' pear ' of grafting and birch-leaf pear equal amount of mixture.
Figure 6 shows that micrografting scion and stock GAI RT-PCR product restriction enzyme digestion electrophoresis detection result.The M:DNA molecular weight standard; 1: ' pear ' stem apex; 2: pear ' blade; 3: pear ' stem section phloem; 4: the grafting mouth; 5: birchleaf pear rootstock stem section phloem; 6: birch-leaf pear stem section xylem; Y: ' pear ' of grafting not; D: the birch-leaf pear of grafting not; M ': not ' pear ' of grafting and birch-leaf pear equal amount of mixture.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Choose stock birch-leaf pear (Pyrus betulaefolia Bunge) and scion ' pear ' (Pyrus bretschneideri ' Yali ') tissue cultured seedling spire, constant light source, light intensity 1500lx, illumination 14h, 23～25 ℃ of room temperatures, relative humidity 85%, 30～40d subculture once.
The vegetable material substratum
' pear ' MS+1.0mgL
-16-BA+0.1mgL
-1IBA
Birch-leaf pear MS+0.5mgL
-16-BA+0.1mgL
-1IBA
Liquid nitrogen is handled-80 ℃ of refrigerators preservations in back and is used for gene clone.Under aseptic condition, the birch-leaf pear tissue cultured seedling behind the numerous 30d of expansion is decaptitated, stay about 1.5cm long shoot section, along the top rip cutting; Get ' pear ' tissue cultured seedling of height of seedling 1cm, 2-3 sheet leaf is stayed on the top, and base portion is whittled into " V " type, inserts the stock otch, fixes with masking foil, and Graft is at substratum MS+0.5mgL
-16-BA+0.1mgL
-1Grow among the IBA.
(1) 1g blade liquid nitrogen grinding changes in the 2.0mL centrifuge tube, add 800 μ l2 * CTAB (2%CTAB, 10mM Tris-HCl, 1.4M NaCl, 1%PVP), the mixing that vibrates slightly is put in 20-40min in 65 ℃ of water-baths, during jog several times.
(2) reduce to room temperature after, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1), centrifugal 10 minutes of room temperature 12000rpm.
(3) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1), centrifugal 10 minutes of 12000rpm.
(4) add 2 times of volume dehydrated alcohols in the supernatant ,-20 ℃ of precipitation 30min.
(5) 12000rpm is centrifugal 10 minutes, abandons supernatant, and precipitation washes twice with 75% ethanol, dries precipitation, is dissolved in an amount of distilled water.
(6) if need, add 1 μ l RNA enzyme ratio in 100 μ l and go RNA to handle, 37 ℃ 30 minutes.With CI extracting one time
Use ethanol sedimentation again, precipitation is dissolved in an amount of distilled water.
(7) 1% agarose gel electrophoresis detect integrity, and ultraviolet spectrophotometer is surveyed 260nm place absorbancy and calculated DNA concentration.
Gene conserved regions design primer according to the Arabidopis thaliana of announcing among the GenBank, pumpkin, apple plant GAI such as ' Fuji '.
Amplification GAI gene uses primer as follows:
Upstream primer: 5 ' TTGATTTCCGAGCCCTACCC-3 ',
Downstream primer: 5 '-AACTCGGTCATCGCTCACTGA-3 '.
Above-mentioned primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.
PCR reaction system: Prime STAR
TMThe HS archaeal dna polymerase (Takara company, DR010S), Takara LA PCR
TMIn vitro Cloning Kit (Takara company, DRR015)
5 * Prime STAR
TMDamping fluid 10 μ l
dNTP?mixture(2.5mM?each) 4μl
Primer?F/R(10μmol/L) 1μl
Templete 1μl
Prime STAR
TMHS DNA polymerization (2.5U/ μ l) 0.5 μ l
Distilled water 32.5 μ l
Amount to 50 μ l
The PCR response procedures is as follows:
94 ℃ of pre-sex change 3min;
94℃40s；
61℃30s；
72℃1min；
30 circulations.
