CN102304523B - Tubulin gene in chamomile and use thereof - Google Patents
Tubulin gene in chamomile and use thereof Download PDFInfo
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Abstract
The invention discloses an alpha-tubulin homologous gene (ClTUA gene) in chamomile, which is obtained by the following steps: extracting the total RNA of chamomile, performing reverse transcription, synthesizing cDNA, obtaining the homologous sequence (SEQ ID No.1) of the alpha-tubulin in chamomile by using homologous cloning technique, and performing full-length amplification of the sequence to obtain the alpha-tubulin homologous gene (ClTUA gene) in chamomile. The alpha-tubulin homologous gene (ClTUA gene) in chamomile, which is obtained by the invention, can express stably in all growth stages of chamomile and under all kinds of non-biological stress conditions and is suitable to be used as a reference gene in study on expression of genes in chamomile; in addition, the abundance of the expression of the alpha-tubulin gene is in an unsaturated state, and by using the alpha-tubulin gene as the reference gene, the result of the analysis on expression is more accurate; and thus, the alpha-tubulin gene is suitable to be used as a reference gene for studying the stress tolerance of chamomile and the gene expression in the growth process of the chamomile.
Description
Technical field
The present invention relates to separation, preparation, purifying and the application of a kind of DNA of genetic engineering, particularly a kind of separation of gene of coded plant protein, preparation purifying and application belong to molecular biological field of genetic engineering.
Background technology
Camomile [Chrysanthemum lavandulifolium (Fisch.ex Trautv.) Makino] has another name called lavandulaleaf dendranthema herb, sweet mother chrysanthemum, it is the diplont of composite family Chrysanthemum, be the cultivating chrysanthemum (wild relatives of C. * morifolium), be a kind of short day halophytes that is widely distributed in mountain region, China North China, obtain further investigation as the model plant in the cultivating chrysanthemum research at present.Inquire into its associated biomolecule knowledge topic, can provide bibliography for the mechanism of resolving the main fancy points formation of cultivating chrysanthemum, also lay the foundation for the research of chrysanthemum molecular breeding.At present degeneration-resistant about camomile and become the research of colored molecule mechanism to make some progress, but reference gene only has actin gene Actin and 26S rRNA gene available.Therefore, excavate the more reference gene of camomile, be conducive to improve stability, the reproducibility and reliability of gene expression pattern research.
When transcriptional level carried out Quantitative analysis of gene expression, it was the most general, reliable method that application house-keeping gene (Housekeeping genes) is proofreaied and correct the target gene expression amount as reference gene (Reference genes).The so-called constant expression of any house-keeping gene all is constant under the cell of certain type or empirical factor effect, desirable house-keeping gene should be the gene of constitutive expression in the metabolic processes of involved in plant basis, such as the gene in protein metabolism approach and the carbohydrate metabolism approach.Present more use mainly contain α in glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), plant elongation factor gene (EF-1a), PolyUb gene (polyubiquitin, polyUB), actin gene (Actin) and the tubulin, 'beta ' family gene (α/β-tubulin).
Tubulin (tubulin) is a kind of skelemin of cell, participate in cell endoplasm circulation, shape maintains, motion, division, differentiation, matter transportation, signal transduction and polarity such as build up at the important physiological activity, can be divided into α, β, γ, δ, the multiple subfamily such as ε, wherein α-tubulin albumen and β-tubulin albumen forms a kind of heterodimer albumen, its gene presents constitutive expression usually, but, studies show that in a large number α-tubulin gene is non-constitutive expression in the various plants Different Organs, for example: α-tubulin gene plays a role with minigene family in higher plant, Arabidopis thaliana, willow, number in grape and the barley is respectively 6,8,7,5, these genes can be divided into two classes (I and II) according to the polymorphism of its coded amino acid C end, this the two class α-expression pattern of tubulin gene in plant has very big-difference, such as the I class α in the trembling poplar-tubulin gene, except PtTUA7 (being the homologous gene of ClTUA gene), all the other members all in xylem gene expression abundance the highest, and II class PtTUA gene all gene expression abundance is very low in each organ; I class α in the Arabidopis thaliana-tubulin gene (AtTUA2/4) constitutive expression in each organ, and the AtTUA1 in the II genoid only expresses in flower section; In the barley, the HvTUA2/4 gene is partly given birth to tissue expression at leaf, and HvTUA1/5 expressed in mitosis anaphase, and HvTUA3 only expressed when metaphase, microfilament formed.
At present, in the degeneration-resistant correlative study with becoming the gene expression analysis such as flower molecule mechanism of camomile, usually select actin gene Actin and 26S rRNA gene as reference gene.Yet, more and more studies have shown that and only use the accuracy that causes experiment of attending a meeting in the conduct of Actin gene to reduce.In addition, a lot of researchs have shown that all ribosome-RNA(rRNA) is not suitable for confidential reference items and uses in gene expression research, especially the mRNA in the different tissues is different with the rRNA content ratio, causes the test-results mistake, for example during mitotic division, 18SrRNA obviously reduces or stops to express.
