CN102154484A - Tobacco glandular hair cDNA (complementary Deoxyribonucleic Acid) microarray for expression profile and preparation and application thereof - Google Patents
Tobacco glandular hair cDNA (complementary Deoxyribonucleic Acid) microarray for expression profile and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a tobacco glandular hair cDNA (complementary Deoxyribonucleic Acid) microarray for an expression profile and preparation and application thereof. A preparation method of the microarray comprises the following steps of: separating tobacco leaf glandular hairs, extracting total RNA (Ribonucleic Acid) and constructing a tobacco glandular hair cDNA library; randomly sequencing the cDNA library to obtain a tobacco glandular hair EST (Expressed Sequence Tag) database and amplifying an EST sequence by undergoing a PCR (Polymerase Chain Reaction) to obtain the tobacco glandular hair cDNA microarray for the expression profile; and analyzing tobacco glandular hairs and glandular hair removed leaf gene expression by using the microarray to obtain up-regulated expression ESTs in glandular hairs and screen a specific expression gene. The microarray obtained by the invention has the advantages of high efficiency, sensitivity, high flux, high accuracy and high specificity, and is suitable for researching tobacco glandular hairs, cultivating measures, environmental stress and the like. By adopting the microarray, the expression modes of a large number of genes can be detected at one time, and the gene expression change rules of a plurality of samples are comprehensively analyzed under the same testing condition.
Description
Technical field
The present invention relates to the gene chip field, be specifically related to one grow tobacco glandular hairs cDNA chip of expression spectrum and the preparation with the application.
Background technology
The plant glandular hairs are as the specialization structure of plant epidermis cell, because of its vital role that is risen in processes such as plant pest defence, opposing environment stress and secondary metabolite synthesize receives much attention.Along with the functional genomics development of technology, growth, metabolism and the excretory molecular mechanism of utilizing chip technology to study the plant glandular hairs becomes a big focus of plant biology research field gradually.Arabidopis thaliana is because of its complete genomic information and perfect chip product, and there is many favourable condition in the molecular biology research of its glandular hairs.But because Arabidopsis is in typical nonsecreting type glandular hairs, thereby these favourable conditions can't be in glandular hairs substance metabolism and secretion field widespread use.
The tobacco glandular hairs belong to typical secretor type glandular hairs, and its main secretory product is glycolipid and diterpene-kind compound, and are closely related with the quality and the resistance of tobacco leaf.The research about the tobacco glandular hairs at present focuses mostly at aspects such as glandular hairs type, density, weave construction, chemical ingredientss, and for the tobacco glandular hairs grow and the molecular mechanism of substance metabolism also lacks further investigation.Though the report that has some to utilize Protocols in Molecular Biology that the specific gene of tobacco glandular hairs is cloned and expressed, present gene expression pattern to the tobacco glandular hairs carries out the panorama type analysis and still lacks valid approach.This and tobacco gene group are huge, and order-checking is not finished as yet, tobacco gene poor information among the Genebank, and commercialization tobacco gene chip imperfection is relevant.At present, the tobacco chip of Agilent that occurs on the market and Nibergen mainly designs according to the U.S. and the calculated est sequence of European tobacco gene group.These chips lack corresponding annotation information, also lack distinctive est sequence in the glandular hairs simultaneously, thereby are not suitable for the gene expression profile research of tobacco glandular hairs.About the research report of tobacco glandular hairs gene expression profile, mainly adopt cDNA library random sequencing technological method to finish at present." chromium is to the influence research of tobacco glandular hairs the genetic expression " (Harada that for example delivers recently, 2010) this article author has set up two glandular hairs cDNA libraries (handling and contrast), and two libraries are carried out random sequencing respectively, by sequencing result being compared and analyzing, find the gene expression difference between handling and contrasting.The main drawback of this method is to check order respectively to different cDNA library, thereby order-checking cost height, is not suitable for a large amount of samples are studied.
