CN106282167A - A kind of cloning expression method of Semen avenae nudae TIFY9 gene - Google Patents
A kind of cloning expression method of Semen avenae nudae TIFY9 gene Download PDFInfo
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Abstract
The invention provides a kind of Semen avenae nudae TIFY9 gene cloning and expression method, specifically providing and obtain the reverse transcription PCR method of TIFY9 gene cDNA, the PCR method of TIFY9 gene and the expression of TIFY9 gene from Semen avenae nudae RNA reverse transcription, screening varieties and breeding for next step provide useful selection.
Description
Technical field
The present invention relates to gene clone and field of protein expression, specifically disclose clone and the expression of TIFY9 gene.
Background technology
TIFY is distinctive gene family in a newfound plant of class, and this family gene is owing to containing in coding protein sequence
There is TIF (F/Y) the XG aminoacid sequence of high conservative, so being named as TIFY family.Current research shows TIFY class base
Because receiving the abduction delivering of the abiotic stress such as mechanical damage, ozone.
In arabidopsis, have been found that JAZ10/TIFY9 can be coerced and jasmonic abduction delivering by wound.In Oryza sativa L.
TIFY family gene has 20 members, wherein has 9 to be induced by jasmonic, and almost all can be non-by such as salt, arid, low temperature etc.
Biotic is induced, and this shows that there is close relationship in TIFY family, for including TIFY9 with saline and alkaline, arid etc.
TIFY gene family carries out physiological Study and the agricultural production tool that in-depth study is coerced for the salt tolerant of plant, mechanical damage
There is important meaning.
Although from the beginning of 2007, people have carried out certain grinding for the TIFY family gene in arabidopsis and Oryza sativa L.
Studying carefully, but the research in this important cereal crops of Semen avenae nudae still belongs to blank, this carries out gene clone and albumen to TIFY9
Expression will provide great convenience for the research in Semen avenae nudae of this gene, the Zengcheng for these cereal crops provides useful side
Help.
Summary of the invention
The purpose of the present invention includes:
A kind of reverse transcriptional PCR method of Semen avenae nudae TIFY9 gene cDNA is provided；
Provide the PCR method of a kind of TIFY9 gene；
A kind of recombinant expression plasmid pET28a-TIFY9 is provided；
The preparation method of a kind of recombinant expression plasmid pET28a-TIFY9 is provided；
The expression of a kind of recombinant expression plasmid pET28a-TIFY9 is provided.
Concrete, the invention discloses a kind of reverse transcriptional PCR method of Semen avenae nudae TIFY9 gene cDNA, comprise the following steps:
(1) Semen avenae nudae 320 and/or the total serum IgE of the happiness 8 seed fresh blades of Seedling strain are extracted；
(2) use Reverse Transcription box to carry out reverse transcription operation, use 20uL reaction system: add in microcentrifugal tube
The total serum IgE 5uL that step (1) is extracted, adds the Oligo (dT) of 5 × RT Reaction Mix, 1.4uL of 4uL, 1uL
The 8.6uL of TUREscript H-RTase/RI Mix and 8.6uL, mixing, centrifugal；
(3) 42 DEG C are heated 50 minutes, and 65 DEG C are heated 15 minutes；
(4) cDNA is obtained ,-20 DEG C of preservations.
Wherein, step (1) uses Plant RNA reagent box to extract total serum IgE；Step (2) uses Reverse Transcription box to carry out instead
Transcribe.
Present invention also offers the PCR method of a kind of TIFY9 gene, comprise the following steps:
(1) reaction system as shown in table 1, adds TIFY9 gene cDNA and primer 1, primer 2 in microcentrifugal tube,
The nucleotide sequence of primer 1 and primer 2 is respectively as shown in sequence table SEQ NO ID.1, SEQ NO ID.2；
(2) PCR reaction condition: enter circulation after 95 DEG C of 5min denaturations；Cycling condition is: 94 DEG C 1 minute, 58 DEG C 1 point
Clock, 72 DEG C 1 minute；After circulating 30 times, extend 10 minutes in 72 DEG C.
