CN107475780A - Structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress - Google Patents
Structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress Download PDFInfo
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Abstract
The present invention relates to biology field, structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress are specifically disclosed.Method comprises the following steps：(1) Pomacea canaliculata low temperature stress is handled；(2) Pomacea canaliculata sample treatment；(3) Pomacea canaliculata total tissue RNA is extracted；(4) reverse transcription synthesis cDNA；(5) cDNA is purified；(6) cDNA digestions and purifying；(7) joint connects；(8) subtractive hybridization；(9) subtractive PCR identifications and purifying；(10) PCR primer is connected with pMD18 carrier Ts；(11) connection product conversion escherichia coli DH5a；(12) library is identified；Pomacea canaliculata SSH libraries under low temperature stress constructed by the inventive method, laid the foundation for a large amount of expressing genes for finding and studying biological invasion Pomacea canaliculata.Meanwhile SSH libraries of the invention have important theory and practical significance for the resistance to inverse mechanism for furtheing investigate biological invasion Fu Shou class.
Description
Technical field
The present invention relates to biology field, more particularly to the structure in Pomacea canaliculata SSH libraries under a kind of low temperature stress,
Authentication method and application.
Background technology
Pomacea canaliculata (Pomacea) also known as Ampullaria gigas, apple spiral shell, it is international pernicious harmful organism.The spiral shell originates in south
America Amazon River basin, phase at the beginning of the eighties in last century introduce Asia, and because of mismanagement and taste is bad is lost one's parents；Pomacea canaliculata is fast
Speed is diffused into field, and some areas are overflowed, and serious harm is caused to aquatic crops such as rice, wild rice stems.Pomacea canaliculata has
Extremely strong reproductive capacity, diffusion and rate of propagation are fast, turn into the serious agricultural pest of most of provinces and regions on the south the Changjiang river at present.
Pomacea canaliculata has been widely distributed in most of provinces and regions on the south 30 ° of China's north latitude now, including Zhejiang, Fujian, Guangdong, Hainan, wide
West, Yunnan, Guizhou, Hunan, Jiangxi, Chongqing, Sichuan, Anhui Province etc., population density is very huge, agricultural production is caused seriously
Influence.
In phagocytic process, due to the change of invasion ground ambient environmental conditions, Pomacea canaliculata can be by different environment stresses
(such as high temperature, freezing, drying, hyperosmosis and oxidation).In order to resist these environment stresses, biology is during evolution
Some cleverly protection mechanisms are gradually formed, to maintain their annidation or ensure that they can be in unfavorable conditions
Under survive.In order to adapt to environmental change in face of adverse circumstance different reactions can occur for Pomacea canaliculata, typically by gene table
The reconstruction that reaches and change its physiological status, include synthesis and the stress protein of molecular chaperones (molecular chaperones)
Synthesis, such as heat shock protein (Heat shock protein, Hsp), cold shock protein (Cold shock protein, Csp) and
Antifreeze protein (Antifreeze protein, AFP) etc., or change enzymatic activity, protein and membrane structure state.
Suppressed subtractive hybridization (Suppression Subtractive Hybridization, SSH) technology is
Diatchenko etc. established in 1996, and the Technology application hybridization kineticses principle, the i.e. single-stranded cDNA of high abundance are in annealing
The speed for producing homologous hybridization is faster than low-abundance single-stranded cDNA, in the cDNA of test group (tester) and driving group (driver)
After denaturation during repeatability, so that differentiated single-stranded cDNA relative amounts reached basically identical in abundance originally.
Simultaneously as the cDNA of test group two parts of decile will produce 5 before being hybridized added with different joints, therefore when hybridizing
Kind different types of molecule, when entering performing PCR amplification using the complementary primer of joint sequences different from two, only target sequence
Effectively expanded, non-targeted sequence is also easy to produce the structure of similar " panhandle ", nothing because both ends have an inverted repeats, during annealing
Method is matched with primer, and amplification is suppressed.1996, since Diatchenko etc. reports SSH technologies, the technology extensively should
For in field of insect research, be mainly used in studying difference grow period and histoorgan, different genotype insect and
Changes in gene expression analysis under stressful environmental.