Expression profiles of gene chip (DNA microarrays for geneexpression profiles) is a kind of of gene chip, to the mRNA in different cell or tissues source in reverse transcription reaction respectively the fluorescent mark with different colours become probe (to use Cy3-dUTP (green) mark control group rnRNA at present always, Cy5-dUTP (redness) mark sample sets mRNA), the gene that probe mixes on back and chip or the mocroarray plate carries out strictness hybridization, pass through the fluorescent scanning chip of different wave length again, to scan gained every bit fluorescent signal value imports computer automatically and carries out information processing, provide fluorescence intensity level and the ratio (ratio value) thereof of each point under different wave length, simultaneous computer gives colour developing figure intuitively.Its hybridization point of gene that is high expression level in sample takes on a red color, and on the contrary, its hybridization point of the gene of high expression level is green in control group, and the suitable displaing yellow of expression level in two groups, these signals have just been represented gene transcription expression in the sample.Gene expression profile can directly detect kind and the abundance of mRNA, can analyze up to ten thousand expression of gene simultaneously and change, and discloses and expresses the mutual relationship that changes between the gene.
Summary of the invention
The technical problem to be solved in the present invention provides the glandular hairs cDNA chip of expression spectrum that grows tobacco, can utilize this chip to carry out the glandular hairs gene expression research, thereby identify the expression of tobacco glandular hairs specific gene, give the preparation and the application method of tobacco glandular hairs cDNA chip of expression spectrum simultaneously.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
The one glandular hairs cDNA chip of expression spectrum that grows tobacco, comprise slide glass, on its plane, be coated with positively charged film, top layer at this film is provided with dot matrix, the sampling point of arranging on the dot matrix, it is characterized in that, on this dot matrix, be placed with and contain the PCR product that derives from tobacco glandular hairs cDNA clone, and contain positive control and negative control sampling point.
Described tobacco glandular hairs cDNA clone's PCR product is to make by the following method:
(1) the total RNA of glandular hairs extracts: select physically well develop, healthy tobacco leaf, adopt and freeze the brush method and collect the blade face glandular hairs, freeze the pure back of depositing and extract the total RNA of tobacco glandular hairs;
(2) cDNA library construction: the full-length cDNA library of adopting the SMART method to construct the tobacco glandular hairs;
(3) cDNA library random sequencing and EST library are set up: random choose cDNA clone, utilize ADI3700 to carry out 5 ' end order-checking, obtain the tobacco ordered sequence, adopt 100bp again, 90% principle obtains the unigene number to its merger unigene, by Blastx EST is carried out sequence alignment and functional annotation, choosing the corresponding clone of ESTs, is that universal primer carries out pcr amplification with M13+ and M13-, with the Virahol method of purification PCR product is carried out purifying.
Described sampling point is that matrix is arranged, single matrix 76 * 114 sampling points of arranging, and each sampling point is provided with 3 repetitions in the chip matrix.
Described positive control sampling point is a tobacco actin gene.
Described negative control sampling point is people's gene and the blank point sample diluent that does not have homology with tobacco.
The preparation method of above-mentioned tobacco glandular hairs cDNA chip of expression spectrum is as follows:
(1) the total RNA of glandular hairs extracts: select physically well develop, healthy tobacco leaf, adopt and freeze the brush method and collect the blade face glandular hairs, freeze the pure back of depositing and extract the total RNA of tobacco glandular hairs;
(2) cDNA library construction: the full-length cDNA library of adopting the SMART method to construct the tobacco glandular hairs;
(3) cDNA library random sequencing and EST library are set up: random choose cDNA clone, utilize ADI3700 to carry out 5 ' end order-checking, obtain the tobacco ordered sequence, adopt 100bp again, 90% principle obtains the unigene number to its merger unigene, by Blastx EST is carried out sequence alignment and functional annotation, choosing the corresponding clone of ESTs, is that universal primer carries out pcr amplification with M13+ and M13-, with the Virahol method of purification PCR product is carried out purifying;
(4) some coremaking sheet: will go up step gained PCR product and be dissolved in the point sample diluent, and with point sample instrument it be selected by the point sample scheme again and be formed on the point sample substrate; The chip that point sample is finished is put and is carried out hydration, drying in the point sample instrument, and it is standby to put 4 ℃ of preservations again after confining liquid sealing, water flushing, drying at room temperature.