Table 1 PCR reaction system
Additionally, present invention also offers a kind of recombinant expression plasmid pET28a-TIFY9, its nucleotide sequence such as sequence table
Shown in SEQ NO ID.5.
Meanwhile, present invention also offers the preparation method of a kind of recombinant expression plasmid pET28a-TIFY9, including following step
Rapid:
(1) TIFY9 gene BamHI Yu Xhol that pET28a carrier and amplification obtain carries out double digestion, and enzyme action condition is such as
Shown in table 2；
Table 2 pET28a carrier and the enzyme action condition of TIFY9 gene
(2) take pET28a carrier digestion products and TIFY9 gene digestion products that step (1) obtains, carry out 1% fine jade respectively
Use glue to reclaim test kit after lipolysaccharide electrophoresis and purpose fragment is cut glue recovery；
(3) take 0.2mL sterilizing Eppendorf pipe one, be separately added into pET28a that step (2) obtains (+) enzyme action reclaims
Fragment 4.5 μ l, TIFY9 enzyme action reclaim fragment 0.5 μ l, T4DNA ligase buffer 5.0 μ l, 16 DEG C connect overnight.
On the other hand, present invention also offers the expression of a kind of recombinant expression plasmid pET28a-TIFY9, including with
Lower step:
(1) recombinant expression plasmid pET28a-TIFY9 is converted expression Host Strains BL21 (DE3) competent cell；To simultaneously
Extract empty carrier pET28a (+) convert express Host Strains BL21 (DE3) make positive control；BL21 (DE3) competent cell is not
Conversion group makees negative control；
(2) BL21 being transformed into pET28a-TIFY9 of the upper growth of picking LB solid medium (containing Kan100ug/mL)
(DE3) colony inoculation is in 5mL LB fluid medium (containing Kan100ug/mL)；
(3) 37 DEG C of shaken cultivation to OD600 be about 0.5 time, take 1mL and be inoculated in the LB liquid training containing Kan in 1:100 ratio
Support in base；
(4) the 100mL culture fluid of cultivation being divided into 2 parts, a copy of it adds IPTG abduction delivering, and another part is not added with IPTG and makees
For comparison；Adding IPTG to final concentration of 1mmol/L, rotating speed is 220rpm, continues 25 DEG C and cultivates 16h；
(5) bacterium solution 1mL after IPTG induction is taken, centrifugal, abandon supernatant, after precipitation brine, add 100ul physiology
Saline and 5 × SDS sample-loading buffer 25ul, 100 DEG C are boiled 10min, centrifugal, take supernatant and carry out SDS-PAGE；To not inducing bacterium
Liquid does same process, observes expression；Simultaneously by unconverted for LB liquid culture and empty carrier pET28a (+) BL21 that converts makees
For comparison.
Present invention firstly discloses the method extracting RNA clone's TIFY9 gene from highland barley plant, provide according to the present invention
Method can effectively obtain and express TIFY9 gene, breed of variety and screening for next step provide useful selection.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
On the premise of the present invention above-mentioned basic fundamental thought, it is also possible to make the amendment of other various ways, replace or change.
The specific implementation method of form by the following examples, is doing the most specifically the foregoing of the present invention
Bright, but the scope that should not be construed as the above-mentioned theme of the present invention is only limitted to Examples below.All real based on foregoing of the present invention institute
Existing technology belongs to the scope of the present invention.
Accompanying drawing explanation
Fig. 1 digestion verification experimental patterns
M:DNA molecular weight marker
BamHI Yu the XhoI double digestion product of 1:pET28-TIFY9
Fig. 2 a, Fig. 2 b PET28-TIFY9 sequencing result and the sequence alignment of known genes of interest
14 TIFY9 10020455 are genome TIFY9 gene
14-1 T7 is recombinant expression plasmid Pet28a-TIFY9
The SDS-PAGE spectrum of Fig. 3 pET28a-TIFY9 protein expression in BL21
M: standard protein molecular weight
1: the Pet28a-TIFY9 bacterium containing carrier is not induced
2: induce containing carrier Pet28a-TIFY9 bacterium
3: induce supernatant containing carrier Pet28a-TIFY9 bacterium
4: carrier Pet28a-TIFY9 bacterium induced precipitation
It it is below detailed description of the invention
Oneth RNA extraction and cDNA reverse transcription PCR
1.RNA extracting and reverse transcription
1.1 with Semen avenae nudae 320 and like 8 germination growth seedlings
1.2 take fresh blade plant extract test kit extracting RNA and detect,
1.3 extraction steps are as follows:
1.3.1 instrument and reagent
Key instrument: SCILOGEX D3024R centrifuge, analytikjena-Easycycler；Scientz-48 high pass
Amount tissue grinder instrument；
Main agents: Reverse Transcription box (TUREscript 1st Stand cDNA SYNTHESIS Kit) (Ai De
Come, article No. PC1802)；2 × premixed liquid (Germany DBI pcrMix).
