CN104694556A - Antrodia camphorate FPPS (farnesyl diphosphate synthase) protein gene as well as cloning and sequencing method thereof - Google Patents
Antrodia camphorate FPPS (farnesyl diphosphate synthase) protein gene as well as cloning and sequencing method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology and discloses an antrodia camphorate FPPS (farnesyl diphosphate synthase) protein gene as well as a cloning and sequencing method thereof. The antrodia camphorate FPPS protein gene is a synthetic FPP (farnesyl diphosphate). Particularly, the nucleotide sequence of the antrodia camphorate FPPS protein gene is shown in SEQ ID NO: 1, and polypeptide with a nucleotide sequence shown in SEQ ID NO: 2 is coded by the nucleotide sequence of the antrodia camphorate FPPS protein gene. The invention further provides the cloning and sequencing method of the antrodia camphorate FPPS protein gene, which comprises the following steps: tissue separation, separation of total RNA, cloning of Full-length cDNA and sequencing. A sequencing result is analyzed through molecular biological software, a phylogenetic tree is constructed, and the nucleotide sequence and the type of the antrodia camphorate FPPS protein gene can be verified.
Description
Technical field
The invention belongs to gene engineering technology field, particularly a kind of Antrodia camphorata FPPS protein gene and Cloning and sequencing method thereof.
Background technology
Farnesyl pyrophosphoric acid synthetase (farnesyl diphosphate synthase, FPPS) short chain allyl group transferring enzyme (prenyltransferase) family is belonged to, this enzyme is the homodimer of 78-80kDa, catalysis geranyl tetra-sodium (geranyl diphosphate in terpene synthesis, GPP) with isopentenyl pyrophosphate (diphosphate, or dimethylallylpyrophosphate (dimethylallyl diphosphate IPP), DMAPP) condensation becomes farnesyl pyrophosphate (farnesyl diphosphate, FPP), this enzyme is a key enzyme in terpene route of synthesis, its catalysis end product farnesyl pyrophosphate (farnesyl diphosphate, FPP) be that many important secondary metabolites are as steroidal, dolichol, participate in the biosynthetic precursor of the ubiquinone of Mitochondrial electron transmission etc., simultaneously under the effect of sesquiterpene cyclase, FPP can also generate the sesquiterpene derivative comprising plant protecting chemical.
Triterpenes is the natural compounds that nature is often found, and the kind of terpene, more than more than 40000, is prevalent in vegitabilia and mycota, and they play the part of very important role in the growth of plant and microorganism and defence.Vegeto-animal terpene, by one-level pathways metabolism, is the key precursor thing of synthesis steroid compound, main relevant with the physiological regulation function of cell, is that cell maintains the very important biochemical substances of Homeostasis function.Except physiological regulating control function, terpene is also relevant with the defense function of organism.Monoterpene (monoterpenoids), sesquiterpene (sesquiterpenoids), diterpene (diterterpenoids), sesterterpene (sesterpenoids), and triterpene (triterpenoids) all belongs to secondary metabolism thing, the metabolite of these tool bitter tastes resists excessively swallowing of insect usually with plant or mushroom relevant, some terpene then has the Antibiotic activity suppressing microorganism growth, organism can be helped to support antimicrobial invasion and attack, therefore terpene plays the part of very important role in the ecology of plant or mushroom, also be the equilibrium function due to these ecologies, terpene is made to become very important medicinal ingredients source.Triterpene compound is terpene chemical composition maximum in camphor tree sesame sporophore, wherein nearly kind more than 10 by camphor tree sesame peculiar.Experiment confirms, the effects such as these triterpene compounds have anticancer, hypotensive.
As secondary metabolite, triterpene compound is by mevalonate pathway, is produced MF59 by method ester tetra-sodium, is being changed into 2,3-oxidosqualene, eventually passes a series of oxidation, reduction and cyclization are formed.Farnesyl pyrophosphoric acid synthetase (farnesyl diphosphate synthase, FPPS) first tapping point in mevalonate pathway is in, a key enzyme in isoprenoid biosynthetic process, the content of FPPS and active height can affect triterpenoid output number.