72 ℃ of last 2min that extend.
The PCR product detects: make 1% sepharose according to the target fragment size, the 0.1%TAE electrophoretic buffer, the about 15min of 70-110v voltage electrophoresis, bromination second pyridine dyeing, ultraviolet lamp detects PCR product clip size down, can get two bands (Fig. 1) that size is identical.
Ke Ruitai Bioisystech Co., Ltd reclaims test kit in the use, and method is with reference to its company's specification sheets:
1. cut and contain the pulsating agarose of target DNA, excise unnecessary gel as far as possible, add the sol solutions PN of 350 μ l.
2. 10min is placed in 50 ℃ of water-baths, and constantly gentleness spins upside down centrifuge tube, guarantees that blob of viscose dissolves fully.If still undissolved blob of viscose is arranged, can replenish some sol solutionses or place several minutes, until dissolving fully.
3. solvent glue is cooled to room temperature, is added to and reclaims in the post, the centrifugal 60s of 13000rpm outwells the waste liquid in the collection tube, and repetitive operation once.
4. the operation in repeating 3. once.
5. add 700 μ l rinsing liquid PW (having added dehydrated alcohol), the centrifugal 60s of 13000rpm outwells the waste liquid in the collection tube.
6. add 500 μ l rinsing liquid PW (having added dehydrated alcohol), the centrifugal 60s of 13000rpm outwells the waste liquid in the collection tube.
7. adsorption column is put back in the collection tube, the centrifugal 2min of 13000rpm removes rinsing liquid as far as possible.
8. take out adsorption column, put into a clean centrifuge tube, on super clean bench, dry up,, place 2min under the room temperature, the centrifugal 1min of 13000rpm at the elutriant that the adding 30 μ l of adsorption column film central authorities have been heated to 68 ℃.
9. get 5 μ l and reclaim product electrophoresis detection recovering effect, it is standby to put into-20 ℃ of preservations.
1. from-70 ℃ of refrigerators, take out bacterial classification, tap the back gently with the toothpick of having sterilized before not thawing fully and on the 90mm substratum of getting ready, rules, about 37 ℃ of incubated overnight 16h, seal when being cultured to visible single bacterium colony in 4 ℃ of refrigerators, preserve standby;
2. prepare LB liquid nutrient medium 300ml, be sub-packed in the triangular flask (50ml/ bottle) of 6 200ml, autoclave sterilization 20min is cooled to about 55 ℃, gets LB liquid nutrient medium 50 μ l in each triangular flask and all is loaded in the same glass test tube of having sterilized.Substratum seals in 4 ℃ of refrigerators standby in the triangular flask;
3. use sterilized rifle head picking list bacterium colony on the cultured bacterium plate of 90mm, the rifle head is pushed in the glass test tube that the LB liquid nutrient medium is housed, seal, in 37 ℃ of isothermal vibration beds more than the 228rpm incubated overnight 16h;
4. get 50 μ l respectively from cultured liquid bacterium liquid in standby 6 triangular flasks that liquid nutrient medium is housed, with ratio inoculation in 1: 100, it was 0.6 that 225rpm is cultured to OD600, is approximately about 3h;
5. bacterium liquid branch is installed in 12 aseptic polypropylene centrifuge tubes of 50ml, place 10min on ice, 4000rpm then, 4 ℃ of centrifugal 10min receive bacterium;
6. abandon supernatant, be inverted centrifuge tube 1min with thorough removal supernatant.Every pipe adds the 0.1M CaCl of 25ml precooling
2, mixing is put 30min on ice gently;
7. 4000rpm, 4 ℃ of centrifugal 10min;
8. abandon supernatant, be inverted centrifuge tube 1min with thorough removal supernatant.Every pipe adds the 0.1M CaCl of 25ml precooling
2, mixing gently;
9. this moment, competence can directly be used, if need long-time the preservation, can add concentration and be 80% glycerine, and to make the glycerine final concentration be 20%, in-70 ℃ of preservations.
