CN104342426B - A kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application - Google Patents
A kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application Download PDFInfo
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- 241001533085 Aquilaria sinensis Species 0.000 title claims abstract description 60
- 238000004114 suspension culture Methods 0.000 title claims abstract description 60
- 101710093888 Pentalenene synthase Proteins 0.000 title claims abstract description 42
- 101710115850 Sesquiterpene synthase Proteins 0.000 title claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 title abstract description 32
- 241001265525 Edgeworthia chrysantha Species 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 230000014509 gene expression Effects 0.000 abstract description 17
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 5
- 241000271309 Aquilaria crassna Species 0.000 description 20
- 239000000523 sample Substances 0.000 description 18
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- FQTLCLSUCSAZDY-UHFFFAOYSA-N (+) E(S) nerolidol Natural products CC(C)=CCCC(C)=CCCC(C)(O)C=C FQTLCLSUCSAZDY-UHFFFAOYSA-N 0.000 description 2
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- 229910020820 NaAc-HAc Inorganic materials 0.000 description 2
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- WASNIKZYIWZQIP-AWEZNQCLSA-N nerolidol Natural products CC(=CCCC(=CCC[C@@H](O)C=C)C)C WASNIKZYIWZQIP-AWEZNQCLSA-N 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
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- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical class N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- 229940005657 pyrophosphoric acid Drugs 0.000 description 1
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- 208000023504 respiratory system disease Diseases 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 235000007586 terpenes Nutrition 0.000 description 1
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- 238000010792 warming Methods 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application.Suspension culture of Aquilaria sinensis sesquiterpene synthase, its amino acid sequence is as shown in SEQ ID NO.2.Suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s SesTPS1, its nucleotide sequence is as shown in SEQ ID NO.1.Applications of the suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s SesTPS1 in the Edgeworthia chrysantha level of detection suspension culture of Aquilaria sinensis tree body.In view of the report at present on suspension culture of Aquilaria sinensis sesquiterpene synthase gene is less, therefore suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s SesTPS1 provided by the present invention and its suspension culture of Aquilaria sinensis different parts expression detection suspension culture of Aquilaria sinensis tree body in Edgeworthia chrysantha level there is important application value, to improve Edgeworthia chrysantha quality be of great practical significance.
Description
Technical field：
The invention belongs to gene biological field, and in particular to a kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene
And application.
Background technology：
Effect that agalloch eaglewood tool promoting qi circulation and relieving pain, warming middle energizer to arrest vomiting, gas of receiving are relievingd asthma, in treatment disease of digestive system, respiratory system disease
Disease, cardiovascular and cerebrovascular disease, the nervous system disease have significant curative effect, also have in terms of antitumor, wind resistance diseases caused by dampness and anti-aging
Preferably act on (Liu the army and the people, Xu Hong China Home-made occluder progress Chinese medicines, 2005,28 (7):627-632).Suspension culture of Aquilaria sinensis is passed through
Agalloch eaglewood can be formed after mechanical induction, chemical damage or fungal induction, and the main component in agalloch eaglewood includes sesquiterpenoids.
The transcript profile sequencing result of suspension culture of Aquilaria sinensis shows that suspension culture of Aquilaria sinensis sesquiterpene synthase is key enzyme (Wu Hongqing, king of agalloch eaglewood biosynthesis
Of heap of stone, He Xin, Bai Ling, Gao Xiaoxia, severe cold are quiet, Zhang Weimin suspension culture of Aquilaria sinensis sesquiterpene synthase Gene As s-SesTPS clone and biology
Informatics and expression analysis, 2014,45 (1)：94-101).Artificial agilawood is compared with natural agilawood, its main pharmacodynamics composition sesquialter
Terpenoid it is relatively low (Gao X X, Xie M R, Liu S F, Guo X L, Chen X Y, Zhong Z J, Wang L,
Zhang W M.Chromatographic fingerprint analysis of metabolites in natural and
artificial agarwood using gas chromatography-mass spectrometry combined with
chemometric methods.Journal of Chromatography B,2014,967:264-273), therefore its quality
Also it is not so good as natural agilawood, and sesquiterpene synthase is the key enzyme of sesquiterpenoids formation, therefore to suspension culture of Aquilaria sinensis sequiterpene
The clone of synthase gene and identify and can provide theoretical direction to lift artificial Edgeworthia chrysantha technology, for the quality of lifting artificial agilawood
It is of great significance.