Farnesyl pyrophosphoric acid synthetase as a kind of important transferring enzyme, current FPPS enzyme gene separating clone from the plants such as capsicum, paddy rice, Arabidopis thaliana, corn, tree cotton, guayule, sagebrush, peppermint, Herba Artemisiae annuae, lupine.And understand few at present at mushroom class terpene substance metabolism route of synthesis, Antrodia camphorata is precious medicinal fungi, especially containing more terpene chemical composition, is well suited for the research carrying out terpene substances metabolism route of synthesis.The present invention is with reference to other species FPPS gene order, cloning and sequencing obtains the cDNA sequence of Antrodia camphorata FPPS gene, and tetraploid rice is carried out to the CDS sequence of different plant species FPPS gene, be intended to understand and the constructional feature of checking FPPS gene, for studying FPPS gene from now on and mushroom class terpene metabolic pathway of synthesizing provides basic data.
Summary of the invention
One object of the present invention is to provide a kind of Antrodia camphorata FPPS protein gene.
Another object of the present invention is the Cloning and sequencing method providing above-mentioned Antrodia camphorata FPPS protein gene.
For solving the problems of the technologies described above, embodiments of the present invention provide a kind of Antrodia camphorata FPPS protein gene, this gene is a kind of synthesis farnesyl pyrophosphate gene, specifically, the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, the nucleotide sequence of this gene is as shown in SEQ ID NO:1.
Meanwhile, the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, its nucleotide sequence coded polypeptide with the aminoacid sequence shown in SEQ ID NO:2.
Embodiments of the present invention also provide the Cloning and sequencing method of above-mentioned Antrodia camphorata FPPS protein gene, and the method includes the steps of:
(1) separate tissue: get Antrodia camphorata mycelia, rinses with redistilled water and removes substratum and impurity, then by organize sample put into rapidly liquid nitrogen freezing after in-70 DEG C of preservations;
(2) separation of total serum IgE: total serum IgE is extracted in frozen organizing in sample from above-mentioned steps (1), by spectrophotometric determination rna content, and adopts agarose electrophoretic analysis to carry out the qualification of total serum IgE;
(3) Cloning and sequencing of full length cDNA sequence: with the total serum IgE obtained in above-mentioned steps (2) for template, design and synthesize the reverse primer shown in the forward primer shown in SEQ ID NO:3 and SEQ ID NO:4, carry out Reverse transcription-PCR amplification, to obtain Antrodia camphorata FPPS protein gene cDNA sequence, cDNA sequence is cloned, obtain recombinant plasmid
SEQ ID NO:3：5’-ATGACCACGAAGGATGAAC-3’；
SEQ ID NO:4:TTTTTTTTTTTTTTT, i.e. Oligo (dT) 15.
(4) check order: the recombinant plasmid in above-mentioned steps (3) is checked order.
Preferably, in the Cloning and sequencing method of the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, in step (2), Trizol reagent is used to extract from during the organizing sample and extract total serum IgE of Antrodia camphorata bacterium.
Preferably, in the Cloning and sequencing method of the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, the inverse transcription reaction liquid composition of Reverse transcription-PCR amplification is carried out: damping fluid (2 × Bca 1st Buffer) 10 μ l, the MgSO of 25Mm in step (3)
44 μ l, Nucleotide (dNTP) 1 μ l, 40U/ μ l enzyme inhibitors (Rnase Inhibitor) 0.5 μ l, 22U/ μ l ThermoScript II (BcaBEST Polymerase) 1 μ l, forward primer 0.5 μ l shown in SEQ ID NO:3, reverse primer 0.5 μ l shown in SEQ ID NO:4, RNA template (RNA Sample) 1 μ l, without enzyme water (Rnase Free dH
2o) 1.5 μ l are totally 20ul.
Preferably, in the Cloning and sequencing method of the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, the reverse transcription reaction condition of carrying out Reverse transcription-PCR amplification in step (3) is: 65 DEG C 1 minute, 30 DEG C 5 minutes, at the uniform velocity heat up in 15 ~ 30 minutes, 65 DEG C 25 minutes, 98 DEG C 5 minutes, 5 DEG C 5 minutes.
Preferably, in the Cloning and sequencing method of the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, the pcr amplification reaction condition of carrying out Reverse transcription-PCR amplification in step (3) is: 97 DEG C of sex change 5 minutes; Then with 95 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 1 minute is a circulation, carry out 30 times circulation; Last 72 DEG C extend 10 minutes, preserve at 4 DEG C.
Preferably, in the Cloning and sequencing method of the Antrodia camphorata FPPS protein gene that embodiments of the present invention provide, step comprises the step that cDNA sequence is cloned in (3): reclaim DNA to Reverse transcription-PCR amplified fragments, the DNA fragmentation be recovered to is connected with carrier T, be transformed in competent cell, then carry out the PCR qualification and the cultivation that transform bacterium colony, reclaim recombinant plasmid, enzyme is carried out to the recombinant plasmid be recovered to and cuts qualification and order-checking.