1.3.2 experimental technique and step
1.3.2.1 the extraction of RNA in tissue samples
RNA extracts test kit and uses jena innuPREP RNA Mini Kit.
1.3.2.2 the synthesis (reverse transcription) of cDNA
Aidlab company Reverse Transcription box (TUREscript 1st Stand cDNA SYNTHESIS Kit) is used to enter
Row reverse transcription operates, employing 20uL reaction system (each reaction 500ul system is for follow-up PCR):
Reverse transcription reaction condition is as follows:
After reaction terminates, obtain cDNA ,-20 DEG C of preservations.
The PCR amplification of 2.GIFY9 gene
2.1 reaction systems as shown in table 1, add TIFY9 gene cDNA and primer 1, primer 2 in microcentrifugal tube,
The nucleotide sequence of primer 1 and primer 2 is respectively as shown in sequence table SEQ NO ID.1, SEQ NO ID.2；
2.2 PCR reaction conditions: enter circulation after 95 DEG C of 5min denaturations；Cycling condition is: 94 DEG C 1 minute, 58 DEG C 1 point
Clock, 72 DEG C 1 minute；After circulating 30 times, extend 10 minutes in 72 DEG C.
Table 1 PCR reaction system
3.PCR product electrophoresis and editor
3.1 instruments and reagent
Key instrument: power supply, electrophresis apparatus, imaging system.
Main agents: agarose, 500ml 1XTAE, marker, Gold view.
3.2 experimental techniques and step:
3.2.1 prepare 1% agarose gel: weigh 0.5g agarose and be placed in conical flask, add 50ml1 × TAE. electromagnetism
Stove heated and boiled is all melted to agarose for 3 times, shakes up, 1.0% agarose gel liquid.
3.2.2 prepared by offset plate: takes lucite inside groove (glue groove) wash clean in electrophoresis tank, dries, put into glue glass
Glass plate. inside groove is placed in horizontal level, and puts comb well in fixed position. the agarose gel liquid being cooled to about 65 DEG C is mixed
Even pour into carefully on inside groove glass plate, make glue slowly launch, until whole glass pane surface forms uniform glue-line. under room temperature
Stand until gel solidifies (30min) completely, the gentliest pull out comb, take off adhesive tape, gel and inside groove are put in electrophoresis tank. add
Adding 1 × TAE electrophoretic buffer was not to having offset plate.
3.2.3 sample-adding: respectively sample is added with 10ul micropipettor in the sample sulculus of offset plate (2ul), often add
One sample, should change a feed head, with anti-pollution, does not break the gel face around sample well during sample-adding.
3.2.4 electrophoresis: the gel slab after sample-adding is energized immediately and carries out electrophoresis, and voltage 130V, 13min, sample is (black by negative pole
Color) move to positive pole (red) direction.
3.2.5 observing photograph: observe under uviol lamp, DNA existence then demonstrates red fluorescence band, uses gel imaging
System is taken pictures preservation.