The PCR product of getting purifying adds pMD18-T carrier and damping fluid (Takara company, D101A, Japan) (with reference to pMD18-T carrier connection description book)
Linked system:
Connect liquid I 5 μ l
PCR product 4.5 μ l
pMD18-T 0.5μl
Cumulative volume 10 μ l
Mixing, centrifugal slightly, more than 16 ℃ of water-bath 12h.The mol ratio of carrier and gene fragment 1: 4.
1. get 50 μ l competent cells, place on ice, fully the dissolving after gently with cell suspension.
2. add 5 μ l and connect liquid, mixing is placed 30min on ice gently.
3. 42 ℃ of heat shock 90s place 2min on ice.
4. add 500 μ l LB substratum, in 37 ℃ of 200rpm shaking culture 1h.
5. the centrifugal 1min of 4000rpm under the room temperature sops up 400 μ l supernatant liquors with the rifle head, with remaining substratum with cell suspension.
6. bacterium is coated on and is added with on the antibiotic solid LB of the ammonia benzyl substratum.
7. flat board is inverted overnight incubation in 37 ℃.
Picking list bacterium colony hickie and locus coeruleus insert mixing in the 50 μ l LB+Amp liquid nutrient mediums, carry out PCR as template and identify.The positive (product that PCR reclaims behind the purifying is a template) and negative (locus coeruleus bacterium liquid is template) and single primer are set simultaneously to compare; Agarose gel electrophoresis detects and inserts clip size.Select positive colony bacterium liquid, check order by Beijing promise slug gene.Sequencing result shows that the fragment of two clauses and subclauses is 1987bp, 634 amino acid of encoding, intronless.
Embodiment 7 birch-leaf pears and ' pear ' GAI gene biological information science are analyzed
Carry out the prediction of homology comparison, molecular weight in the software that institute's calling sequence is provided on websites such as http://www.ncbi.nlm.nih.gov; Carry out multisequencing compare of analysis, evolutionary tree drafting analysis with softwares such as DNAMAN (Lynnon BioSoft) and BioEdit.
Blast comparison result shows on NCBI: ' pear ' and birch-leaf pear GAI aminopeptidase gene acid sequence and other plant have a higher similarity, wherein with the similarity the highest (97%) of ' loud, high-pitched sound is drawn ' MdRGL (1a) and apple ' Fuji ', next is followed successively by malus xiaojinensis, Hubei Chinese flowering crabapple and ' loud, high-pitched sound is drawn ' MdRGL (1b).The aminoacid sequence of ' pear ' and birch-leaf pear GAI genes encoding includes 5 structural domains (Fig. 2), consistent with structural domain LHR I, VHIID, LHR II, PFYRE and the SAW of GAI gene in the known other plant, prove that the sequence that the clone obtains is an encoding D ELLA protein gene.Therefore, with its difference called after YL-GAI and DL-GAI.
Make up the systematic evolution tree of 14 kind of plant GAI aminopeptidase gene acid sequences such as YL-GAI, DL-GAI by DNAMAN.This evolutionary tree shows, the aminoacid sequence that is obtained is nearest with ' loud, high-pitched sound is drawn ' MdRGL (1a), apple ' Fuji ' MfGAI and malus xiaojinensis MxGAI sibship, nearer with Hubei Chinese flowering crabapple MhGAI1, ' loud, high-pitched sound is drawn ' MdRGL (1b) relation, far away with pumpkin CmGAIP, CmGAIPB and grape VvGAI1 sibship.And with Arabidopis thaliana AtGAI, AtRGA1 and brassica plant BoGAI, BrGAI sibship (Fig. 3) farthest.The systematic evolution tree that this research obtains is consistent with the botany classification result to a certain extent.Show that the evolution that amino acid whose sequence is evolved with plant is consistent, equal plant is in same branch on evolutionary tree, and sibship far away be in different branches, and amino acid whose homology percentage also far diminishes with the change of species sibship.