At present, a variety of different floristic sesquiterpenoids synthase genes are by successful clone, but agalloch eaglewood sequiterpene
The biosynthesis research of synthase related gene is simultaneously few, and only clone obtains the sesquiterpene synthase gene that principal product is guaiene
(Kumeta Y,Ito M.Characterization ofδ-guaiene synthases from cultured cells of
Aquilaria,responsible for the formation of the sesquiterpenes in agarwood[J]
.Plant Physiol,2010,154(4):1998-2007；Yue Yuechong, model swallow duckweed plant terpenes synzyme and its metabolic regulation
Progress gardening journals, 2011,3 (2)：379-388).
The content of the invention：
First purpose of the present invention is to provide a kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase.
The suspension culture of Aquilaria sinensis sesquiterpene synthase of the present invention, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.2.
Second purpose of the invention is to provide the suspension culture of Aquilaria sinensis sesquiterpene synthase for encoding above-mentioned suspension culture of Aquilaria sinensis sesquiterpene synthase
Gene A s-SesTPS1, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.1.
Third object of the present invention is to provide suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 in detection suspension culture of Aquilaria sinensis
Application in the Edgeworthia chrysantha level of tree body.
Present invention clone has obtained suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1, and phylogenetic analysis result shows this
Gene belongs to the TPSa subtribes of angiosperm sesquiterpene synthase genomic constitution, with the genetic homology highest gene be δ-more
The wooden alkene synthase gene of wound, its amino acid sequence homology is also only 54%, therefore is speculated as a kind of new suspension culture of Aquilaria sinensis times
Hemiterpene synthase gene.The gene is analyzed in suspension culture of Aquilaria sinensis Edgeworthia chrysantha position (containing a large amount of agalloch eaglewood), transition part by quantitative fluorescent PCR
The expression of position (containing a small amount of agalloch eaglewood, the position between Edgeworthia chrysantha and non-Edgeworthia chrysantha) and non-Edgeworthia chrysantha position (no agalloch eaglewood), can
The Edgeworthia chrysantha level of detection suspension culture of Aquilaria sinensis tree body is acted on, important molecular biology foundation is provided to evaluate agalloch eaglewood quality.
Suspension culture of Aquilaria sinensis sesquiterpene synthase provided by the present invention has following physicochemical property：The coded by said gene albumen
Molecular weight is 64.14kD, and isoelectric point is 5.14, is acidic protein, and its half-life period, mammal granulophilocyte was in vitro
30h, is more than 20h in yeast body, and 10h is more than in Escherichia coli body.Unstability index is 44.57, is labile protein.Should
The albumen of coded by said gene can be catalyzed substrate farnesyl pyrophosphate generation sesquiterpenoids, in being agalloch eaglewood biosynthesis pathway
Key gene.
In view of the report at present on suspension culture of Aquilaria sinensis sesquiterpene synthase gene is less, therefore suspension culture of Aquilaria sinensis provided by the present invention
Sesquiterpene synthase Gene A s-SesTPS1 and its suspension culture of Aquilaria sinensis different parts expression detection suspension culture of Aquilaria sinensis tree body in
Edgeworthia chrysantha level has important application value, is of great practical significance to improving Edgeworthia chrysantha quality.
Brief description of the drawings：
The agarose gel electrophoresis that Fig. 1 extracts RNA sample by suspension culture of Aquilaria sinensis different parts is identified：1 is non-Edgeworthia chrysantha sample；2
For transition sample；3 be Edgeworthia chrysantha position sample
Fig. 2 identifies for suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 agarose gel electrophoresis：1 is with RT-PCR
The sequence for the suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 that obtained cDNA is obtained as template amplification；2 be with whitewood
Fragrant genome is the sequence that template amplification is obtained；3 be the cDNA that RT-PCR is obtained；4 be DL2000marker；
Fig. 3 is plant terpene synthase gene family system chadogram；
Fig. 4 is that suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 is analyzed in the expression of suspension culture of Aquilaria sinensis different parts：
A.qPCR analyzes expression, and wherein agalloch eaglewood sample, transition sample and whitewood's sample is respectively Edgeworthia chrysantha position sample, transition sample
Product, non-Edgeworthia chrysantha sample；B. agarose gel electrophoresis is identified, wherein 1,2,3,4,5,6,7,8 be respectively that reference gene Histone exists
Suspension culture of Aquilaria sinensis Edgeworthia chrysantha position, intermediate location, non-Edgeworthia chrysantha position, blank control, As-SesTPS1 genes are in suspension culture of Aquilaria sinensis Edgeworthia chrysantha position, mistake
Cross position, non-Edgeworthia chrysantha position, the expression of blank control.