Second: reclaim PCR primer order-checking sub-clone abduction delivering, and the structure of recombinant strains
1. the recovery of genes of interest DNA fragmentation and qualification
1.1 the PCR primer purification of TIFY9 gene target DNA fragment reclaims
With 10g/L agarose gel electrophoresis separation pcr amplification product, gel reclaims test kit and reclaims purpose fragment.Step
As follows:
(1) smash to pieces after cutting the agarose containing purpose fragment, by weight/volume ratio 1:3 (agarose weight: NE combines liquid
Volume) add NE combine liquid.Liquid is combined as 200mg agarose adds 600mL NE；
(2) 50～60 DEG C of water-baths 3～5min, glue melts completely, can turn upside down therebetween 2～3 times；
(3) solution is proceeded in centrifugal purification post, stand 5min make DNA fully be combined with pellosil, 13000r/min from
The heart 10～20 seconds, outwell the waste liquid in collecting pipe；
(4) adding 80% ethanol of 500 μ l in centrifugal purification post, 13000r/min is centrifuged 10～20 seconds, outwells collection
Waste liquid in pipe；
(5) step (4) is repeated；
(6) 13000r/min recentrifuge 1min, removes residual ethanol as far as possible；
(7) centrifugal purification post is placed in new centrifuge tube, placement 2～3min of uncapping, makes ethanol be evaporated completely；
(8) by aseptic TE solution 50 DEG C preheating, in centrifugal purification post, add 40～50 μ l, stand 3～5min, make eluting
Pellosil is fully impregnated with by liquid；
(9) 13000r/min is centrifuged 1min, and solution at the bottom of pipe is required DNA.
The qualification of 1.2 genes of interest
Use this area conventional method, TIFY9 genes of interest is checked order, the nucleotide sequence of genes of interest such as sequence table
Shown in SEQ NO ID.3.
Disclosed in prior art, TIFY9 gene order is as shown in sequence table SEQ NO ID.4
Through sequence alignment, obtain high-purity through PCR method of the present invention, only had the TIFY9 gene of a few sudden change.
2 prepare competent cell BL21 (DE3)
(1) one single bacterium colony of picking (diameter 2-3mm) from 37 DEG C of flat boards cultivating 16～20h, forwards one to and contains
In 100mL LB culture medium, after 37 DEG C of violent shaken cultivation 2h, survey OD600 to about 0.5 every 30min；
(2) antibacterial is transferred to one aseptic, in ice-cold 50mL polypropylene tube, make culture be cooled to 0 DEG C；
(3) it is centrifuged 10min in 4 DEG C with 4000r/min, to reclaim cell；
(4) pour out culture fluid, pipe is inverted 1min, so that last trace culture fluid flows to end；
(5) every 50mL initial incubation liquid 20mL, 0.1mol/L CaCl2 and 10mL, 0.1mol/L MgCl2 is put on ice
Put 30min, the resuspended every part of precipitation of solution, put 10min in mixture of ice and water；
(6) it is centrifuged 10min in 4 DEG C with 4000r/min, to reclaim cell；
(7) pour out supernatant, pipe is inverted 1min, so that last trace supernatant flows to end；
(8) every 50mL initial incubation liquid 2mL ice-cold 0.1mol/L CaCl2 solution resuspended every part of cell precipitation.
Cell preserves 12～24h at 4 DEG C, to improve transformation efficiency.
More than operate and the most aseptically carry out.
3. recombinant expression plasmid pET28a (+) preparation of-TIFY9, identify and express
3.1 will containing plasmid pet128 (+) strain culturing after, extract plasmid, and with BamHI and XhoI double digestion, enzyme
The system of cutting is shown in Table 2, and 37 DEG C of effect 2h, gel reclaims test kit and reclaims target DNA.
TIFY9 gene PCR purification is reclaimed product BamHI and XhoI double digestion, and enzyme action system is shown in Table 4-5,37 DEG C of works
With 2h, gel reclaims test kit and reclaims target DNA.
Table 2 plasmid and the endonuclease reaction system of PCR primer
4 enzyme action recovery product pET28a (+) reclaim connection and the qualification of fragment with TIFY9 enzyme action
4.1 connect:
Take 0.2mL sterilizing Eppendorf pipe one, the pET28a being separately added in step 3 (+) enzyme action reclaim fragment with
TIFY9 enzyme action reclaims fragment, and after adding each composition by table 3,16 DEG C connect overnight, obtain recombinant expression plasmid pET28a-TIFY9.
Table 3 coupled reaction system
4.2 order-checkings and qualification
Use digestion verification that genes of interest is verified, the qualification result of digestion verification experiment, see Fig. 1.Restriction enzyme mapping
Display, genes of interest obtains two bands of TIFY9 and 28a after BamHI Yu XhoI double digestion, it was demonstrated that pET28-TIFY9 carrier
Successfully construct.