The application of above-mentioned tobacco glandular hairs cDNA chip of expression spectrum on screening tobacco glandular hairs specific expression gene specifically may further comprise the steps:
(1) RNA extracts and purifying: employing Trizol single stage method is extracted the tobacco glandular hairs respectively and is removed the total RNA of glandular hairs blade;
(2) fluorescent probe mark: reverse transcription mark cDNA probe and purifying, mix fluorescent mark dCTP, with Cy5-dCTP mark glandular hairs sample in a chain is synthetic, the Cy3-dCTP mark removes the glandular hairs leaf sample, carry out reactionary slogan, anti-communist poster simultaneously, DNA purifying final vacuum is drained, and is standby;
(3) chip hybridization: adopt ordinary method to carry out chip hybridization;
(4) data gathering: with the chip after the laser scanner scans hybridization, and the original signal that chip obtained is carried out piece calibration and data homogenization handle, calculate the differential expression value Ratio of each gene point in this test, Ratio 〉=2.0 are up-regulated expression, express for downward modulation Ratio≤0.5, filters out difference expression gene thus.
The present invention has actively useful effect:
1. the chip prepared of the present invention has the advantage of high-throughput, high precision, high specificity, thereby can a large amount of expression of gene patterns of one-time detection, and can be under same test conditions, the changes in gene expression rule of a plurality of samples of analysis-by-synthesis, be applicable to that tobacco glandular hairs and cultivation step, environment-stress etc. concern the research of aspect, compare with cDNA order-checking means, have efficient, economic, characteristics fast.
2. set up detectable quality control system,, guaranteed the specificity of detected result, can reach effect efficient, sensitive, accurate, the parallel detection of high-throughput as negative, positive, blank.
3. tobacco glandular hairs cDNA chip of expression spectrum of the present invention is based upon on the basis, tobacco glandular hairs cDNA library, and most of sequence carried out functional annotation, compare with the chip in other tobacco EST source, pointed aspect candidate gene, accuracy and the property released, and having improved the low abundance gene possibility that is separated to specifically expressing in the glandular hairs, is the strong instrument of tobacco glandular hairs gene expression research and new gene excavating.
Description of drawings
Fig. 1 is a tobacco glandular hairs cDNA chip of expression spectrum scintigram.
Fig. 2 is that gene chip expression result of the present invention and RT-PCR result compare collection of illustrative plates.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Test method among the following embodiment if no special instructions, is ordinary method; Used test materials and reagent among the following embodiment, if no special instructions, all available from routine biochemistry reagent shop.
The embodiment 1 one glandular hairs cDNA chip of expression spectrum that grows tobacco, comprise slide glass, on its plane, be coated with positively charged film, top layer at this film is provided with dot matrix, the sampling point of arranging on the dot matrix, be placed with on this dot matrix and contain the PCR product that derives from 2830 cDNA of tobacco glandular hairs clone, its length is at 400～1000 bp, and contains positive control and negative control; Sampling point is that matrix is arranged, single matrix 76 * 114 sampling points of arranging, and each sampling point is provided with 3 repetitions in the chip matrix.Positive control is a tobacco actin gene, and negative is people's gene and the blank point sample diluent that does not have homology with tobacco to reference.
The preparation method of above-mentioned tobacco glandular hairs cDNA chip of expression spectrum specifically may further comprise the steps:
(1) the total RNA of the collection of tobacco glandular hairs and glandular hairs extracts:
With tobacco bred K326 is material, select physically well develop, healthy leaves, adopt and to freeze brush method (Erming, 2001) and carry out the collection of blade face glandular hairs, before collecting with the selected blade of flushing with clean water.Blade in liquid nitrogen frozen five minutes is selected the moderate mane brush of hardness for use, gets glandular hairs along master pulse from phyllopodium in frozen pipe to blade tip direction brush, preserves in the liquid nitrogen container.The total RNA of glandular hairs extracts and carries out according to the working specification on the Trizol reagent specification sheets of GIBCO BRL company.0.56g glandular hairs tissue is put into the stone roller alms bowl, in liquid nitrogen, be ground to Powderedly, go to the homogenate pipe, add 5.5 mL Trizol homogenate; The chloroform extracting, isopropanol precipitating, 75% washing with alcohol precipitation is dried, and MilliQ water dissolution precipitation is standby after ultra-violet analysis and the electrophoresis detection.