The cDNA sequence of the FPPS protein gene in the present invention be by with total serum IgE in Antrodia camphorata hypohostroma for template, with reference to the homologous sequence design primer of the FPPS protein gene of the species such as glossy ganoderma, Bai Can, rainbow conk, Poria cocos, carry out reverse transcription PCR and the new gene sequence that obtains.The Antrodia camphorata FPPS protein gene cDNA sequence obtained is shown in SEQ ID NO.1.Encoding sequence (CDS) in Antrodia camphorata FPPS protein gene cDNA sequence is shown in SEQ ID NO.2 by the protein coding sequence that universal codons is translated into.
In SEQ ID NO.1, RT-PCR product length is about 1160bp, and electrophoresis result is shown in accompanying drawing 1, and wherein 23-25 position is initiator codon ATG, 1115-1117 position be terminator codon TAA, 23-1117 position is coded protein region (CDS).At SEQ ID NO.1, Antrodia camphorata FPPS protein gene coding 364 amino acid.The fungi FPPS protein gene coding amino acid sequence homology comparative results alignd in Clustal X such as Antrodia camphorata and white ginseng, glossy ganoderma, rainbow conk, Poria cocos are shown in accompanying drawing 2.To the CDS sequence construct phylogenetic tree of 14 FPPS protein genes of 14 species comprising SEQ IDNO.2, found that: FPPS albumen is nearest with the evolutionary distance of Poria cocos, the sequence that indirect proof obtains is really FPPS protein gene.
Accompanying drawing explanation
Fig. 1 is the RT-PCR product electrophorogram in embodiment 1, and wherein, swimming lane 1 is Marker:DL2000; Swimming lane 2,3,4,5 is pcr product, swimming lane 6:DL2000;
Fig. 2 is the amino acid homology comparison chart in embodiment 2;
Fig. 3 is the phyletic evolution tree graph built in embodiment 2.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.But, persons of ordinary skill in the art may appreciate that in each embodiment of the present invention, proposing many ins and outs to make reader understand the application better.But, even without these ins and outs with based on the many variations of following embodiment and amendment, each claim of the application technical scheme required for protection also can be realized.
The clone of embodiment 1 Antrodia camphorata FPPS protein gene cDNA sequence
1. separate tissue (Isolation)
No. 20, the road Antrodia camphorata substratum that makes people rich from Chen Jia town, Chongming district, Qin Shengyuan bio tech ltd, Shanghai is located in be adopted be in the Antrodia camphorata mycelia in vegetative period, uses ddH
2o rinses and removes the impurity such as substratum, by organize sample put into rapidly liquid nitrogen freezing after in-70 DEG C of preservation, bring back laboratory and prepare extraction total tissue RNA.
2. the separation (Total RNA isolation) of total serum IgE
(1) preparation work of RNA is extracted
The NaOH soaked overnight of used in glass products 0.1M, rinses repeatedly with tap water, then uses distilled water flushing 2 times, and 180 DEG C are toasted 4 hours.
Homogenizer, electrophoresis chamber with 3% hydrogen peroxide dipping 20-30 minute, then with 0.1% DEPC water rinse. due to non-deactivation DEPC on PCR reaction have impact, the SDS process of available 0.5%.
The DEPC water soaking of Tip head, EP effective 0.1% spends the night, and autoclaving makes DEPC inactivation.
Distilled water and solution DEPC process, namely every 100ml water or solution add 0.1mlDEPC liquid, left at room temperature over night, autoclaving 30 minutes DEPC inactivations.
(2) total tissue RNA is extracted
The sample of organizing getting 100mg frozen in liquid nitrogen is placed with homogenate in the homogenate organ pipe of 1ml Trizol (trizal reagent, invitrogenBRL, USA).Centrifugal 10 minutes of 4 DEG C of 12000g.Aspirate supernatant, 15-30 DEG C of incubation 5 minutes.Add 0.2ml chloroform, cover lid, with hand concuss 15 seconds.15-30 DEG C of incubation 2-3 minute.Centrifugal 15 minutes of 4 DEG C of 12000g.Aspirate supernatant, adds 0.8ml Virahol, mixes.15-30 DEG C of incubation 10 minutes, centrifugal 10 minutes of 4 DEG C of 12000g, bottom centrifuge tube, white precipitate is RNA.Outwell supernatant, add 1ml 75% washing with alcohol RNA, centrifugal 10 minutes of 4 DEG C of 7500g.Seasoning or the middle packing of vacuum-drying RNA. water-soluble (RNase free) ,-70 DEG C of preservations.