There is the investigator to carry out the screening of Dendranthema candidate reference gene, such as (reference: Gu C such as Gu, Chen S M, Liu Z l, et al.Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress.Mol Biotechnol, doi:10.1007/s12033-011-9394-6) studies show that EF1-α gene is applicable when research chrysanthemum response biology is coerced, and larger in water stress Expression In The Process quantitative changeization; The PP2A gene then in contrast, it is relatively poor to coerce in the process performance at biology, constant in heat, water stress Expression In The Process; And widely used reference gene Actin above-mentioned coerce all show in the process relatively poor.Therefore, excavate out the more camomile reference gene of stably express, will be conducive to improve stability, the reproducibility and reliability of camomile gene expression analysis research.
Summary of the invention
Primary and foremost purpose of the present invention be for the problem that above-mentioned prior art exists provide a kind of each growth and development stage of camomile and camomile all can stably express under various growing environments camomile α-tubulin homologous gene, its gene order fragment and they are as the application of internal memory gene.Camomile α of the present invention-tubulin homologous gene can not only be expressed in the stages consistence of camomile growth, and can consistence express in various adverse environments, can be used for analyzing the adversity gene expression of camomile.
For realizing purpose of the present invention, one aspect of the present invention provides a kind of camomile [Chrysanthemum lavandulifolium (Fisch.ex Trautv.) Makino] tubulin homologous gene (α-tubulin homologous gene) fragment (EST series fragment), and its base is shown in the SEQ ID NO:1.
The present invention at first grows up functional leaf as raw material take camomile, extracts total RNA, then carries out the reverse transcription of RNA, and synthetic cDNA then according to known Microtubules in plants albumen homology gene design degenerated primer, take synthetic camomile as template, carries out the RT-PCR amplification; At last amplified production is separated, namely obtain camomile tubulin homologous gene (α-tubulin homologous gene) sequence fragment (EST series fragment).
Wherein, the degenerated primer of design is as follows:
ClTUA-JBs：5′-TCTGTTGTYGAGCCYTACAACAGTGT-3′；(SEQ?ID?NO：5)
ClTUA-JBx：5′-TAGTTRATWCCRCACTTGAAYCC-3′。(SEQ?ID?NO：6)
Wherein, Y, R, W are the degeneracy base, Y=C/T (1: 1); R=A/G (1: 1); W=A/T (1: 1).
Provide on the other hand the purposes of a kind of camomile microtubule protein gene series fragment as the Dendranthema reference gene, particularly as the purposes of camomile reference gene.
Further aspect of the present invention provides a kind of camomile microtubule protein gene (ClTUA gene), and its base is SEQ ID NO:4.
Another aspect provides the purposes of a kind of camomile microtubule protein gene (ClTUA gene) as the Dendranthema reference gene, particularly as the purposes of camomile reference gene.
The present invention uses the RACE technology to obtain the full length sequence of this gene according to camomile microtubule protein gene est sequence, called after ClTUA gene, bioinformatic analysis show that ClTUA full length gene cDNA sequence is 1580bp, open reading frame 1350bp wherein, 449 amino acid of encoding.Its speculating acid sequence is carried out the systematic evolution tree analytical proof, and it is the homologous sequence of plant I class α-tubulin gene.Use camomile reference gene ClActin and the positive contrast of 26S rRNA gene, utilize RT-PCR research ClTUA gene to coerce expression Changing Pattern between the sample of lower and the different growth and development stages of camomile at salt, arid, hot and cold, dormin and Whitfield's ointment.The result shows: the ClTUA gene is not only expressed stable under abiotic stress, and in different growth and development stage samples stably express; The expression stability of camomile ClTUA gene is better than ClActin and camomile 26SrRNA gene, can be used as the reference gene of genetic expression in research camomile resistance and the growth and development process.
Camomile microtubule protein gene of the present invention (ClTUA gene) with and the expression amount of gene line row fragment (EST series fragment) in the plant camomile, have higher expression level, excavate out the more camomile reference gene of stably express, will be conducive to improve stability, the reproducibility and reliability of camomile gene expression analysis research.
Description of drawings
Fig. 1 is the gel electrophoresis figure by the camomile α of Homology-based cloning acquisition-tubulin homologous gene sequence fragment;
Fig. 2 is the gel electrophoresis figure of camomile α-tubulin homologous gene (ClTUA gene) 3 ' RLM-RACE product;
Fig. 3 is the gel electrophoresis figure of camomile α-tubulin homologous gene (ClTUA gene) 5 ' RLM-RACE product;
Fig. 4 is the gel electrophoresis figure of camomile α-tubulin homologous gene (ClTUA gene) total length amplified production;
Fig. 5 is camomile Actin after salt stress is processed, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Fig. 6 is camomile Actin behind the Whitfield's ointment Stress treatment, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Fig. 7 is camomile Actin after arid is processed, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Fig. 8 is camomile Actin behind the cold Stress treatment, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Fig. 9 is camomile Actin after heat stress is processed, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Figure 10 is that dormin (ABA) is induced the rear camomile Actin of processing, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed;
Figure 11 is each growth and development stage blade Actin of camomile, the gel electrophoresis figure that 26S rRNA and α-tubulin homologous gene (ClTUA gene) is expressed.