(2) tobacco blade face glandular hairs cDNA library construction:
Adopt the full-length cDNA library of SMART (Switching Mechanism At 5 ' end of RNA Transcript) technique construction tobacco glandular hairs.LD PCR method synthetic double chain cDNA, according to test kit (Clontech, SMART cDNA Library Construction Kit, 634901) interpolation reagent and primer are described, be reflected on the HYBAID PCR EXPRESS PCR instrument that is preheating to 95 ℃ and carry out, 95 ℃ of sex change 15 sec, 68 ℃ prolong 6 min, totally 30 circulations.Negate answers product 5 mL to carry out electrophoresis detection on 1.1% sepharose.
The dscDNA that draws 50 mL amplification (in the centrifuge tube of 2～3mg) to 0.5 mL sterilization, adds Proteinase K, through phenol: chloroform: behind primary isoamyl alcohol extracting, the ethanol sedimentation, use restriction enzyme
SfiThe I enzyme is cut, and then by the separation and purification of CHROMA SPIN-400 pillar, the cDNA that collects successively through separation and purification 10 manages totally, and 1.1% agarose gel electrophoresis detects cDNA content and clip size.
PBluescript II SK carrier is transformed, transform the sequence between EcoR I and the Not I as Sfi I A and Sfi I B joint sequence.After enzyme is cut, the cDNA orientation after the separation and purification is connected to (Sfi I A → Sfi I B) on this carrier.Test kit is pressed in ligation, and (#EL0012) explanation is carried out for Fermentas, T4 DNA Ligase, and cDNA mixes according to 1:3 with carrier concn.
Get 200 mL competence host bacterium (DH-5 α) to the 50mL centrifuge tube of sterilization, add above-mentioned 1uL and connect liquid, ice bath 45 min behind the mixing gently, 42 ℃ of water-bath heat shock 90 sec, add 2 mL LB substratum, behind 37 ℃ of cultivation 45min, centrifugal 10 min of 3000rpm collect thalline, and 250 mL LB substratum are resuspended.Get 200 mL bacterium liquid and coat the LB solid medium that contains Apr-IPTG/x-gal, 37 ℃ are spent the night, and calculate colony number and blue hickie ratio, and calculate recombination fraction, storage capacity and the library titre in library according to colony number
For Rapid identification is carried out in the library, choose 24 clones immediately and carry out 5 ' end sequencing, and sequencing result is carried out the unigene merger.In 24 ordered sequences that obtain, there are 23 to be unigene, the unigene ratio is 95.8%.Simultaneously sequence is carried out the total length analysis, have 21 sequences that relevant homologous information is arranged in 24 sequences, wherein 14 is total length, and the integrity ratio is 66.7%.Illustrate that constructed tobacco glandular hairs cDNA library has higher quality.
Tobacco blade face glandular hairs cDNA library constructing method also can referring to document " structure in tobacco glandular hairs full-length cDNA library " (Xiamen University's journal (natural science edition), in November, 2006, the 45th the volume the 6th phase, 859-861).
(3) cDNA library random sequencing and EST library are set up:
Random choose cDNA clone utilizes ADI3700 to carry out 5 ' end order-checking.According to phred software QC10 standard, carrier shielding (parameter is-minmatch 14 – penalty-2 – minscore 30), get Chang Du ≧ 500, obtain 5139 of tobacco ordered sequences.Adopt 100bp again, 90% principle is to these 5139 sequence merger unigene, and obtaining the unigene number is 2830.By Blastx EST is carried out sequence alignment and functional annotation.Found that among 2831 unigene that 987 (34.9 %) can't find matching sequence, its Unknown Function; Other 1844 (65.1 %) individual gene is submitted to amigo website (http://amigo.geneontology.org) carries out functional annotation and classification, find the wherein 61.5% ESTs coding albumen relevant, account for 16.8%, 16.1%, 12.9%, 6.5%, 4.4% and 0.6% respectively with biology adjusting, transhipment, irritant reaction, signal transduction, growth, sequence that growth is relevant with metabolism.