At present, report is not had no also using expression conditions of the SSH technical research Pomacea canaliculata under low temperature stress.Cause
This, establish the structure in Pomacea canaliculata SSH libraries under a kind of low temperature stress, authentication method to study biological invasion Pomacea canaliculata invasion machine
System has great importance.
The content of the invention
Present invention aim to address problems of the prior art, and provide Pomacea canaliculata SSH under a kind of low temperature stress
Structure, the authentication method in library.The present invention is specifically to adopt the following technical scheme that realization：
Structure, the authentication method in Pomacea canaliculata SSH libraries, its step are as follows successively under low temperature stress：
(1) by Pomacea canaliculata at a temperature of 6 ± 0.2 DEG C Stress treatment 1h hours；
(2) take the fresh Pomacea canaliculata tissue 50mg~100mg of (1) Stress treatment to be rinsed 3~5 times with DEPC water, put rapidly
Preserved in liquid nitrogen；
(3) under the conditions of liquid nitrogen by Pomacea canaliculata tissue grinder into fine-powdered, be then transferred into containing 0.3~0.5mL cells
In the centrifuge tube of lysate, the total serum IgE of Pomacea canaliculata tissue is extracted；
(4) total serum IgE of extraction in (3) is synthesized to the cDNA of Pomacea canaliculata tissue by the reverse transcription of SMART methods；
(5) cDNA obtained in (4) is purified；
(6) digestion and purifying are carried out to the cDNA obtained in (5)；
(7) joint connection is carried out to the cDNA obtained in (6)；
(8) subtractive PCR identifications and purifying are carried out to the cDNA obtained in (7)；
(9) PCR primer obtained in (8) is connected with pMD18-T carriers；
(10) connection product in (9) is converted into escherichia coli DH5a；
(11) library is identified.
Above steps is preferably realized in the following way：
The cDNA of described SMART methods reverse transcription synthesis Pomacea canaliculata tissue comprises the following steps：
4.1) total serum IgE, the A of 1 μ L, 3 ' SMART CDS Primer II described in 1-3.5 μ L are added in PCR pipe, then
4.5 μ L are complemented to DEPC water, are mixed, centrifugation；
4.2) by PCR pipe in 70 DEG C of warm bath 3min, then in 42 DEG C of warm bath 2min；
4.3) reaction mixture is prepared, each reaction dosage includes in its agent formulations：2μL5×Frist-Strand
Buffer、0.25μL 100mM DTT、1μL 10mM dNTP Mix、1μL 12μm SMARTer II A
Oligonucleotide, 0.25 μ L RNase Inhibitor and 1 μ L 100U SMARTScribe Reverse
Transcriptase；
4.4) RNA/primer mixed liquors are added in the reaction mixture described in 5.5 μ L in each pipe, are mixed, instantaneously from
The heart；
4.5) 42 DEG C of warm bath 1.5h are subsequently placed in；
4.6) 70 DEG C of warm bath 10min, terminating reaction are placed in again；
4.7) plus 40 μ L water are into each pipe, mix, brief centrifugation, obtain cDNA the first chain reaction dilutions；
4.8) the μ L of the first chain reactions of cDNA dilution 10 are taken to insert in PCR reaction tubes；
4.9) build reaction mixture, in mixture system agent formulations include 74 μ L deionized waters, 10 μ L10 ×
Advantage 2PCR Buffer, 2 50 × dNTP of μ L 10mM, 2 μ L, 12 μm of 5 ' PCR Primer II A and 2 μ L 50 ×
Advantage 2Polymerase Mix, mix, centrifugation；
4.10) during 4.8) reaction mixture in 4.9) is added in PCR reaction tubes, mix；
4.11) PCR reaction tube mixed liquors are placed in the enterprising performing PCR amplified reaction of PCR instrument again, PCR amplification conditions are：95℃
1min；21 circulations：95℃15s,65℃30s,68℃6min；Reaction is placed in -20 DEG C of refrigerators and preserved after terminating.
The described A sequences of 3 ' SMART CDS Primer II are：
5'-AAGCAGTGGTATCAACGCAGAGTACT(20)N-1N-3', N=A, C, G, or N-1=A, G, or C；
Described SMARTer II A Oligonucleotide sequences are：
5'-AAGCAGTGGTATCAACGCAGAGTA-3'；
5 ' described PCR Primer II A sequences are：5'-CTAATACGACTCACTATAGGG-3'.