The extraction of embodiment 8 total RNA
CTAB method (Zhang Yugang etc., 2005) is adopted in the extraction of the total RNA of vegetable material, extracts total RNA from scion, stock blade and stem section, and with 30 μ l DEPC water dissolution, electrophoresis detection postposition-80 ℃ refrigerator is preserved standby.
The removal of DNA among the RNA:
1. in 500 μ L thin-walled PCR pipes, add:
RNA 2μg
The DNA enzyme buffer liquid 5 μ d of no RNA enzyme
RNA enzyme inhibitors (40U/ μ l) 2 μ l
The DNA enzyme 2 μ l of no RNA enzyme
DEPC water complements to 50 μ l
2. handle 30min for 37 ℃; Add 100 μ L and do not have the water (0.1%DEPC processing) of RNA enzyme; Add equal-volume chloroform/primary isoamyl alcohol (24: 1) mixing, ice bath 10min;
3. 4 ℃ of centrifugal 10min of 10000rpm; Get supernatant, add equal-volume chloroform/primary isoamyl alcohol (24: 1) mixing, the 10min ice bath; 4 ℃ of centrifugal 10min of 10000rpm;
4. get supernatant, add 10 μ l 10M NH
4AC adds the dehydrated alcohol of 2.5 times of volumes simultaneously, and-20 ℃, 30min; 4 ℃, the centrifugal 20min of 12000rpm; Abandon supernatant, precipitation adds an amount of 75% ethanol upsprings, and the centrifugal 10min of 7500rpm washs 2 times; Vacuum exhaustion ethanol;
5. be deposited in low temperature katabatic wind machine and dry up, being dissolved in 50 μ L does not have in the water of RNA enzyme, and-70 ℃ of preservations are standby.
Embodiment 9 cDNA are synthetic
Reverse transcription system (25 μ l) and process: in the 200ul of no RNA enzyme thin-walled tube, add following reagent: total RNA 2 μ l, Oligo dT 1 μ l, DEPC water 12 μ l, the mixture 5000rpm mixing of 15ul is placed 70 ℃ of 5min altogether, take out and be positioned on ice at once, add again with the lower section: M-MLV 1 μ l, 5 * M-MLV damping fluid, 2.5 μ l, RNA enzyme inhibitors 0.6 μ l, dNTP (10mM) 1.25 μ l, DEPC water 4.65 μ l.
Add the above mixture of 25ul system altogether, 5000rpm mixing, 42 ℃ of 1h, 70 ℃ of 10min, stopped reaction.
Resulting cDNA is as the template of following pcr amplification.
The GAI pcr amplification of embodiment 10 birch-leaf pears and pears cDNA
YL-GAI and DL-GAI gene nucleotide series similarity are up to 99%, and Auele Specific Primer is difficult to design, utilize the method for RT-PCR to identify infeasible.So the contriver is according to the nucleotide sequence that has obtained, utilize DNAMAN analysis stock and scion nucleotide sequence glucose-6-phosphate dehydrogenase to cut the site, relatively the difference between the restriction enzyme site is selected the stock restriction enzyme site special to scion, designs primer with Primer5.0 and DNAMAN at its two ends.Promptly by to the analysis of two gene restriction enzyme sites, we filter out a special restriction enzyme HapII, and (Takara company D1053A), is about to the fragment that ' pear ' and birch-leaf pear are cut into different sizes.The transmission of adopting enzyme to cut the method validation GAI gene mRNA of evaluation.
Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.