(4) point sample film-making:
The corresponding clone of 2830 ESTs is chosen, and is that universal primer carries out pcr amplification with M13+ and M13-, and PCR system volume is 80 μ l, and wherein the dNTP final concentration is 0.2mM, and the primer final concentration is 0.2 μ M, Mg
2+Final concentration is 1.5mM, and rTaq enzyme 4U is about cDNA clone 2-10ng.The PCR response procedures is as follows:
① 94℃ 30?sec；
② 94℃ 30?sec；
③ 56℃ 30sec；
④ 72℃ 2min；
⑤ go?to?② 35Cycles；
⑥ 72℃ 6min；
7. 10 ℃ (or 4 ℃) for ever.
PCR finishes, and confirms the quality and the output of PCR product by 1% agarose gel electrophoresis.
With the Virahol method of purification PCR product is carried out purifying, purification step is as follows: get 8 μ l NaAc (pH=7.0 respectively, 3mol/L) add successively in the PCR product plate with 85 μ l Virahols, mixing, in-20 ℃ of refrigerator-freezers or refrigerator, precipitate and spend the night (or-20 ℃ leave standstill more than 3 hr), precipitation finishes, 96 orifice plates are placed whizzer, the centrifugal 30min of 4500 rpm, abandoning supernatant is inverted 5min, the ethanol 100 μ l of adding 75% in every hole, the centrifugal 20min of 4500 rpm in the rearmounted 96 orifice plate whizzers of mixing gently, abandoning supernatant is more than the room temperature standing and drying 4hr.
Point coremaking sheet: exsiccant PCR product in the 96 hole PCR plates is dissolved in the 16 μ l left and right sides point sample diluents (the star gene chip companies is won in Shanghai), fully dissolving.With 12 road micropipets the sample in the 96 hole PCR plates is moved in 384 orifice plates (Genetix company) in certain sequence.Open the OmniGrid point sample instrument, load onto MicroQuill 2000 point needles (Majer Precision company), design program by the point sample scheme, put 384 hole sample panel and treat point sample substrate (the star gene chip companies is won in Shanghai), working procedure begins point sample.
The chip that point sample is finished is put hydration 30 min in the point sample instrument, drying at room temperature 2 hr; With standby after confining liquid (the star gene chip companies is won in Shanghai) sealing 15min, water flushing, the drying at room temperature.
Each is cloned in same substrate different positions and repeats point sample three times, does positive control with tobacco actin gene, does not have the people's gene of homology with tobacco and does negative control, and it is standby to put 4 ℃ of preservations after the chip quality inspection is qualified.
Utilize the method for above-mentioned tobacco glandular hairs cDNA chip screening glandular hairs specific expression gene:
(1) RNA extracts and purifying
Employing Trizol single stage method is extracted glandular hairs respectively and is removed the total RNA of glandular hairs blade.Adopt the total RNA of QIAGEN RNeasy test kit purifying, the MessageAmpTM II aRNA Amplification Kit that the linear amplification of RNA adopts Ambion company to produce, RNA Amplification for Array Analysis test kit.The strict test kit specification sheets of pressing is operated.
(2) fluorescent probe mark
Concrete steps are as follows:
1. in 1.5 mL Eppendorf pipes of sterilization, add following reagent (the reaction final volume is 50 μ L, and following reagent is RNase-free) successively:
ddH
2O 23?μL
Random primer 5 μ L
aRNA 3?μg
The vibration mixing places 70 ℃ of water-bath 10 min, after the taking-up, places on ice rapidly.
2. add following reagent respectively:
Reversed transcriptive enzyme damping fluid 10 μ L
DTT?5?μL
dNTPs?4?μL
3. then in the darkroom, add following reagent:
Reversed transcriptive enzyme 2 μ L
Cy5-dCTP or Cy3-dCTP 3 μ L
4. beat tube wall with the mixing sample with the finger bullet, maniluvium 2 min.The Eppendorf pipe is placed 42 ℃ of water-bath 2 hr.
5. add labelled reagent I 4 μ l successively in the Eppendorf pipe, add labelled reagent II 4 μ l behind 65 ℃ of water-bath 10 min, mixing merges control group, experimental group, and lucifuge, vacuum are drained to the 50 μ L.
6. use DNA purification column (or ethanol sedimentation) purify DNA.
7. add labelled reagent III 8 μ l, vacuum is drained.