The specific embodiment mode
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Illustrate: the genetic recombination of using learns a skill and is routine techniques in this area in the following specific embodiment.The technology that in following examples, does not describe in detail, all carry out according to the related Sections in following laboratory manual or the document or part, comprise: the people such as Sambrook, Molecular Cloning, ALaboratory Manual (the 3rd edition .Cold Spring Harbor Lab Press, 2001).Wang Guanlin, Fang Hongjun." plant genetic engineering philosophy and technique " (the 2nd edition.Science Press, 2002)
One, material and reagent
1, the preparation of each growth phase test materials of camomile
(1) seed material
Gather the camomile seed as seed material in the Beijing Forestry University nursery in December, 2009, the seed collecting plant strain growth is good, and seed maturity is full, and the kind skin is intact; Seed material places rapidly the liquid nitrogen quick-frozen afterwards in collection, is stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
(2) spire, grow up functional leaf, stem section material, root system material
It is among 2.5% the chlorine bleach liquor that the camomile seed is soaked in concentration of volume percent, use aseptic water washing 3-5 time behind the soaking disinfection 10min, blot surface-moisture on the filter paper with aseptic drying, be seeded in vermiculite: in the flowerpot of turfy soil (1: 1), place phytotron to cultivate.
The culture condition of phytotron is: relative humidity 80%, constant temperature 20-240C, intensity of illumination 80-200umol/M2/S, periodicity of illumination are 16h dark/8h illumination cultivation.
Gather respectively first pair of true leaf growing behind the seed germination as the spire material; When plant to be planted grows to 10-12 to true leaf, gather as from the top terminal bud downwards the 5-6 of number to true leaf as the functional leaf material of growing up; Gather 10-12 to the stem section of true leaf plant as stem section material; Plant is carefully taken out from flowerpot, clean compost and gather root system (comprising that main root, fibrous root gather together) as the root system material, this moment, root system was long 10-15 centimetre.Spire material, the functional leaf of growing up, stem section, root system material place rapidly the liquid nitrogen quick-frozen afterwards in collection respectively, are stored in-80 ℃ of Ultralow Temperature Freezers, and be for subsequent use.
(3) short day inducer blade, bud, petal, the rear old and feeble blade of blooming
Adopt the culture condition identical with " spire material " culture condition to cultivate the camomile seed, plant strain growth behind seed germination is to 4-5 during to true leaf, a part of plant is moved to the cultivation of short day phytotron, and (condition is: relative humidity 80%, constant temperature 20-24C, intensity of illumination 80-200umol/M
2/ S, periodicity of illumination are 12h dark/12h illumination cultivation, and this moment, the camomile blade was finished " virgin phase ", can experience short day), after cultivating 50d, plant strain growth to 10-12 to true leaf after, gather from terminal bud downwards the 5-6 of number to true leaf as short day inducer blade material; During 60d to be cultivated, camomile buddings, gathers bud (diameter 5mm); Continue to cultivate about 10-20d, the head inflorescence of camomile is fully open, and gather petal as material this moment; Continue to cultivate after two months, camomile has been finished and has bloomed, tie kind, and this moment, blade was tending towards old and feeble (plant leaf is counted 20-24 to true leaf), gather the 5-6 that counts downwards from terminal bud to true leaf as old and feeble blade and blade sheet material.
Above-mentioned each material places rapidly respectively the liquid nitrogen quick-frozen after collection, then be stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
(4) various Stress treatment materials
Adopt culture condition cultivation camomile seed identical with " the functional leaf material of growing up " culture condition, the plant strain growth behind seed germination carries out various Stress treatments to 4-5 during to true leaf, and concrete operation step is as follows
(4-1) salt stress is processed material
Carry out salt stress with the NaCl solution of 200mM pouring cave dish seedling and process, wherein, the irrigation amount of NaCl solution be the 200ml/ basin/time, every 24h pouring is once; The 2-3 that counts downwards from terminal bud that gathers the pouring of NaCl solution 0h, 2h, 12h, 24h processes material to the true leaf blade as salt stress, be respectively salt stress-1,2,3,4, each salt stress material, after collection, place rapidly respectively the liquid nitrogen quick-frozen, be stored in-80 ℃ of Ultralow Temperature Freezers, for subsequent use.
(4-2) Whitfield's ointment Stress treatment material
Carry out Stress treatment with the salicylic acid solution of 3mM pouring cave dish seedling, wherein, irrigation amount be the 100ml/ basin/time, every 24h pouring is once; Gather salicylic acid solution pouring 0h, 2h, 12h, 24h from terminal bud downwards the 2-3 of number to the true leaf blade as Whitfield's ointment Stress treatment material, be respectively Whitfield's ointment and coerce-1,2,3,4, respectively coerce material, after collection, place rapidly respectively the liquid nitrogen quick-frozen, be stored in-80 ℃ of Ultralow Temperature Freezers, for subsequent use.