(3) qualification of total tissue RNA
By spectrophotometer measuring rna content and by the quality of agarose electrophoretic analysis RNA, concrete grammar is as follows: electrophoresis chamber RNA scavenging solution (100mM NaOH, 1mM EDTA) soak 4-5 hour, then it is stand-by to pour 1 × TBE into after repeatedly rinsing several with DEPC treated water.Sepharose 1 × TBE prepares.10ul sample-loading buffer (2 × TBE, 13% phenanthrene can, 0.1% bromjophenol blue, 7M urea) in add more than 1ul total tissue RNA, 65 DEG C of sex change put into frozen water 2-3 minute after 10 minutes immediately.Point sample is electrophoresis in sepharose.
3. the clone (Cloning of Full-length cDNA) of full-length cDNA
(1) design of primers and synthesis
According to the FPPS protein gene mRNA sequence ORF flanking homologous sequence of the species such as rainbow conk (Accession No:XM_008046437), glossy ganoderma (AccessionNo:EU399545.1), Poria cocos that GenBanK delivers, design following primer for the pcr amplification that is masterplate with Antrodia camphorata FPPS albumen cDNA, to obtain Antrodia camphorata FPPS protein gene cDNA sequence.Primer Primer Premier 5.0 designed, designed, synthesized by Sani bio tech ltd, Shanghai, the nucleotide sequence of this primer pair is respectively SEQ ID NO:3 (forward) and SEQ ID NO:4 (oppositely), and sequence information is as follows:
SEQ ID NO:3 (forward): 5 '-ATGACCACGAAGGATGAAC-3 '
SEQ ID NO:4 (oppositely): TTTTTTTTTTTTTTT, i.e. Oligo (dT) 15.
(2) RT-PCR amplification
Reverse transcription is carried out by precious biological Reverse Transcription box (the BcaBEST RNA PCR Kit Ver.1.1) requirement in Dalian.Inverse transcription reaction liquid forms: 10ul 2 × Bca 1st Buffer, 4ul 25Mm MgSO
41ul dNTPMixture, 0.5ul Rnase Inhibitor (40U/ul), 1ul BcaBEST Polymerase (22U/ul), reverse primer 0.5ul shown in forward primer 0.5ul shown in SEQ IDNO:3, SEQ ID NO:4,1ul RNASample, 1.5ul Rnase Free dH2O is totally 20ul.Reverse transcription reaction condition: 65 DEG C 1 minute, 30 DEG C 5 minutes (in 15 minutes-30 minutes at the uniform velocity heat up), 65 DEG C 25 minutes, 98 DEG C 5 minutes, 5 DEG C 5 minutes.Pcr amplification condition: 97 DEG C of sex change 5 minutes; 95 DEG C 30 seconds → 55 DEG C 30 seconds → 72 DEG C 1 minute, so carry out 30 times circulation; 72 DEG C extend 10 minutes, 4 DEG C of preservations.RT-PCR product electrophorogram as shown in Figure 1.
(3) Cloning and sequencing
The recovery of pcr amplified fragment.Fragment reclaims is undertaken by the explanation of Shanghai China Shun a small amount of glue recovery test kit, and operating process is as follows: the Agarose plug cut off containing DNA little as far as possible, puts into 1.5mlEP pipe.The ratio adding 300ulS1 liquid in every 100mg agarose adds S1 liquid, 50 DEG C of water-baths 10 minutes.The agar liquid glucose dissolved is moved into adsorption column, and centrifugal 1 minute of 10000g, outwells liquid in pipe.In adsorption column, add 500ul W1 liquid, centrifugal 15 seconds of 10000g, outwells liquid in pipe.In adsorption column, add 500ul W1 liquid, leave standstill after 1 minute, centrifugal 15 seconds of 10000g, outwells liquid in pipe.Centrifugal 1 minute.Adsorption column is put into a clean 1.5mlEP pipe, add 30ulT1 liquid in adsorption film central authorities, leave standstill after 1 minute, centrifugal 1 minute of 10000g, in EP pipe, liquid is the DNA of recovery.
Reclaim the connection of fragment with carrier T.Connect with TaKaRa pMD18-T Vector test kit, operating process is as follows: in EP pipe, prepare following ligation liquid: pMD 18-T Vector (50ng/ul) 0.5ul, DNA20-40ng, Solution 15ul, adds dH2O to 10ul.By above-mentioned reaction solution 16 DEG C of reactions, 30 minutes (also can spend the night), product is used for transformed competence colibacillus cell.