Reverse transcription mark cDNA probe and purifying mix fluorescent mark dCTP in a chain is synthetic, with Cy5-dCTP mark glandular hairs sample, the Cy3-dCTP mark removes the glandular hairs leaf sample, carries out reactionary slogan, anti-communist poster simultaneously.DNA purifying final vacuum is drained, and is standby.
(3) chip hybridization
Concrete steps are as follows:
1. add 6.5 μ L hybridizing reagent I in the probe tube of draining, fully mixing makes the probe dissolving.Add 6.5 μ L hybridizing reagent II again, mixing is standby.
2. the slide with prehybridization takes out, and washes away cover glass with ddH2O.
3. probe is placed 95 ℃ of water-bath sex change 2 min; Slide places 95 ℃ of water-bath sex change 30 sec, and slide takes out and soaks dehydrated alcohol 30 sec, and probe places on ice rapidly after taking out.
4. probe is placed on the chip, cover, place the hybridization cabin,, put into 42 ℃ of hybridization casees and hybridize spend the night (16～18 h) with the Parafilm sealing with cover glass.
5. with 0.5% washings, 1 flushing slide, remove cover glass.
6. prepare two staining jars, the reagent 2 of developing a film of 0.5% the reagent 1+2% that develops a film, 5% the reagent 3 of developing a film are housed respectively, put into 60 ℃ of water-baths.
7. slide is immersed successively washing 10 min in above two staining jars.
8. with 0.5% washings, 1 flushing slide, dry back scanning.
(4) data gathering
Chip scans with the ScanArray4000 laser scanner, gets scintigram shown in Figure 1.Adopt GenePix Pro 3.0 image analysis software (Axon Instruments company) that chip image is analyzed, after the chip hybridization scanning, the original signal that chip obtained is carried out piece calibration and data homogenization processing with analysis software.Homogenization is handled according to following 2 principles screening effective gene point: the first, and the Cy3 of this gene point, Cy5 signal value are all greater than 200, and perhaps one of them is greater than 800; The second, the Ratio value of this gene point is obtained r=ln (Ratio) then between 0.1～10, calculate the mean value R of whole effective gene point r again, and the homogenization coefficient of test is EXP (R) with regard to the inverse that equals R.The Cy3 signal value of all gene points is multiplied by the homogenization coefficient, draw adjusted Cy3*, calculate the differential expression value Ratio=Cy5/Cy3* of each gene point in this test, Ratio 〉=2.0 are up-regulated expression, express for downward modulation Ratio≤0.5, filters out difference expression gene thus.
(5) differential gene RT-PCR checking
Select portion gene to carry out the RT-PCR checking according to the chip hybridization interpretation of result.According to primer-design software Primer5 design primer such as table 1.Adopt SuperScript One-Step system to carry out RT-PCR and detect, the program of operating is to specifications carried out.25 μ L systems, 30 circulations, reaction conditions is as follows: 94 ° of C sex change 1min, 50～55 ° of C 2 min that anneal, 72 ° of C extend 2 min.Make interior mark with the Actin gene.
Chip expression of results and RT-PCR result relatively see accompanying drawing 2.
Table 1 RT-PCR primer sequence table
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. the glandular hairs cDNA chip of expression spectrum that grows tobacco, comprise slide glass, on its plane, be coated with positively charged film, top layer at this film is provided with dot matrix, the sampling point of arranging on the dot matrix, it is characterized in that, on this dot matrix, be placed with and contain the PCR product that derives from tobacco glandular hairs cDNA clone, and contain positive control and negative control sampling point.
2. tobacco glandular hairs cDNA chip of expression spectrum according to claim 1 is characterized in that, described tobacco glandular hairs cDNA clone's PCR product is to make by the following method:
(1) the total RNA of glandular hairs extracts: select physically well develop, healthy tobacco leaf, adopt and freeze the brush method and collect the blade face glandular hairs, freeze the pure back of depositing and extract the total RNA of tobacco glandular hairs;
(2) cDNA library construction: the full-length cDNA library of adopting the SMART method to construct the tobacco glandular hairs;
(3) cDNA library random sequencing and EST library are set up: random choose cDNA clone, utilize ADI3700 to carry out 5 ' end order-checking, obtain the tobacco ordered sequence, adopt 100bp again, 90% principle obtains the unigene number to its merger unigene, by Blastx EST is carried out sequence alignment and functional annotation, choosing the corresponding clone of ESTs, is that universal primer carries out pcr amplification with M13+ and M13-, with the Virahol method of purification PCR product is carried out purifying.