Endonuclease reaction system includes in step 6)：43.5μL ds cDNA、5μL 10×Rsa I restriction
Buffer and 1.5 μ L 10U/ μ L Rsa I.
Described joint coupled reaction step is as follows：
7.1) the Tester cDNA for taking 1 μ L Rsa I to digest, add in 5 μ L aqua sterilisas and be diluted；Prepare 5 parts of companies
The Master Mix connect, each of which, which is reacted in dosage, includes 3 μ L Sterile H2O, 2 μ 5 × Ligation of L Buffer and
1μL 400U/μL T4DNA Ligase；Mix, centrifugation；
7.2) positive abatement and the connector interfaces system reversely cut down are established；The connector interfaces system of forward direction abatement includes
Two groups of Tester 1-1 and Tester 2-1, Tester 1-1 groups include the Tester after being diluted in 2 μ L steps 7.1)
CDNA, 2 μ L, 10 μM of Adaptor 1, the Master Mix described in 6 μ L；Tester 2-1 groups include dilute in 2 μ L steps 7.1)
The Master Mix described in Tester cDNA, 2 μ L10 μM Adaptor 2R, 6 μ L after releasing；The connector interfaces reversely cut down
Also two groups of Tester 1-1 and Tester 2-1 is included in system, Tester 1-1 groups are included after being diluted in 2 μ L steps 7.1)
Tester cDNA, 2 μ L, 10 μM of Adaptor 1, the Master Mix described in 6 μ L；Tester 2-1 groups include 2 μ L steps
The Master Mix described in Tester cDNA, 2 μ L, 10 μM of Adaptor 2R, 6 μ L after rapid 7.1) middle dilution；It is wherein positive
The Tester cDNA of abatement are with the cDNA, the Tester cDNA reversely cut down of the Pomacea canaliculata of 6 ± 0.2 DEG C of low temperature stress processing
For with the cDNA of the Pomacea canaliculata of 25 ± 0.2 DEG C of processing；
7.3) each Tester pipes are stayed overnight in 16 DEG C of connections of PCR instrument, 1 μ L EDTA/ is added within second day into reaction system
Glycogen terminating reactions；Make connection enzyme killing in 72 DEG C of reaction 5min in PCR instrument；After centrifugation, -20 DEG C are stored in, for making
For the template of subtractive PCR identifications.
Described subtractive PCR identifications and purification step are as follows：
8.1) carry out the identification of first round PCR：
Prepare PCR reaction solutions, each reaction dosage includes 19.5 μ L Sterile H in reaction solution system2O、2.5μL 10
× PCR reaction buffer, 0.5 μ L 10mM dNTP, 1.0 μ L, 10 μM of PCR Primer 1,1.0 μ L templates, 0.5 μ L
50×Advantage Cdna Polymerase Mix；Mix, centrifugation；
PCR pipe is placed in PCR instrument and reacted as follows：75℃5min；94℃25s；27cycles:94℃10s,66℃
30s,72℃1.5min；
3 μ L PCR primers are taken, are added in the 0.5ml centrifuge tubes containing 27 μ L aqua sterilisas, concussion mixes, and centrifuges, and is
The template of second wheel PCR reactions；
8.2) the second wheel PCR identifications are carried out：
Prepare PCR Master Mix, each react in dosage includes 18.5 μ L Sterile H2O、2.5μL 10×PCR
reaction buffer、0.5μL 10mM dNTP、1.0μL 10μM Nested PCR Primer 1、1.0μL 10μM
Nested PCR Primer 2R, 1.0 μ L templates, 0.5 μ L 50 × Advantage Cdna Polymerase Mix；Mix,
Centrifugation；Mixed liquor is subjected to following PCR reactions：12cycle:94℃10s,68℃30s,72℃1.5min.
The described sequences of PCR Primer 1 are：5'-CTAATACGACTGTAGGGCTAT-3'.
The described sequences of Nested PCR Primer 1 are：5'-TCGAGCGGCCGCCCCTGGACG-3'；Described
Nested PCR Primer 2R sequences are：5'-AGCGTGGTCGCGGCCGT-3'.