Recombinant expression plasmid pET28a-TIFY9 is checked order, and TIFY9 gene order known in the art compares
Right, shown in control experiment result as Figure of description 2, the sequence that clone obtains is consistent with aim sequence.
5 abduction deliverings
5.1 recombinant expression plasmid pET28a-TIFY9 converts expresses Host Strains BL21 (DE3) competent cell.To carry simultaneously
The empty carrier pET28a that takes (+) convert and express Host Strains BL21 (DE3) and make positive control；BL21 (DE3) competent cell does not turns
Change group makees negative control.
The abduction delivering of 5.2 recombiant proteins
The condition that abduction delivering uses is 0.1mM IPTG, 25 DEG C, 16h.
The cultivation of 5.3 recombinant strains and expression
The BL21 being transformed into pET28a-TIFY9 of the upper growth of picking LB solid medium (containing Kan100ug/mL)
(DE3) colony inoculation is in 5mL LB fluid medium (containing Kan100ug/mL)；
37 DEG C of shaken cultivation to OD600 be about 0.5 time, take 1mL and be inoculated in the LB liquid culture containing Kan in 1:100 ratio
In base；
The 100mL culture fluid of cultivation is divided into 2 parts, and a copy of it adds IPTG abduction delivering, and another part is not added with IPTG conduct
Comparison.Add IPTG to final concentration of 1mmol/L, continue shaken cultivation
Change IPTG concentration, inducing temperature and incubation time and obtain optimal abduction delivering condition.
Take bacterium solution 1mL after IPTG induction, centrifugal, abandon supernatant, after precipitation brine, add 100ul physiology salt
Water and 5 × SDS sample-loading buffer 25ul, 100 DEG C are boiled 10min, centrifugal, take supernatant and carry out SDS-PAGE.To not inducing bacterium solution
Do same process, observe expression.
Simultaneously using unconverted for LB liquid culture and empty carrier pET28a (+) BL21 that converts is as comparison.
6. the SDS-PAGE electrophoresis detection of recombinant expression plasmid engineering bacterium expression albumen
(1) Vertial electrophorestic tank is illustratively installed, it is ensured that the sealing adhesive strip sealing to notch.Size according to destination protein and
Electrophoresis tank specification determines separation gel and the concentration concentrating glue and volume；
(2) configuring the separation gel of desired concn, speed is injected into device of gel, reserves perfusion and concentrates glue requisite space, uses
Suction pipe is carefully separating one layer of water of glue surface overlying lid, is disposed vertically at room temperature by gel；
(3), after separation gel polymerization completely, an obvious boundary line occurs between gel and water, pours out cover layer liquid,
Residual liquid is blotted with filter paper；
(4) preparing the concentration glue of desired concn, on the separation gel being polymerized, perfusion concentrates glue, and comb is inserted concentration
In glue, should avoid producing bubble, gel is disposed vertically at room temperature, be polymerized about 45min；
(5), after concentrating glue polymerization completely, comb is carefully taken out.Gel is fixed on electrophoresis tank, upper and lower groove all irrigates
Tris-glycine running buffer；
(6) it is loaded in a predetermined order, turns on the power, constant voltage electrophoresis.First with 60V low-voltage electrophoresis, when dye front enters
After separation gel, voltage is adjusted to 120V, continues electrophoresis until bromophenol blue arrives bottom separation gel；
(7) take out gel after electrophoresis, rinse the electrophoretic buffer on clean glue with deionized water, gel is put into fixing
Liquid is fixed 15min, dyes with Coomassie brilliant blue dye liquor, in 65 DEG C of dyeing 15～20min, then shake with destaining solution de-gently
Color 3～5h, observes protein band and finds, occurs in intended molecular weight 24kDa position significantly inducing band, protein induced table
Reach merit.SDS-PAGE spectrum (sds polyacrylamide gel electrophoresis collection of illustrative plates) is shown in Fig. 3.