Fig. 5 is to be catalyzed substrate farnesyl pyrophosphate product containing sesquiterpene synthase recombinant bacterium supernatant coded by As-SesTPS1
GC-MS qualification figures：A. recombinant bacterium supernatant enzymatic reaction product after recombinant bacterium supernatant enzymatic reaction product B.IPTG inductions is not induced.
Embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment 1：
Suspension culture of Aquilaria sinensis difference Edgeworthia chrysantha component R NA extraction, comprises the following steps：
(1) sample (non-Edgeworthia chrysantha sample, transition sample or Edgeworthia chrysantha position sample) is fully ground in liquid nitrogen, addition contains
The extract solution of beta -mercaptoethanol takes 500 μ L into 1.5mL centrifuge tubes, and concussion is mixed, 12000r/min centrifugation 10min, takes supernatant；
(2) isometric extract I is extracted again, 12000r/min centrifugation 5min, takes supernatant；Add isometric take out
Extract II, acutely concussion, 12000r/min centrifugation 8min take supernatant；
(3) 1/2 volume absolute ethyl alcohol, the 4mol/L LiCl isometric with supernatant, β-sulfydryl second of final concentration 1% are added
Alcohol, overturns and mixes repeatedly, and -30 DEG C stand 4h or stay overnight, 4 DEG C of centrifugation 10min of 12000r/min；
(4) precipitation is dissolved in appropriate DEPC water, adds 1/10 volume 3mol/L NaAc-HAc, pH=5.2, after mixing, plus
Enter 3 times of volume absolute ethyl alcohols, 4 DEG C of centrifugation 10min of -30 DEG C of standings 30min, 12000r/min；Abandoning supernatant, 75% ethanol
Washing precipitation 2 times, precipitation is dissolved in 30 μ L DEPC water, and -80 DEG C save backup.
Above-mentioned improvement guanidinium isothiocyanate-CTAB extract solutions composition be 38% water-saturated phenol, 1mol/L guanidinium isothiocyanates,
2%CTAB, 100mmol/L NaAc-HAc pH=5.2,2mol/L NaCl, 2%PVP；The composition of extract I is water-saturated phenol:
Chloroform: isoamyl alcohol=25: 24: 1；The composition of extract II is chloroform: isoamyl alcohol=24: 1.
Obtained RNA is extracted to identify through agarose gel electrophoresis, as a result as shown in figure 1, obtain respectively non-Edgeworthia chrysantha sample,
The RNA of transition sample, Edgeworthia chrysantha position sample.As a result show that carried RNA 28S and 18S band is limpid in sight, 28S RNA brightness
About 2 times of 18S RNA brightness, and two bands are without substantially hangover.Through UV spectrophotometer measuring OD260/280Size exists
Between 1.8-2.0, RNA integralities preferably, are not degraded, can meet subsequent experimental requirement.
Embodiment 2：The acquisition of suspension culture of Aquilaria sinensis cDNA sequence, specifically includes following steps：
The RNA of the Edgeworthia chrysantha position sample obtained using extraction is template, with Oligo (dT)15It is anti-that random primer carries out RT-PCR
Should, reaction system is as follows：
Used RT-PCR conditions are as follows：
Obtain reverse transcription cDNA
CDNA and suspension culture of Aquilaria sinensis Edgeworthia chrysantha position genome are obtained after the completion of using reverse transcription as template, with primer：5’-
atgtcagctgctcaggtctcac-3’；5 '-tcatatagtaattggatggaccagc-3 ' enter performing PCR amplification, reaction condition
It is as follows：
Gained PCR primer is identified with 1% agarose gel electrophoresis, as a result as shown in Fig. 2 being obtained by template amplification of cDNA
1671bp fragment, its sequence such as SEQ ID NO:Shown in 1, the coded by said gene amino acid sequence such as SEQ ID NO:Shown in 2.