It is as follows that connection product converts the step of escherichia coli DH5a：
The connection product in 5-10 μ L steps (9) is taken to be added in the DH5a competent cells that 100 μ L dissolve on ice, on ice
Place 30min after by mixed liquor in 42 DEG C of water-baths heat shock 1.5min, be then transferred quickly on ice, place 2min；Then
900 μ L are added in the LB fluid nutrient mediums of 37 DEG C of preheatings, 1h is incubated under the conditions of 37 DEG C, 160-200rpm；Then at 4000rpm from
Heart 5min, cell is resuspended after abandoning supernatant；50-100 μ L transformed bacteria solutions are taken to be coated on Amp containing 100mg/L, IPTG and X-gal
Resistance LB square positions, 37 DEG C are incubated overnight, blue hickie screening positive clone.
The beneficial effects of the invention are as follows：
(1) sample size is few needed for, it is not necessary to which mRNA is individually purified from total serum IgE can build SSH libraries.
(2) present invention is quick, effective, repeatability is strong, and the SSH libraries of structure are the weights to fresh water mollusk cDNA library
Supplement.
(3) Pomacea canaliculata is important Strategy of Alien Invasive Species, and the present invention contributes to the flooding mechanism and diffusion road to Pomacea canaliculata
The research in footpath, also provide help for the research of its controlling way.
Brief description of the drawings
Electrophoretogram after Fig. 1 double-strands cDNA synthesis, wherein, M Marker；
Electrophoresis detection figure after Fig. 2 double-strand cDNA digestions；
The agarose gel electrophoresis detection of Fig. 3 subtractive PCR primers；
The electrophoresis detection figure of Fig. 4 Insert Fragments identification.
Embodiment
The present invention is further elaborated and illustrated with reference to the accompanying drawings and detailed description.
Embodiment 1：The structure in Pomacea canaliculata SSH libraries under low temperature stress
Structure, the authentication method in Pomacea canaliculata SSH libraries mainly include the following steps that under low temperature stress:
(1) by Pomacea canaliculata at a temperature of 6 ± 0.2 DEG C Stress treatment 1h.
(2) the fresh Pomacea canaliculata tissue 50mg of Stress treatment is taken, is rinsed 3 times with DEPC water, is immediately placed in liquid nitrogen and preserves.
(3) Pomacea canaliculata total tissue RNA is extracted：Under the conditions of liquid nitrogen then Pomacea canaliculata tissue grinder is shifted into fine-powdered
Into the centrifuge tube containing 0.3-0.5mL cell pyrolysis liquids；Extract the total serum IgE of Pomacea canaliculata tissue.
(4) cDNA of Pomacea canaliculata tissue is synthesized by the reverse transcription of SMART methods.
The chains of cDNA first synthesize：
A, 0.2mL PCR pipes are taken by following preparation RNA/primer mixed liquors：
Mix, gently centrifuge.
Wherein：3’SMART CDS PrimerⅡA：
5'-AAGCAGTGGTATCAACGCAGAGTACT(20)N-1N-3', N=A, C, G, or N-1=A, G, or C；
B, the warm bath 3min at 70 DEG C, the warm bath 2min at 42 DEG C；
C, according to the form below prepares reaction mixture：
SMARTer II A Oligonucleotide(12μm)：5'-AAGCAGTGGTATCAACGCAGAGTA-3'
D, each tube RNA/primer mixed liquors add 5.5 μ L reaction mixture mixed liquors in, gently mix, instantaneously from
The heart；
E, in 42 DEG C of warm bath 1.5h.
F, then at 70 DEG C of warm bath 10min, terminating reaction.
G, plus 40 μ L water are to sample tube, gently mix, brief centrifugation.CDNA the first chain reaction dilutions are obtained, in -20 DEG C
Saved backup in refrigerator.
The synthesis of the chains of cDNA second：
H, the μ L of the first chain reaction of above-mentioned steps (g) dilution 30 are taken to insert in 1.5mL reaction tubes.
I, according to following system construction reaction mixture：
Mix, somewhat centrifuge.
Wherein：5’PCR Primer II A：5'-CTAATACGACTCACTATAGGG-3'
J, above-mentioned i reaction mixture is added in step (h) 1.5mL reaction tubes, mixes, be averagely dispensed into three
In 0.2mL PCR pipe, A pipes, B pipes and C pipes are respectively labeled as.