Claims (7)
1. a reverse transcriptional PCR method for Semen avenae nudae TIFY9 gene cDNA, comprises the following steps:
(1) Semen avenae nudae 320 and/or the total serum IgE of the happiness 8 seed fresh blades of Seedling strain are extracted；
(2) use Reverse Transcription box to carry out reverse transcription operation, use 20uL reaction system: in microcentrifugal tube, add step
(1) the total serum IgE 5uL extracted, adds the Oligo (dT) of 5 × RT Reaction Mix, 1.4uL of 4uL, 1uL
The 8.6uL of TUREscript H-RTase/RI Mix and 8.6uL, mixing, centrifugal；
(3) 42 DEG C are heated 50 minutes, and 65 DEG C are heated 15 minutes；
(4) cDNA is obtained ,-20 DEG C of preservations.
Method the most according to claim 1, it is characterised in that step (1) uses Plant RNA reagent box to extract total serum IgE.
Method the most according to claim 1, it is characterised in that step (2) uses Reverse Transcription box to carry out reverse transcription.
4. a PCR method for TIFY9 gene, comprises the following steps:
(1) reaction system as shown in table 1, adds TIFY9 gene cDNA and primer 1, primer 2, primer 1 in microcentrifugal tube
With the nucleotide sequence of primer 2 respectively as shown in sequence table SEQ NO ID.1, SEQ NO ID.2；
Table 1 PCR reaction system
(2) PCR reaction condition: enter circulation after 95 DEG C of 5min denaturations；Cycling condition is: 94 DEG C 1 minute, 58 DEG C 1 minute,
72 DEG C 1 minute；After circulating 30 times, extend 10 minutes in 72 DEG C.
5. a recombinant expression plasmid pET28a-TIFY9, its nucleotide sequence is as shown in sequence table SEQ NO ID.5.
6. a preparation method of recombinant expression plasmid pET28a-TIFY9, comprises the following steps:
(1) TIFY9 gene BamHI Yu Xhol that pET28a carrier and amplification obtain carries out double digestion, enzyme action condition such as table 2 institute
Show；
Table 2 pET28a carrier and the enzyme action condition of TIFY9 gene
(2) take pET28a carrier digestion products and TIFY9 gene digestion products that step (1) obtains, carry out 1% agarose respectively
Use glue to reclaim test kit after electrophoresis and purpose fragment is cut glue recovery；
(3) take 0.2mL sterilizing Eppendorf pipe one, be separately added into pET28a that step (2) obtains (+) enzyme action reclaims fragment
4.5 μ l, TIFY9 enzyme action reclaim fragment 0.5 μ l, T4DNA ligase buffer 5.0 μ l, 16 DEG C connect overnight.
7. an expression of recombinant expression plasmid pET28a-TIFY9, comprises the following steps:
(1) recombinant expression plasmid pET28a-TIFY9 is converted expression Host Strains BL21 (DE3) competent cell；To extract simultaneously
Empty carrier pET28a (+) convert express Host Strains BL21 (DE3) make positive control；BL21 (DE3) competent cell does not converts
Group makees negative control；
(2) BL21 (DE3) being transformed into pET28a-TIFY9 of the upper growth of picking LB solid medium (containing Kan100ug/mL)
Colony inoculation is in 5mL LB fluid medium (containing Kan100ug/mL)；
(3) 37 DEG C of shaken cultivation to OD600 be about 0.5 time, take 1mL and be inoculated in the LB fluid medium containing Kan in 1:100 ratio
In；
(4) the 100mL culture fluid of cultivation being divided into 2 parts, a copy of it adds IPTG abduction delivering, and another part is not added with IPTG as right
According to；Adding IPTG to final concentration of 1mmol/L, rotating speed is 220rpm, continues 25 DEG C and cultivates 16h；
(5) bacterium solution 1mL after IPTG induction is taken, centrifugal, abandon supernatant, after precipitation brine, add 100ul normal saline
And 5 × SDS sample-loading buffer 25ul, 100 DEG C are boiled 10min, centrifugal, take supernatant and carry out SDS-PAGE；Do not inducing bacterium solution
Same process, observes expression；Simultaneously using unconverted for LB liquid culture and empty carrier pET28a (+) BL21 that converts is as right
According to.
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