(4-3) drought stress is processed material
The camomile seedling is taken out from flowerpot, clean root system with distilled water, be positioned over water ratio and be and carry out drought stress in 50% the wetting absorbent cotton and process; The true leaf blade that the collection arid is processed behind 0h, 2h, 12h, the 24h is processed material as drought stress, is respectively drought stress-1,2, and 3 and 4, material places rapidly respectively the liquid nitrogen quick-frozen, is stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
(4-4) cold Stress treatment material
Place 4 ℃ of incubators to carry out cold Stress treatment the camomile seedling; Culture condition: relative humidity 80%, intensity of illumination 80-200umol/M
2/ S, periodicity of illumination are 16h dark/8h illumination cultivation; Gather true leaf blade behind cold Stress treatment 0h, 2h, 12h, the 24h as cold Stress treatment material, be respectively cold and coerce-1,2,3 and 4, each material places rapidly the liquid nitrogen quick-frozen, then is stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
(4-5) heat stress is processed material
The camomile seedling put carry out heat stress in 40 ℃ of incubators and process; Culture condition: relative humidity 80%, intensity of illumination 80-200umol/M
2/ S, periodicity of illumination are 16h dark/8h illumination cultivation; The true leaf blade that the collection heat stress is processed behind 0h, 2h, 12h, the 24h is processed material as heat stress, is respectively heat stress-1,2, and 3 and 4, each material places rapidly the liquid nitrogen quick-frozen, then is stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
(4-6) dormin (ABA) is induced the Stress treatment material
ABA solution with 200 μ M sprays the camomile blade, carry out ABA and induce Stress treatment, wherein, the amount of spraying be the 50ml/ basin/time, every 24h sprays once; True leaf blade behind collection ABA Stress treatment 0h, 2h, 12h, the 24h is induced the Stress treatment material as ABA, and be respectively ABA and coerce-1,2,3 and 4, each material places rapidly the liquid nitrogen quick-frozen, then is stored in-80 ℃ of Ultralow Temperature Freezers, and is for subsequent use.
2, reagent:
M-MLV ThermoScript II test kit (Promega company); DNA reclaims test kit (Easy Pure Quick Gel Extraction Kit, TransGenBiotech company); The terminal rapid amplifying test kit of cDNA (BD SMART
TMRACE cDNA Amplification Kit, Clontech company); PMD18-T vector carrier (the precious biotinylated biomolecule in Dalian company); Bacillus coli DH 5-α competent cell (TransGenBiotech company); Trizol reagent (the handsome company of the U.S.); RNA settling agent, RNase inhibitor (Beijing Baeyer enlightening Bioisystech Co., Ltd)
Two, test method
1, the extraction of the total RNA of camomile material
The adult functional leaf that 1) will gather places the mortar of precooling after taking out immediately from Ultralow Temperature Freezer, add liquid nitrogen and carry out quick grind into powder;
2) add 800 μ lTrizol reagent in the 2ml centrifuge tube, get above-mentioned ground adult functional leaf powder 1g, join in the centrifuge tube, thermal agitation 15s makes the lysis of each camomile material; After lysate was at room temperature placed 5min, in 4 ℃, centrifugal 15min obtained supernatant liquor under the 12000rpm;
3) supernatant liquor is transferred to the 2ml centrifuge tube after, in each centrifuge tube, add respectively water-saturated phenol (pH=5.0, Beijing Baeyer enlightening company), the mixed solution of chloroform and primary isoamyl alcohol, wherein the volume ratio of chloroform and primary isoamyl alcohol is 24: 1, and the volume of water-saturated phenol is 1: 3 with the ratio of the mixed volume of chloroform and primary isoamyl alcohol; With the vibration of each centrifuge tube evenly, add water-saturated phenol, chloroform and primary isoamyl alcohol mixed solution act as protein and the DNA that removes in the material; At room temperature place 2-3min and namely be divided into three layers, lower floor is phenol phase (DNA is dissolved in the phenol phase), and the middle level is the albumen phase, and the upper strata is water (RNA is dissolved in water).In 4 ℃, centrifugal 10min under the 12000rpm obtains supernatant liquor (water that contains RNA);
4) with step 3) preparation supernatant liquor be transferred to respectively the 2ml centrifuge tube after, adding respectively volume ratio in each centrifuge tube is that 24: 1 chloroform and the mixed solution of primary isoamyl alcohol repeat extracting three times, in order to remove the macromolecular substance such as insoluble protein, polysaccharide, polyphenol in the cell, obtain total RNA extracting solution of each camomile material;
5) in the RNA extracting solution, add (0.7 times of volume) Virahol and 10 μ lRNA settling agents (RNAdown) respectively, under-70 ℃, leave standstill 20min, make the RNA precipitation; At 4 ℃, centrifugal 15min discards supernatant under the 12000rpm, is precipitated with the extracting solution after leaving standstill;
6) with after the 70% washing with alcohol precipitation, again with the absolute ethanol washing precipitation, precipitation is dried, the distilled water of processing with 50 μ l diethylpyrocarbonates (DEPC) (is DEPC ddH
2O) dissolution precipitation adds 1 μ lRNase inhibitor in dissolving RNA precipitation, to prolong the RNA shelf time, obtain the total rna solution of the adult blade of camomile;
7) detecting the total RNA of camomile material with 1.5% agarose gel electrophoresis, use Smart Spec Pluss spectrophotometric determination concentration, quantitatively is 1000ng/ μ L with each camomile material total rna solution, places-70 ℃ of Ultralow Temperature Freezers to save backup.