K, above-mentioned three pipes mixed liquor is placed in PCR instrument and carries out following reactions：Reaction condition is as follows：95℃1min；21
Circulation：95 DEG C of 15s, 65 DEG C of 30s, 68 DEG C of 6min, reaction are placed in -20 DEG C of refrigerators and preserved after terminating.
Fig. 1 show the 1.5% agarose electrophoresis figure of c DNA after amplification, it is seen that 500bp-3000bp sizes, brightness are different
Smear.M is molecular weight standard.
(5) cDNA purifying is carried out.
(6) cDNA digestions and purifying：
To 2 samples (Tester, Driver) while operated：
A, by following system construction reaction system
B, in 37 DEG C of warm bath 1.5h, of short duration centrifugation.
C, 5 μ L are taken to be stored in -20 DEG C, with electrophoretic analysis later.
D, 2.5 μ L 0.5M EDTA terminating reactions are added.
E, 50 μ L phenol are added：Chloroform：Isoamyl alcohol (volume ratio 25：24：1).
F, oscillator fully shaking mixes, 14000rpm room temperatures centrifugation 10min.
G, supernatant is transferred in new 0.5mL pipes, adds 50 μ L chloroforms：Isoamyl alcohol (volume ratio 24：1).
H, oscillator fully shaking mixes, 14000rpm room temperatures centrifugation 10min.
I, supernatant is transferred in new 0.5mL pipes, adds 25 μ L4M NH4OAc and the ethanol of 187.5 μ L 95%.
G, oscillator fully shaking mixes, 14000rpm room temperatures centrifugation 10min.
K, supernatant is carefully abandoned, the ethanol cleaning of the μ L 80% of addition 200 gently.
L, in 14000rpm, room temperature centrifugation 5min.
M, supernatant is carefully abandoned, is air-dried 10 minutes.
N, dried object adds 6.7 μ L water dissolving, takes out 1.2 μ L solution and is transferred to the 1.5mL centrifuge tubes containing 11 μ L water
In be diluted.Remaining -20 DEG C of 5.5 μ L solution saves backup.
Fig. 2 show the 1.5% agarose electrophoresis figure of cDNA after digestion, it is seen that 500bp-2000bp sizes, brightness are different
Smear.M is molecular weight standard.
(7) joint connects：
Joint coupled reaction step is as follows：
1) the Tester cDNA for taking 1 μ L Rsa I to digest, add in 5 μ L aqua sterilisas and be diluted.Forward direction abatement
Tester cDNA are with the cDNA of the Pomacea canaliculata of 6 ± 0.2 DEG C of low temperature stress processing, and the Tester cDNA reversely cut down are with 25
The cDNA of the Pomacea canaliculata of ± 0.2 DEG C of processing；
2) prepare the Master Mix of connection, mix, of short duration centrifugation.
3) above-mentioned mixed liquor configures 5 parts, after the completion of place on ice it is standby.
4) connector interfaces system is established according to following table：
Wherein：The Tester cDNA of dilution 1) Diluted tester cDNA are respectively adopted in.
5) each Tester pipes are stayed overnight in 16 DEG C of connections of PCR instrument, 1 μ L EDTA/ is added within second day into reaction system
Glycogen terminating reactions.
Make connection enzyme killing in 72 DEG C of reaction 5min in PCR instrument.
6) after somewhat centrifuging, -20 DEG C are stored in.
(8) subtractive PCR identifications and purifying, the first round pcr template as follow-up subtractive PCR identifications.
First round PCR：
A, PCR reaction solutions are prepared according to following system, a brief period of time mixes, of short duration centrifugation
B, the mixed liquor of above-mentioned each PCR pipe is covered into 50 μ L atoleines, is placed in PCR instrument and is reacted as follows：75
℃5min；94℃25s；27cycles:94℃10s,66℃30s,72℃1.5min；
C, the 8 above-mentioned PCR primers of μ L are taken, 2.0% agarose electrophoresis checks result.