2200bp or so band is obtained by template of genome.Both are delivered into sequencing company to be sequenced, it is found that both have
The homology of 1671bp this partial nucleotide sequence reaches 100%, illustrates that the sequence obtained using genome as template amplification is removed
Open reading outer frame, contains intron sequences.Phylogenetic analysis result shows that the gene belongs to the synthesis of angiosperm sequiterpene
The TPSa subtribes (Fig. 3) of enzyme gene composition, are suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 (its core by the unnamed gene
Nucleotide sequence is as shown in SEQ ID NO.1), its protease encoded is named as：Suspension culture of Aquilaria sinensis sesquiterpene synthase (its amino acid sequence
Row are as shown in SEQ ID NO.2).
Suspension culture of Aquilaria sinensis sesquiterpene synthase has following physicochemical property：The molecular weight of the coded by said gene albumen is
64.14kD, isoelectric point is 5.14, is acidic protein, and its half-life period, mammal granulophilocyte was 30h in vitro, in yeast
It is more than 20h in vivo, 10h is more than in Escherichia coli body.Unstability index is 44.57, is labile protein.The coded by said gene
Albumen can be catalyzed substrate farnesyl pyrophosphate generation sesquiterpenoids, be the crucial base in agalloch eaglewood biosynthesis pathway
Cause.
Embodiment 3：Expressions point of the suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 in suspension culture of Aquilaria sinensis different parts
Analysis, is comprised the following steps：
RNA using the suspension culture of Aquilaria sinensis Edgeworthia chrysantha position of extraction, intermediate location and non-Edgeworthia chrysantha position is template, by institute in embodiment 1
State condition progress RT-PCR and obtain cDNA sequence；
Using resulting cDNA sequence as template, with primer：5 '-atggtggaaggatcttgatttcaaaaca-3 ',
5 '-acctctcaattgcatctgtgagtagctt-3 ' carry out qPCR amplifications, using Histone genes as internal reference, reference gene
Primer is：5 '-gtaccgctaccggagggaagttgaaga-3 ', 5 '-cttcttgggcgacttggtagccttggt-3 '
Reaction system is as follows：
Reaction condition is as follows：
Calculate expression of the different samples with respect to reference gene Histone, the identification of 1% agarose gel electrophoresis.Identification
As a result as shown in figure 4, suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 high expression in Edgeworthia chrysantha position, has in intermediate location
A small amount of expression, are not almost expressed, the expression at Edgeworthia chrysantha position is intermediate location at whitewood position (non-Edgeworthia chrysantha position)
8.87 times, be 27.66 times of non-Edgeworthia chrysantha position expression, results of statistical analysis shows, the whitewood at suspension culture of Aquilaria sinensis Edgeworthia chrysantha position
Fragrant sesquiterpene synthase Gene A s-SesTPS1 expressions are significantly higher than intermediate location and non-Edgeworthia chrysantha position As-SesTPS1's
Expression (p<0.01).Agarose gel electrophoresis result also indicates that, the suspension culture of Aquilaria sinensis sesquiterpene synthase at suspension culture of Aquilaria sinensis Edgeworthia chrysantha position
Gene A s-SesTPS1 band becomes clear, and intermediate location only has faint band, and non-Edgeworthia chrysantha position and blank control are then not
Show the band (Fig. 4 B) of target gene.Illustrate that suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 is agalloch eaglewood sesquiterpenoids
The key gene of compound biosynthesis, has for the quality that Edgeworthia chrysantha level in detection suspension culture of Aquilaria sinensis tree body and evaluation produce agalloch eaglewood
Important application value.