The PCR reaction system:
5 * Prime STAR
TMDamping fluid 10 μ l
dNTPs(2.5mM) 4μl
Upstream primer and downstream primer (10 μ mol/L) 1 μ l
Prime STAR
TMHS archaeal dna polymerase (2.5U/ μ l) 0.5 μ l
Distilled water 32.5 μ l
Amount to 50 μ l
The PCR reaction conditions:
94℃3min；
30 circulations: 94 ℃ of 30s;
60℃30s；
72℃1min；
72℃6min。
Embodiment 11 PCR product Hap II enzymes are cut
With the PCR product reclaim, purifying, method is carried out enzyme subsequently and is cut with embodiment 4, it is as follows that enzyme is cut system:
The water 3 μ l of nuclease free
10 * damping fluid, 1 μ l
Hap?Ⅱ 1μl
Make 2% sepharose according to the target fragment size, the 0.1%TAE electrophoretic buffer, the about 15min of 70-110v voltage electrophoresis, bromination second pyridine dyeing, ultraviolet lamp detects PCR product clip size down.
The PCR product of finding birch-leaf pear can digested one-tenth 24bp, three bands of 44bp, 292bp, and the PCR product of ' pear ' can digested one-tenth 24bp, 44bp, 45bp, four bands of 248bp (Fig. 4), and the result is consistent with the restriction enzyme mapping of expection.This shows that designed primer and restriction enzyme Hap II can be used to identify the transmission of GAI gene mRNA in the pears.
The evaluation that GAI mRNA transmits between birch-leaf pear and pears after embodiment 12 graftings
Behind the micrografting 1,2,3,4,5,10d, respectively from scion ' pear ' stem apex, blade, stem section phloem, take a sample in grafting mouth and stock birch-leaf pear stem section phloem, the xylem organization, extract total RNA, press embodiment 8,9 after the reverse transcription, 10,11 method is carried out RT-PCR (Fig. 5).With the negative contrast of cDNA of the stem section phloem of ' pear ' and birch-leaf pear self Graft separately, with the positive contrast of cDNA balanced mix of the stem section phloem of ' pear ' and birch-leaf pear self Graft.The RT-PCR product is carried out enzyme with restriction enzyme Hap II cut evaluation (Fig. 6).
The result shows: first three sky after grafting, the specific enzymes slitting band that all shows as is separately respectively organized in stock and scion, but since the 4th day, can see except birch-leaf pear stem section xylem, all the other are respectively organized all has new enzyme slitting to take out of now, be each position of scion ' pear ' except containing autospecific enzyme slitting band, also increased the specific enzymes slitting band of stock birch-leaf pear simultaneously; And the stem section phloem of stock birch-leaf pear has also increased the specific enzymes slitting band of scion ' pear ' simultaneously except containing autospecific enzyme slitting band.But during by the 10th day, do not detect transport phenomenon.' pear ' in the control group and birch-leaf pear self Graft also find no new specific enzymes slitting band.
The sequence explanation:
SEQ ID NO.1 and 2 is the nucleotide sequence of birch-leaf pear GAI and the aminoacid sequence of GAI; SEQ ID NO.3 and 4 is the nucleotide sequence of ' pear ' GAI and the aminoacid sequence of GAI; SEQ ID NO.5 and 6 is that the primer of amplification GAI total length is right; SEQ ID NO.7 and 8 is that the segmental primer of amplification GAI is right.
Claims (8)
1. birch-leaf pear GAI gene, its nucleotide sequence is shown in SEQ ID NO 1.
2. by the albumen of the described genes encoding of claim 1, its aminoacid sequence is shown in SEQ ID NO 2.
3. ' pear ' GAI gene, its nucleotide sequence is shown in SEQ ID NO 3.
4. by the albumen of the described genes encoding of claim 3, its aminoacid sequence is shown in SEQ ID NO 4.
5. the molecular assay method that GAI mRNA molecule transmits between pear anvil fringe comprises the steps:
(1) ' pear ' is scion, and birch-leaf pear is that stock carries out the test-tube plantlet micrografting;
(2) right to analysis requires 1 and the nucleotide sequence of the described gene of claim 3, selects special separately restriction enzyme site, designs primer respectively in the upstream and downstream of this restriction enzyme site;
(3) extract total RNA of stock and scion after birch-leaf pear, ' pear ' and the grafting respectively, reverse transcription becomes cDNA, the gene fragment of pcr amplification GAI;
(4) with selected restriction enzyme the PCR product is carried out enzyme and cut, relatively the enzyme before and after the grafting is cut spectrogram.