D, the 3 above-mentioned PCR primers of μ L are taken, are added in the 0.5ml centrifuge tubes containing 27 μ L aqua sterilisas, concussion mixes, of short duration
Centrifugation.The template of as second wheel PCR reactions.
Second wheel PCR：
Epicycle pcr template is the first round cut back in above-mentioned steps (d)
E, PCR Master Mix according to the form below prepares, fully mix, of short duration centrifugation.Prepare a Master more
Mix。
F, above-mentioned mixed liquor is subjected to following PCR reactions：
12cycle:94℃10s,68℃30s,72℃1.5min；
G, the 8 above-mentioned PCR primers of μ L are taken, 2.0% agarose electrophoresis checks result.
Fig. 3 is secondary PCR product electrophoresis detection figure, and wherein M is Marker.
(9) PCR primer is connected with pMD18-T
PCR primer in (8) after purification is carried out to the connection of carrier according to following reaction system：
Reaction condition：16 DEG C of connection 3-5h.
(10) connection product conversion escherichia coli DH5a
The step is carried out in the following order successively：
A, the connection product in 5-10 μ L steps (9) is taken to be added in the DH5a competent cells dissolved on ice (100 μ L)；
B, place 30min on ice (time can not be lacked)；
C, by mixed liquor in 42 DEG C of water-baths heat shock 1.5min；(not stirred during heat shock)
D, and then it is transferred quickly on ice, place 2min；
E, 900 μ L are added in the LB fluid nutrient mediums of 37 DEG C of preheatings, in 37 DEG C, 1h is incubated under the conditions of 160-200rpm；
F, 4000rpm centrifuges 5min, abandons 800 μ L of supernatant, cell is then resuspended；
G, the resistance LB for taking appropriate (50-100 μ L) transformed bacteria solution to be coated on Amp containing 100mg/L, IPTG and X-gal is put down
Disk, 37 DEG C are incubated overnight, blue hickie screening positive clone.
Fig. 4 is library colony growth situation.
Identify in the library of embodiment 2：
(1) Insert Fragment length is identified
On from the flat board in positive library and reverse library, 24 clones of picking carry out bacterium colony PCR, bacterium colony pcr template respectively
Manufacturing process is as follows:
A, with sterilizing toothpick from the flat board in positive and negative library, random 24 bacterium colonies of picking of difference；
B, thalline is suspended from 100 μ L ddH20, pipettor piping and druming is mixed, and 20min is placed in -20 DEG C of refrigerators；
C, 95-100 DEG C of heating 10min makes bacterial cell disruption；
D, 4,000rpm centrifuges 5min, and supernatant can be used as the use of bacterium colony pcr template；
E, bacterium colony PCR reaction system is as follows:
F, above-mentioned mixed liquor is subjected to following PCR reactions:94℃10min；30cycles:94℃10s,55℃30s,72℃
1.5min；72℃5min
G, electrophoresis detection result.
Fig. 4 is the electrophoresis detection figure of Insert Fragment identification, is followed successively by the PCR productions of the 1-24 clone selected from left to right
Thing.
(2) library capacity is identified
Need to identify library capacity after library construction success, to determine total clone's number (CFU).
The identification of library capacity is calculated according to following formula.
N：Monoclonal number (individual) in flat board；
M:The bacterium solution volume (μ L) being coated on flat board；
V：Library bacterium solution cumulative volume (μ L)；
100 μ L transformed bacteria solution is taken to be coated on the LB flat boards of the section penicillin of ammonia containing 100mg/L, 37 DEG C are incubated overnight.Number
The bacterium colony number gone out on flat board, it is possible to it is determined that calculating library capacity.
It is computed, constructed Pomacea canaliculata capacity is 8400.
Claims (9)
1. structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress, it is characterised in that step is as follows successively：
(1) by Pomacea canaliculata at a temperature of 6 ± 0.2 DEG C Stress treatment 1h hours；
(2) take the fresh Pomacea canaliculata tissue 50mg~100mg of (1) Stress treatment to be rinsed 3~5 times with DEPC water, be immediately placed in liquid
Preserved in nitrogen；
(3) under the conditions of liquid nitrogen by Pomacea canaliculata tissue grinder into fine-powdered, be then transferred into and cracked containing 0.3~0.5mL cells
In the centrifuge tube of liquid, the total serum IgE of Pomacea canaliculata tissue is extracted；
(4) total serum IgE of extraction in (3) is synthesized to the cDNA of Pomacea canaliculata tissue by the reverse transcription of SMART methods；
(5) cDNA obtained in (4) is purified；
(6) digestion and purifying are carried out to the cDNA obtained in (5)；
(7) joint connection is carried out to the cDNA obtained in (6)；
(8) subtractive PCR identifications and purifying are carried out to the cDNA obtained in (7)；
(9) PCR primer obtained in (8) is connected with pMD18-T carriers；
(10) connection product in (9) is converted into escherichia coli DH5a；
(11) library is identified.