2, the reverse transcription of camomile RNA
With M-MLV ThermoScript II test kit the total RNA of various camomile materials is carried out reverse transcription, synthetic cDNA the first chain, concrete steps are: add respectively the total RNA 3 μ l of above-mentioned camomile material in the PCR pipe, Oligo dT 3 μ l add DEPC ddH again
2O to 15 μ l, sex change 5min in 70 ℃ of water-baths, ice bath 1min is little centrifugal immediately, then adds successively: MLV 5 * buffer 5.0 μ l, 10mM dNTP 2.5 μ l, RNase inhibitor 0.5 μ l, MMLVRTase 1.0 μ l, DEPC ddH
2O 1.0 μ l, reaction system is 25 μ l;
The PCR pipe is put into the PCR instrument, hatch 90min in 42 ℃, obtain reverse transcription product cDNA the first chain, reverse transcription product is positioned over-20 ℃ and saves backup;
3, camomile α-tubulin homologous gene segment amplification
The relevant homogenic amino acid conserved sequence and nucleotide sequence (tobacco Nicotiana tabacum alpha-tubulin, the AJ421413.1 that have delivered according to GenBank; Tea tree Camellia sinensis alpha tubulin 1, DQ340766.2; Longan Dimocarpus longan alpha-tubulin, FJ479617.1; Upland cotton Gossypium hirsutum alpha-tubulin, EF151305.1), the design degenerated primer utilizes the RT-PCR technology, separates the homologous sequence of camomile α-tubulin gene from the reverse transcription product that step 2 obtains, and wherein the degenerated primer sequence is:
ClTUA-JBs：5′-TCTGTTGTYGAGCCYTACAACAGTGT-3′；
ClTUA-JBx:5 '-TAGTTRATWCCRCACTTGAAYCC-3 '; Wherein Y, R, W are the degeneracy base, Y=C/T (1: 1); R=A/G (1: 1); W=A/T (1: 1).
The PCR response procedures is as shown in table 1:
The RT-PCR amplified reaction program of table 1 camomile α-tubulin homology segment
The PCR step is: add successively 2.0 μ l10 * PCR buffer in centrifuge tube, the dNTP of 0.4 μ l10mM, 10 μ M ClTUA-JBs1.0 μ l, 10 μ M ClTUA-JBx1.0 μ l, the reverse transcription product 1.0 μ l that step 2 obtains, 3U/ μ l Taq enzyme 0.5 μ l, ddH
2O14.1 μ l amounts to 20 μ l, and wherein Taq E enzyme is that a day root Bioisystech Co., Ltd produces, and dNTP is that Beijing Baeyer enlightening biotech company produces.
As for carrying out the PCR reaction in the PCR instrument, the PCR program is: 94 ℃ of denaturation 5min with centrifuge tube; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s, 35 circulations; 72 ℃ are extended 10min; 10 ℃ stop.
The PCR product uses 1.5% agarose gel electrophoresis to detect, result such as Fig. 1, and use DNA to reclaim test kit (Easy Pure Quick Gel Extraction Kit) and reclaim amplified production band (be among Fig. 1 from top to bottom article one bright band).The amplified production that reclaims is connected on the pMD18-T vector carrier, after connecting product conversion bacillus coli DH 5-α competent cell (TransGenBiotech company), carry out blue hickie screening, the extracting waste bacterial plaque is carried out the pcr amplification evaluation, with positive bacteria liquid order-checking (Beijing AudioCodes biotech firm).
(3) amplified fragments bioinformatic analysis
Use DNAMAN, NCBI/blastx software that this sequence is carried out bioinformatic analysis, comparison result shows: amplified fragments and betula pendula α-tubulin gene (FJ228477.1) has 85% homology, thereby obtain the homologous sequence fragment (est sequence) of α of the present invention-tubulin gene, the est sequence fragment length is 526bp, and nucleotide sequence fragment is denoted as SEQ NO:1.
4, the homogenic RACE amplification of camomile α-tubulin
(1) design of primers
According to above-mentioned est sequence, adopt primer premier 5.0 bioinformatics softwares, design 3 ' and 5 ' terminal specific primer, wherein the amplification method of 3 ' RACE carries out (Meng Li with reference to the people's such as Meng Li in 2007 method, Dai Silan. clone, sequential analysis and the prokaryotic expression thereof of cineraria F3 ' 5 ' H gene cDNA. Molecular Plant Breeding, 2005.3 (6): 780-786); The amplification method of 5 ' RACE is with reference to the BD SMART of Clontech company
TMRACE cDNAAmplification Kit test kit carries out.