3. tobacco glandular hairs cDNA chip of expression spectrum according to claim 2 is characterized in that described sampling point is that matrix is arranged, single matrix 76 * 114 sampling points of arranging, and each sampling point is provided with 3 repetitions in the chip matrix.
4. tobacco glandular hairs cDNA chip of expression spectrum according to claim 2 is characterized in that described positive control sampling point is a tobacco actin gene.
5. tobacco glandular hairs cDNA chip of expression spectrum according to claim 2 is characterized in that, described negative control sampling point is people's gene and the blank point sample diluent that does not have homology with tobacco.
6. the preparation method of the described tobacco glandular hairs of claim 1 cDNA chip of expression spectrum may further comprise the steps:
(1) the total RNA of glandular hairs extracts: select physically well develop, healthy tobacco leaf, adopt and freeze the brush method and collect the blade face glandular hairs, freeze the pure back of depositing and extract the total RNA of tobacco glandular hairs;
(2) cDNA library construction: the full-length cDNA library of adopting the SMART method to construct the tobacco glandular hairs;
(3) cDNA library random sequencing and EST library are set up: random choose cDNA clone, utilize ADI3700 to carry out 5 ' end order-checking, obtain the tobacco ordered sequence, adopt 100bp again, 90% principle obtains the unigene number to its merger unigene, by Blastx EST is carried out sequence alignment and functional annotation, choosing the corresponding clone of ESTs, is that universal primer carries out pcr amplification with M13+ and M13-, with the Virahol method of purification PCR product is carried out purifying;
(4) some coremaking sheet: will go up step gained PCR product and be dissolved in the point sample diluent, and with point sample instrument it be selected by the point sample scheme again and be formed on the point sample substrate; The chip that point sample is finished is put and is carried out hydration, drying in the point sample instrument, and it is standby to put 4 ℃ of preservations again after confining liquid sealing, water flushing, drying at room temperature.
7. the application of the described tobacco glandular hairs of claim 1 cDNA chip of expression spectrum on screening tobacco glandular hairs specific expression gene.
8. application according to claim 7 is characterized in that, may further comprise the steps:
(1) RNA extracts and purifying: employing Trizol single stage method is extracted the tobacco glandular hairs respectively and is removed the total RNA of glandular hairs blade;
(2) fluorescent probe mark: reverse transcription mark cDNA probe and purifying, mix fluorescent mark dCTP, with Cy5-dCTP mark glandular hairs sample in a chain is synthetic, the Cy3-dCTP mark removes the glandular hairs leaf sample, carry out reactionary slogan, anti-communist poster simultaneously, DNA purifying final vacuum is drained, and is standby;
(3) chip hybridization
(4) data gathering: with the chip after the laser scanner scans hybridization, and the original signal that chip obtained is carried out piece calibration and data homogenization handle, calculate the differential expression value Ratio of each gene point in this test, Ratio 〉=2.0 are up-regulated expression, express for downward modulation Ratio≤0.5, filters out difference expression gene thus.
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| CN103308627A (en) * | 2013-07-05 | 2013-09-18 | 川渝中烟工业有限责任公司 | Method for measuring effect factors of microorganism preparation for reducing protein content in tobaccos |
| CN113215077A (en) * | 2021-06-15 | 2021-08-06 | 山东中医药大学 | Method for obtaining honeysuckle glandular hair tissue |
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| 《中国优秀硕士学位论文全文数据库农业科技辑》 20090415 冀浩 烟草腺毛基因表达谱比较研究 1-50 1-8 第2009年卷, 第4期 * |
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| CN103308627A (en) * | 2013-07-05 | 2013-09-18 | 川渝中烟工业有限责任公司 | Method for measuring effect factors of microorganism preparation for reducing protein content in tobaccos |
| CN113215077A (en) * | 2021-06-15 | 2021-08-06 | 山东中医药大学 | Method for obtaining honeysuckle glandular hair tissue |
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