The conversion of plasmid.The competent cell of preservation is taken out, ice bath hydrotropy from-70 DEG C of refrigerators.100ul competent cell adds 10ul and connects product, ice bath 30 minutes.In 42 DEG C of water-bath heat stress 90 seconds, to insert immediately in ice bath 2 minutes.Add 400ul LB liquid medium, 37 DEG C bring back to life 50 minutes.Getting 100ul is laid on LB flat board, dull and stereotyped ammonia benzyl mould Amp (100mg/ml) containing 0.1% (V/V).37 DEG C of constant temperature culture 10-15 hour.
Transform PCR qualification and the cultivation of bacterium colony.Transform with marking pen mark the single bacterium colony cultivated, dip single bacterium colony with the toothpick of sterilizing.Dentiscalprum head is placed in the EP pipe being added with PCR reaction solution (not containing template) and rocks, make single bacterium colony serve as template.Increase by the PCR condition obtaining this fragment.Whether PCR primer carries out agarose gel electrophoresis together with Marker, differentiate in carrier T containing object fragment.If obtain object fragment; the choicest of available sterilizing rifle is taken at the single bacterium colony corresponding with it on flat board, puts into 40ml LB liquid nutrient medium, first adds the benzylpenicillin sodium (100mg/ml) of 0.1% (V/V) in a liquid; 37 DEG C spend the night shake bacterium cultivate, reclaim for plasmid.
The recovery of plasmid.Plasmid reclaims with Shanghai China Shun mini-scale plasmid extracting and purifying test kit, and operation steps is as follows: add in 1.5mlEP pipe by transforming the bacterium liquid cultivated, 4000 revs/min centrifugal, outwells liquid and obtain bacterial precipitation.A bacterium liquid can be added again centrifugal when bacterial precipitation is inadequate.In bacterial precipitation, add 250ulP1 liquid, vibration suspends.Add 250ulP2 liquid, gentleness shakes up, and room temperature leaves standstill 4 minutes.Add 350ulP3 liquid, gentleness shakes up.Centrifugal 10 minutes, supernatant liquor is carefully moved into adsorption column, centrifugal 15 seconds, outwell liquid.In adsorption column, add 500ulW1, centrifugal 15 seconds, outwell liquid.In adsorption column, add 500ulW1, leave standstill 1 minute, centrifugal 15 seconds, outwell liquid.Centrifugal 1 minute.Adsorption column is put into a clean 1.5mlEP pipe, add 25-30ulT1 liquid in adsorption film central authorities, leave standstill after 1 minute, in centrifugal 1 minute .EP pipe, liquid is the plasmid of recovery.
The enzyme of plasmid cuts qualification.In order to prove to reclaim plasmid really for inserting the recombinant plasmid of object fragment, adopting the qualification of double digestion method. this research concrete operations are as follows: scheme according to TaKaRa pMD18-T Vector, select restriction endonuclease Bam H I and Hin d III. ensure the point of contact without these two kinds of enzymes in object fragment.Endonuclease reaction system composed as follows: Bam H I 1ul, Hin d III 1ul, 10 × K Buffer 2ul, DNA≤1ul, adds dH2O to 20ul.30 DEG C are reacted 3 hours.Agarose gel electrophoresis detects.
The order-checking of recombinant plasmid.Get 20ul recombinant plasmid in EP pipe, sealed membrane seals, and gives Shanghai Sani biotech company to check order.
Embodiment 2: Antrodia camphorata FPPS protein gene coding sequence homology compares and builds with phylogenetic tree
1, Antrodia camphorata FPPS protein gene coding sequence homology compares
GenBank searches for and obtains the FPPS protein gene coding protein sequence of 13 kinds of fungies such as glossy ganoderma, yeast, Bai Zhi, brown rot fungus, Poria cocos, by the SEQ IDNO.1 sequence of its present invention's acquisition obtained with cloning and sequencing together, at molecular biology software MEGA4 enterprising line order row tetraploid rice (see accompanying drawing 2).Comparative result shows: SEQ ID NO.2 sequence and Poria cocos FPPS protein amino acid sequence homology are 80.00%.