2. according to the method for claim 1, it is characterised in that described SMART methods reverse transcription synthesis Pomacea canaliculata tissue
CDNA comprises the following steps：
4.1) total serum IgE, the A of 1 μ L, 3 ' SMART CDS Primer II described in 1-3.5 μ L are added in PCR pipe, then uses DEPC
Water complements to 4.5 μ L, mixes, centrifugation；
4.2) by PCR pipe in 70 DEG C of warm bath 3min, then in 42 DEG C of warm bath 2min；
4.3) reaction mixture is prepared, each reaction dosage includes in its agent formulations：2μL5×Frist-Strand
Buffer、0.25μL 100mM DTT、1μL 10mM dNTP Mix、1μL 12μm SMARTer II A
Oligonucleotide, 0.25 μ L RNase Inhibitor and 1 μ L 100U SMARTScribe Reverse
Transcriptase；
4.4) RNA/primer mixed liquors are added in the reaction mixture described in 5.5 μ L in each pipe, are mixed, brief centrifugation；
4.5) 42 DEG C of warm bath 1.5h are subsequently placed in；
4.6) 70 DEG C of warm bath 10min, terminating reaction are placed in again；
4.7) plus 40 μ L water are into each pipe, mix, brief centrifugation, obtain cDNA the first chain reaction dilutions；
4.8) the μ L of the first chain reactions of cDNA dilution 10 are taken to insert in PCR reaction tubes；
4.9) build reaction mixture, in mixture system agent formulations include 74 μ L deionized waters,
10μL10×Advantage 2PCR Buffer、2μL 10mM 50×dNTP、2μL 12μm 5’PCR PrimerII A
With 2 μ L 50 × Advantage 2Polymerase Mix, mix, centrifugation；
4.10) during 4.8) reaction mixture in 4.9) is added in PCR reaction tubes, mix；
4.11) PCR reaction tube mixed liquors are placed in the enterprising performing PCR amplified reaction of PCR instrument again, PCR amplification conditions are：95℃
1min；21 circulations：95℃15s,65℃30s,68℃6min；Reaction is placed in -20 DEG C of refrigerators and preserved after terminating.
3. according to the method for claim 1, it is characterised in that the described A sequences of 3 ' SMART CDS Primer II are：
5'-AAGCAGTGGTATCAACGCAGAGTACT(20)N-1N-3', N=A, C, G, or N-1=A, G, or C；
Described SMARTer II A Oligonucleotide sequences are：5'-AAGCAGTGGTATCAACGCAGAGTA-3'；
5 ' described PCR Primer II A sequences are：5'-CTAATACGACTCACTATAGGG-3'.
4. according to the method for claim 1, it is characterised in that endonuclease reaction system includes in step 6)：43.5μL ds
CDNA, 5 μ 10 × Rsa of L I restriction buffer and 1.5 μ L 10U/ μ L Rsa I.