A:3 ' RACE amplimer:
3 ' RACE is the constructional feature of utilizing the terminal polyA of mRNA, add that at 3 of dT primer ' end the anchor primer (AP) about 20bp carries out reverse transcription, design gene order Auele Specific Primer (ClTUA-3RACE) is as upstream primer, additional anchor primer (AUAP) carries out 3 of goal gene ' end amplification as downstream primer.
The AP primer: 5 '-GGCCACGCGTCGACTAGTAC (T) 16-3 '
The AUAP primer: 5 '-GGCCACGCGTCGACTAGTAC-3 '
The ClTUA-3RACE primer: 5 '-CAGTTTGTGGACTGGTGCCC-3 '
B:5 ' RACE amplimer:
5 ' CDS primer: BD SMART
TMRACE cDNAAmplification Kit.
ClTUA-5RACE-GSP?1：5′-GTATGTGGGACGCTCAATGTC-3′
(2) 3 ' RLM-RACEs
Add total RNA 3 μ l of the adult functional leaf of camomile of said extracted in the PCR pipe, AP primer 3 μ l add DEPC ddH again
2O to 15 μ l, sex change 5min in 70 ℃ of water-baths, ice bath 1min is little centrifugal immediately, then add successively: MLV 5 * buffer5.0 μ d, 10mM dNTP 2.5 μ l, RNase inhibitor 0.5 μ l, MMLVRTase 1.0 μ l, DEPC ddH2O 1.0 μ d, reaction system is 25 μ l;
The PCR pipe is put into the PCR instrument, hatch 90min in 42 ℃, obtain reverse transcription product cDNA the first chain (3 '-RACE-Ready cDNA), reverse transcription product is positioned over-20 ℃ and saves backup;
Carry out the pcr amplification of 3 ' RACE as template take synthetic this reverse transcription product cDNA the first chain (3 '-RACE-Ready cDNA).The pcr amplification reaction program of 3 ' RACE is as shown in table 2:
The pcr amplification reaction program of the homogenic 3 ' RACE of table 2 camomile α-tubulin
Concrete steps are: add successively 2.0 μ l10 * PCRbuffer in centrifuge tube, the dNTP of 0.4 μ l10mM, 10 μ MAUAP primers, 1.0 μ l, 10 μ M ClTUA-3RACE primers, 1.0 μ l, above-mentioned 3 ' RACE-Ready cDNA1.0 μ l, 3U/ μ l Taq enzyme 0.5 μ l, ddH
2O14.1 μ l amounts to 20 μ l.
As for carrying out the PCR reaction in the PCR instrument, the PCR program is: 94 ℃ of denaturation 5min with centrifuge tube; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 40s, 35 circulations; 72 ℃ are extended 5min; 10 ℃ stop.
Amplified production carries out electrophoresis detection with 1.2% sepharose, the results are shown in Figure 2.Behind the DNA of TransGenBiotech company recovery test kit (Easy Pure Quick Gel Extraction Kit) recovery 3 ' RLM-RACE product, to reclaim fragment is connected on the pMD 18-T carrier (the precious biotech firm in Dalian), transformed competence colibacillus intestinal bacteria TOP10 (TransGenBiotech company), carry out blue hickie screening, the extracting waste bacterial plaque is carried out pcr amplification and is identified, chooses positive colony check order (worker company is given birth in Shanghai).Sequencing result shows: 3 ' end sequence length is 476bp, nucleotide sequence SEQ ID NO:2.
(3) 5 ' RLM-RACEs
Adopt the BD SMART of Clontech company
TMRACE cDNA Amplification Kit test kit carries out 5 ' RLM-RACE, according to the test kit specification sheets, grow up total RNA of functional leaf of the camomile of extracting as the reverse transcription primer, take step 1 with 5 ' CDS primer carries out synthetic the first chain cDNA (5 '-RACE-Ready cDNA) of reverse transcription as template;
In the centrifuge tube of 200 μ L, add total RNA of the adult functional leaf of 5 ' CDS primer, 1 μ L BD SMARTIIAoligo and 3 μ L camomiles of 1 μ L, after slight concussion shakes up, behind 70 ℃ of water-bath sex change 2min, be put in immediately and cool off 2min in the ice chest; The of short duration centrifugal composition that makes is assembled the pipe end; Then add 2 μ l, 5 * First-Strand Buffer, 1 μ l DTT (20mM), 1 μ ldNTP mixture (10mM), 1 μ l BD PowerScript Reverse Transcriptase, totally 10 μ l behind the mixing, heat 90min in 42 ℃; After adding 100 μ L Tricine-EDTABuffer, in 72 ℃ of heating 7min, the first chain cDNA that obtains synthesizing (5 '-RACE-Ready cDNA), be stored in-20 ℃ and descend for subsequent use;
As template, as the PCR primer, carry out 5 ' RLM-RACE with the ClTUA-5RACE-GSP1 primer with the first synthetic chain cDNA (5 '-RACE-Ready cDNA), wherein,
The PCR reaction system is: 2.5 μ l 5 '-RACE-Ready cDNA, 1 μ l ClTUA-5RACE primer, 5 μ l10 * BD Advantage 2PCR Buffer, 1 μ l10mM dNTP Mix, 1 μ l50 * BD Advantage 2Polymerase Mix, 34.5 μ l PCR-Grade Water, totally 50 μ l;
The PCR reaction conditions is: at 94 ℃ of denaturation 5min; Loop parameter is: front 5 circulations: 94 ℃ of sex change 30s, and 70 ℃ of annealing 30s, 72 ℃ are extended 3min; Rear 20 circulations: 94 ℃ of sex change 30s, 65 ℃ of annealing 30s, 72 ℃ are extended 3min; Obtain 5 ' RLM-RACE product.