6. molecular assay method according to claim 5 is characterized in that, described restriction enzyme is the Hap II.
7. molecular assay method according to claim 5 is characterized in that, the described sample that is used to extract the total RNA of scion is selected from 3～4 days blade of grafting, stem apex, stem section phloem or grafting mouth.
8. as according to the application of each described molecular assay method of claim 5～7 in the evaluation that GAI mRNA molecule between other pear plant anvil fringes transmits.
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| CN104604539A (en) * | 2015-01-30 | 2015-05-13 | 山东省林业科学研究院 | Grafting method for arabidopsis |
| CN107460200A (en) * | 2016-06-02 | 2017-12-12 | 中国农业大学 | The method for identifying molecules that a kind of HMGR gene mRNAs transmit between pear anvil fringe |
| CN108330182A (en) * | 2017-01-18 | 2018-07-27 | 中国农业大学 | A kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe |
| CN108611354A (en) * | 2018-05-11 | 2018-10-02 | 中国农业大学 | A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe |
| CN111500592A (en) * | 2020-03-20 | 2020-08-07 | 中国农业大学 | Identification method for GAPC1 gene mRNA transportation between rootstocks and ears of Cucurbitaceae |
| CN111593056A (en) * | 2020-04-23 | 2020-08-28 | 中国农业大学 | Molecular identification method for transporting NAC2 gene mRNA between cucurbitaceae rootstock ears |
| CN112029774A (en) * | 2020-08-17 | 2020-12-04 | 中国农业大学 | Chaperonin for enhancing plant phloem RNP signal communication and application |
| CN116286856A (en) * | 2023-01-10 | 2023-06-23 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Gene for coding DELLA6 protein and application thereof in judging mango dwarf variety |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104604539A (en) * | 2015-01-30 | 2015-05-13 | 山东省林业科学研究院 | Grafting method for arabidopsis |
| CN107460200A (en) * | 2016-06-02 | 2017-12-12 | 中国农业大学 | The method for identifying molecules that a kind of HMGR gene mRNAs transmit between pear anvil fringe |
| CN107460200B (en) * | 2016-06-02 | 2020-07-28 | 中国农业大学 | Molecular identification method for transmitting HMGR gene mRNA between pear rootstock and scion |
| CN108330182A (en) * | 2017-01-18 | 2018-07-27 | 中国农业大学 | A kind of Fluorescence Identification method that mRNA molecules transmit between plant anvil fringe |
| CN108330182B (en) * | 2017-01-18 | 2020-04-10 | 中国农业大学 | Fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion |
| CN108611354A (en) * | 2018-05-11 | 2018-10-02 | 中国农业大学 | A kind of method for identifying molecules that FT mRNA molecules transmit between congener anvil fringe |
| CN108611354B (en) * | 2018-05-11 | 2020-06-16 | 中国农业大学 | Molecular identification method for transmitting FT mRNA molecules between rootstock and scion of congeneric plant |
| CN111500592A (en) * | 2020-03-20 | 2020-08-07 | 中国农业大学 | Identification method for GAPC1 gene mRNA transportation between rootstocks and ears of Cucurbitaceae |
| CN111593056A (en) * | 2020-04-23 | 2020-08-28 | 中国农业大学 | Molecular identification method for transporting NAC2 gene mRNA between cucurbitaceae rootstock ears |
| CN112029774A (en) * | 2020-08-17 | 2020-12-04 | 中国农业大学 | Chaperonin for enhancing plant phloem RNP signal communication and application |
| CN112029774B (en) * | 2020-08-17 | 2021-05-25 | 中国农业大学 | Chaperonin for enhancing plant phloem RNP signal communication and application |
| CN116286856A (en) * | 2023-01-10 | 2023-06-23 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Gene for coding DELLA6 protein and application thereof in judging mango dwarf variety |
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