5. according to the method for claim 1, it is characterised in that described joint coupled reaction step is as follows：
7.1) the Tester cDNA for taking 1 μ L Rsa I to digest, add in 5 μ L aqua sterilisas and be diluted；Prepare 5 parts of connections to use
Master Mix, each of which reaction dosage in includes 3 μ L Sterile H2O, 2 μ 5 × Ligation of L Buffer and 1 μ L
400U/μL T4DNA Ligase；Mix, centrifugation；
7.2) positive abatement and the connector interfaces system reversely cut down are established；The connector interfaces system of forward direction abatement includes
Two groups of Tester 1-1 and Tester 2-1, Tester 1-1 groups include the Tester after being diluted in 2 μ L steps 7.1)
CDNA, 2 μ L, 10 μM of Adaptor 1, the Master Mix described in 6 μ L；Tester 2-1 groups include dilute in 2 μ L steps 7.1)
The Master Mix described in Tester cDNA, 2 μ L, 10 μM of Adaptor 2R, 6 μ L after releasing；The joint connection reversely cut down
Also two groups of Tester 1-1 and Tester 2-1 is included in system, Tester 1-1 groups are included after being diluted in 2 μ L steps 7.1)
Tester cDNA, 2 μ L, 10 μM of Adaptor 1, the Master Mix described in 6 μ L；Tester 2-1 groups include 2 μ L
The Master Mix described in Tester cDNA, 2 μ L, 10 μM of Adaptor 2R, 6 μ L after being diluted in step 7.1)；Wherein just
It is with the cDNA, the Tester reversely cut down of the Pomacea canaliculata of 6 ± 0.2 DEG C of low temperature stress processing to the Tester cDNA of abatement
CDNA is with the cDNA of the Pomacea canaliculata of 25 ± 0.2 DEG C of processing；
7.3) each Tester pipes are stayed overnight in 16 DEG C of connections of PCR instrument, 1 μ LEDTA/ is added within second day into reaction system
Glycogen terminating reactions；Make connection enzyme killing in 72 DEG C of reaction 5min in PCR instrument；After centrifugation, -20 DEG C are stored in, for making
For the template of subtractive PCR identifications.
6. according to the method for claim 1, it is characterised in that described subtractive PCR identifications and purification step are as follows：
8.1) identification of first round PCR is carried out：
Prepare PCR reaction solutions, each reaction dosage includes 19.5 μ L Sterile H in reaction solution system2O、2.5μL 10×PCR
Reaction buffer, 0.5 μ L 10mM dNTP, 1.0 μ L, 10 μM of PCR Primer 1,1.0 μ L templates, 0.5 μ L 50 ×
Advantage Cdna Polymerase Mix；Mix, centrifugation；
PCR pipe is placed in PCR instrument and reacted as follows：75℃5min；94℃25s；27cycles:94℃10s,66℃30s,
72℃1.5min；
3 μ L PCR primers are taken, are added in the 0.5ml centrifuge tubes containing 27 μ L aqua sterilisas, concussion mixes, centrifugation, and as second
Take turns the template of PCR reactions；
8.2) the second wheel PCR identifications are carried out：
Prepare PCR Master Mix, each react in dosage includes 18.5 μ L Sterile H2O、2.5μL 10×PCR
reaction buffer、0.5μL 10mM dNTP、1.0μL 10μM Nested PCR Primer 1、1.0μL 10μM
Nested PCR Primer 2R, 1.0 μ L templates, 0.5 μ L 50 × Advantage Cdna Polymerase Mix；Mix,
Centrifugation；Mixed liquor is subjected to following PCR reactions：12cycle:94℃10s,68℃30s,72℃1.5min.
7. according to the method for claim 6, it is characterised in that the described sequences of PCR Primer 1 are：5'-
CTAATACGACTGTAGGGCTAT-3'。
8. according to the method for claim 6, it is characterised in that the described sequences of Nested PCR Primer 1 are：5'-
TCGAGCGGCCGCCCCTGGACG-3'；Described Nested PCR Primer 2R sequences are：5'-
AGCGTGGTCGCGGCCGT-3'。
9. according to the method for claim 1, it is characterised in that it is as follows that connection product converts the step of escherichia coli DH5a：
Take the connection product in 5-10 μ L steps (9) to be added in the DH5a competent cells that 100 μ L dissolve on ice, place on ice
After 30min by mixed liquor in 42 DEG C of water-baths heat shock 1.5min, be then transferred quickly on ice, place 2min；Then add
900 μ L are incubated 1h in the LB fluid nutrient mediums of 37 DEG C of preheatings under the conditions of 37 DEG C, 160-200rpm；Centrifuged then at 4000rpm
5min, cell is resuspended after abandoning supernatant；50-100 μ L transformed bacteria solutions are taken to be coated on the anti-of Amp containing 100mg/L, IPTG and X-gal
Property LB square positions, 37 DEG C are incubated overnight, blue hickie screening positive clone.
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