Amplified production carries out electrophoresis detection with 1.2% sepharose, the results are shown in Figure 3.Behind the DNA of TransGenBiotech company recovery test kit (Easy Pure Quick Gel Extraction Kit) recovery 5 ' RLM-RACE product, to reclaim fragment is connected on the pMD18-T carrier, transformed competence colibacillus intestinal bacteria TOP10, carry out blue hickie screening, the extracting waste bacterial plaque is carried out pcr amplification and is identified, chooses positive colony check order (worker company is given birth in Shanghai); Sequencing result shows: 5 ' end sequence length is 753bp, nucleotide sequence SEQ ID NO:3.
(4) EST complete sequence assembly unit
Use the DNAMAN biosoftware to carry out comparison, the translation of sequence, complete sequence assembly unit; After the assembly unit of DNAMAN software, obtained camomile α-tubulin homologous gene, called after ClTUA gene (SEQ IDNO:4), full length gene is 1580bp, 449 amino acid of open reading frame 1350bp coding wherein, and 5 ' UTR and 3 ' UTR be long 81bp and 149bp respectively.
5, the homogenic cDNA total length amplification of camomile α-tubulin
According to assembly unit obtain camomile α-tubulin full length gene sequences Design total length amplimer is as follows:
ClTUA-fullsequenceS：5′-TTCACTCTTTTACCCAACTCTCTTT-3′
ClTUA-fullsequenceX：5′-TCCCCCCTCCACCTCCCCCTAACAG-3′
Camomile take the synthetic preparation of step 2 grows up functional leaf strand cDNA as template, carries out pcr amplification take above-mentioned sequence as primer, and camomile α-tubulin homogenic cDNA total length amplification reaction system and response procedures are as shown in table 3.
The homogenic cDNA total length of table 3 camomile α-tubulin pcr amplification reaction program
The PCR response procedures is: as for carrying out the PCR reaction in the PCR instrument, the PCR program is: 94 ℃ of denaturation 5min with centrifuge tube; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s, 35 circulations; 72 ℃ are extended 5min; 10 ℃ stop.
Amplified production carries out electrophoresis detection with 1.2% sepharose, the results are shown in Figure 4; Behind the full amplified production of the DNA of TransGenBiotech company recovery test kit (Easy Pure Quick Gel Extraction Kit) recovery, to reclaim fragment is connected on the pMD18-T carrier, transformed competence colibacillus intestinal bacteria TOP10, carry out blue hickie screening, the extracting waste bacterial plaque is carried out pcr amplification and is identified, chooses positive colony check order (worker company is given birth in Shanghai).
Sequencing result shows: extension increasing sequence length is 1580bp, and nucleotides sequence is classified SEQ ID NO:4 as.Comparison result shows that the nucleotide sequence of the full length sequence of amplification and the complete sequence that the assembly unit of DNAMAN software obtains is identical, namely obtains camomile α-tubulin gene, called after ClTUA gene.