2, there is tree structure in FPPS protein gene coding sequential system
GenBank searches for and obtains the coding protein sequence of the FPPS protein gene of 13 kinds of species such as glossy ganoderma, yeast, Bai Zhi, brown rot fungus, Poria cocos, add the SEQ ID NO.2 sequence that the present invention obtains, utilize molecular biology software MEGA4 to construct phylogenetic tree (see accompanying drawing 3).Result shows: Antrodia camphorata FPPS protein gene is nearest with the sibship of Poria cocos; The sequence that indirect proof obtains is really FPPS protein gene.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above realize specific embodiments of the invention, and in actual applications, various change can be done to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (8)
1. an Antrodia camphorata FPPS protein gene, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
2. Antrodia camphorata FPPS protein gene according to claim 1, is characterized in that, the described nucleotide sequence coded polypeptide with the aminoacid sequence shown in SEQ ID NO:2.
3. a Cloning and sequencing method for Antrodia camphorata FPPS protein gene, is characterized in that, comprise following steps:
(1) separate tissue: get Antrodia camphorata mycelia, rinses with redistilled water and removes substratum and impurity, then by organize sample put into rapidly liquid nitrogen freezing after in-70 DEG C of preservations;
(2) separation of total serum IgE: total serum IgE is extracted in frozen organizing in sample from above-mentioned steps (1), by spectrophotometric determination rna content, and adopts agarose electrophoretic analysis to carry out the qualification of total serum IgE;
(3) clone of full length cDNA sequence: with the total serum IgE obtained in above-mentioned steps (2) for template, design and synthesize the reverse primer shown in the forward primer shown in SEQ ID NO:3 and SEQ ID NO:4, carry out Reverse transcription-PCR amplification, to obtain Antrodia camphorata FPPS protein gene cDNA sequence, cDNA sequence is cloned, obtains recombinant plasmid;
(4) check order: the recombinant plasmid in above-mentioned steps (3) is checked order.
4. the Cloning and sequencing method of Antrodia camphorata FPPS protein gene according to claim 3, is characterized in that, uses Trizol reagent to extract in described step (2) from when organizing sample and extract total serum IgE.
5. the Cloning and sequencing method of Antrodia camphorata FPPS protein gene according to claim 3, it is characterized in that, consisting of of the reverse transcription reaction system of Reverse transcription-PCR amplification is carried out: 2 × Bca 1st damping fluid 10 μ l, the MgSO of 25Mm in described step (3)
4the enzyme inhibitors 0.5 μ l of 4 μ l, dNTP 1 μ l, 40U/ μ l, the ThermoScript II 1 μ l of 22U/ μ l, the forward primer 0.5 μ l shown in SEQ ID NO:3, the reverse primer 0.5 μ l shown in SEQ ID NO:4, RNA template 1 μ l, without enzyme water 1.5 μ l, is totally 20 μ l.
6. the Cloning and sequencing method of Antrodia camphorata FPPS protein gene according to claim 3, it is characterized in that, the reverse transcription reaction condition of carrying out Reverse transcription-PCR amplification in described step (3) is: 65 DEG C 1 minute, 30 DEG C 5 minutes, at the uniform velocity heat up in 15 ~ 30 minutes, 65 DEG C 25 minutes, 98 DEG C 5 minutes, 5 DEG C 5 minutes.
7. the Cloning and sequencing method of Antrodia camphorata FPPS protein gene according to claim 3, is characterized in that, the pcr amplification reaction condition of carrying out Reverse transcription-PCR amplification in described step (3) is: 97 DEG C of sex change 5 minutes; Then with 95 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 1 minute is a circulation, carry out 30 times circulation; Last 72 DEG C extend 10 minutes, preserve at 4 DEG C.
8. the Cloning and sequencing method of Antrodia camphorata FPPS protein gene according to claim 3, it is characterized in that, described step comprises the step that cDNA sequence is cloned in (3): reclaim DNA to Reverse transcription-PCR amplified fragments, the DNA fragmentation be recovered to is connected with carrier T, be transformed in competent cell, then carry out the PCR qualification and the cultivation that transform bacterium colony, reclaim recombinant plasmid, enzyme is carried out to the recombinant plasmid be recovered to and cuts qualification and order-checking.
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| CN105907768A (en) * | 2016-05-05 | 2016-08-31 | 福建农林大学 | Antrodia cinnamomea housekeeping gene sequence, qRT-PCR (quantitative real-time polymerase chain reaction) amplification primer, amplification method and screening method |
| CN105907768B (en) * | 2016-05-05 | 2019-04-09 | 福建农林大学 | Antrodia camphorata house-keeping gene sequence, qRT-PCR amplimer, amplification method and screening technique |
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