The RT-PCR expression analysis of test example camomile ClTUA gene, camomile Actin gene and 26S rRNA gene
Get the various camomile materials of the preparation in above-mentioned embodiment " preparation of each growth phase experiment material of the camomile " step, extract total RNA of each camomile test materials according to operation steps described in the step 1 in the test method " extraction of the total RNA of camomile material ", carry out the reverse transcription of RNA according to operation steps described in the step 2 " reverse transcription of camomile RNA ", synthetic the first chain cDNA that obtains various camomile materials, respectively take the first chain cDNA of various camomile materials as template, carry out the RT-PCR amplification, respectively to the α of the camomile among the present invention-tubulin gene (ClTUA gene), camomile Actin gene and 26S rRNA gene carry out the RT-PCR expression analysis, wherein:
(1) amplimer
The primer of A:ClTUA gene amplification is:
Upstream primer: 5 '-TTTCGTCATACGCTCCTGTA-3 '
Downstream primer: 5 '-TGGATGGTTCTCTTGGTCTT-3 '
The primer of B:Actin gene amplification is:
Upstream primer: ClActins:5 '-AGGCTGGATTTGCTGGAGATG-3 '
Downstream primer: ClActinx:5 '-CGACCTTGATCTTCATGCTGC-3 '
The primer of C:26S rRNA gene amplification is:
Upstream primer: Cl26Ss:5 '-ACGGCACTTGCACATGGGTTAG-3 '
Downstream primer: Cl26Sx:5 '-ACTGAGTCGTTTCCAGGGTGGG-3 '
(2) reaction system of gene amplification is response procedures
Amplification program and the reaction system of A:Actin gene are as shown in table 4
The pcr amplification reaction program of table 4 camomile Actin gene
The PCR step is: add successively 2.0 μ l10 * PCR buffer in centrifuge tube, the dNTP of 0.4 μ l10mM, 10 μ M ClActins primers, 1.0 μ l, 10 μ M ClActinx primers, 1.0 μ l, the first chain cDNA (template) of various camomile materials, 3U/ μ l Taq enzyme 0.5 μ l, ddH
2O14.1 μ l amounts to 20 μ l.As for carrying out the PCR reaction in the PCR instrument, the PCR program is: 94 ℃ of denaturation 5min with centrifuge tube; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 50s, 28 circulations; 72 ℃ are extended 5min; 10 ℃ stop.
Amplification program and the reaction system of B:26S rRNA gene are as shown in table 5
The pcr amplification reaction program of table 5 camomile 26S rRNA gene
The PCR step is: add successively 2.0 μ l10 * PCR buffer in centrifuge tube, the dNTP of 0.4 μ l10mM, 10 μ M Cl26Ss primers, 1.0 μ l, 10 μ M Cl26Sx primers, 1.0 μ l, the first chain cDNA (template) of various camomile materials, 3U/ μ l Taq enzyme 0.5 μ l, ddH
2O14.1 μ l amounts to 20 μ l.As for carrying out the PCR reaction in the PCR instrument, the PCR program is: 94 ℃ of denaturation 5min with centrifuge tube; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 50s, 28 circulations; 72 ℃ are extended 5min; 10 ℃ stop.
C: identical described in the step 2 of the amplification program of camomile ClTUA gene and reaction system and embodiment.
The amplified production of various camomile materials carries out electrophoresis detection with 1.2% sepharose, and the result is respectively shown in Fig. 5-11.
Electrophoresis result figure shows:
1, camomile Actin, 26S rRNA and ClTUA gene coerce at salt stress, Whitfield's ointment, arid, coldly coerce, all can consistence express under the adverse environmental factor that heat stress and ABA coerce, illustrates that the adversity gene that these three genes all can be used for analyzing camomile expresses;
2, camomile Actin gene is consistent with the expression amount in the petal at spire, stem section, the functional leaf of growing up, short day inducer blade, bud, but the expression amount in seed, old and feeble blade and root system is lower, illustrates that it is in each growth and development stage unstable expression of camomile; In addition, camomile Actin gene expression abundance in spire, stem section, petal, bud is very high, and the accuracy that causes easily expressing quantitative result when the target gene abundance is low reduces;
3, camomile 26S rRNA gene is not expressed in the camomile seed, expression amount is lower in spire, short day inducer blade, petal, and high abundance is expressed in stem section, bud, old and feeble blade and root system, camomile 26S rRNA gene is described in each growth and development stage unstable expression of camomile, is not suitable as reference gene and uses;
4, camomile ClTUA gene equal energy stably express in above-mentioned all camomile materials, and the expression amount abundance state that do not reach capacity, illustrate that the ClTUA gene is constitutive gene, can be used as the reference gene of gene expression analysis on each growth and development stage of camomile, the expression analysis result is more accurate.
5, select plural reference gene to do with reference to the test-results that helps to obtain more accurately when carrying out gene expression analysis, camomile ClTUA gene will help the accuracy when carrying out the Dendranthema gene expression analysis.
Claims (3)
1. a camomile [Chrysanthemum lavandulifolium (Fisch.ex Trautv.) Makino] reference gene, it is characterized in that being: its base is shown in the SEQ ID NO:4.
2. gene claimed in claim 1 is characterized in that as the purposes of the reference gene of genetic expression in the research camomile resistance: described resistance is that salt stress-resistant, anti-Whitfield's ointment are coerced, drought-resistantly coerced, anti-ly coldly coerce, heat resistanceheat resistant is coerced or anti-ABA coerces.
3. gene claimed in claim 1 is as the purposes of the reference gene of genetic expression in the research camomile biological development process.
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| Chunsun Gu等.Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress.《Mol Biotechnol》.2011,第49卷(第2期),192-197. * |
| Sumei Chen等.Analysis of Expressed Sequence Tags (ESTs) Collected from the Inflorescence of Chrysanthemum.《Plant Mol Biol Rep》.2009,第27卷(第4期),503-510. * |
| 彭玉华等.野生植物资源甘菊的研究进展.《中国园艺文摘》.2010,第26卷(第10期),63-65. * |
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