Embodiment 4：The catalysis substrate farnesyl pyrophosphate life of As-SesTPS1 coded by said gene suspension culture of Aquilaria sinensis sesquiterpene synthase
Into sesquiterpenoids nerolidol, specific experiment scheme is as follows：
1) expression of the As-SesTPS1 in Escherichia coli
CDNA (particular sequence is as shown in the SEQ ID NO.1) full length sequence for cloning obtained As-SesTPS1 genes is used
NcoI and XhoI is in 37 DEG C of double digestion 2h, PCR primer QIAquick Gel Extraction Kit recovery product, by pET28a with NcoI and XhoI in 37 DEG C
Double digestion 2h, PCR primer QIAquick Gel Extraction Kit recovery product, then in that will reclaim obtained digestion products in 22 DEG C of connection 5h, turns
Change bacillus coli DH 5 alpha competent cell, picking monoclonal expands culture, the plasmid for extracting gained bacterium solution carries out double digestion identification.
Sequencing is sent by positive colony.
Gained positive colony is converted to e. coli bl21 (DE3) competent cell, and picked clones expand culture, gained sun
Property clone (As-SesTPS1 genes being inserted in pET28a carriers, in conversion entrance e. coli bl21 (DE3)) be seeded to
LB fluid nutrient mediums, add final concentration of 1mM IPTG (thioisopropyl galactoside), same batten when OD is 0.5 or so
IPTG is added without in the LB fluid nutrient mediums of the recombinant bacterium containing As-SesTPS1 of part culture, continues to cultivate after 3h in 37 DEG C, centrifugation
Thalline is collected, ultrasonication, 8000rpm centrifugation 10min discard precipitation and retain supernatant.IPTG recombinant bacterium is not added with as control.
Electrophoresis Sample is prepared, Western blot identifications are carried out by primary antibody of Anti-His mouse resource monoclonal antibody, recombinant bacterium is not induced
Any band is not obtained in supernatant, and the band for obtaining 65kD or so is can detect in recombinant bacterium supernatant, with theoretical prediction such as SEQ
The sesquiterpene synthase molecular weight of As-SesTPS1 codings shown in ID NO.2 is consistent, illustrates restructuring sesquiterpene synthase success
Expression.
2) the GC-MS identifications of catalysis substrate farnesyl pyrophosphate and its product：
Supernatant not induce recombinant bacterium, with farnesyl pyrophosphate (FPP) for substrate, contains as control after detection induction
As-SesTPS1 recombinant bacteriums break the enzyme activity of supernatant after bacterium, and enzymatic reaction system is as follows:
Total system is 200 μ l, containing Tris-HCl buffer (25mM, pH 7.0), 10% glycerine, 100mM MgS04,
The 5mM μ l recombinant bacterium supernatants of DTT, 46 μM of FPP, 50.After fully mixing 1h is incubated at 30 DEG C.Waved with SPME SPME absorption
Stimulating food matter 0.5h.
Catalysate is detected using gas chromatograph-mass spectrometer.Specific testing conditions are as follows：
Instrument：Agilent7890-5975 GC-MSs are analyzed, chromatographic column：30m×0.25mm×0.25μm
VF-5MS quartz capillary columns.
Ionization mode:EI, electron energy 70eV, carrier gas are helium, and flow velocity is 1mL/min, and 250 DEG C of injector temperature rises
Beginning temperature is 80 DEG C, rises to 180 DEG C with 5 DEG C/min, then rise to 250 DEG C with 20 DEG C/min.30~500amu. of quality of scanning scope
Agalloch eaglewood feature product is not detected by recombinant bacterium supernatant enzymatic reaction product as shown in figure 5, not inducing, and through IPTG
It can be examined in the recombinant bacterium supernatant enzymatic reaction product (suspension culture of Aquilaria sinensis of gene code containing As-SesTPS1 sesquiterpene synthase) of induction
Measure and agalloch eaglewood characteristic component nerolidol.Illustrate that the restructuring suspension culture of Aquilaria sinensis sesquiterpene synthase through induced expression can be with catalysis method Buddhist nun
Base pyrophosphoric acid generates agalloch eaglewood characteristic component, therefore As-SesTPS1 genes can be used as the characterizing gene for identifying agalloch eaglewood quality.
Claims (1)
1. applications of the suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 in detection suspension culture of Aquilaria sinensis tree body Edgeworthia chrysantha level, described
Suspension culture of Aquilaria sinensis sesquiterpene synthase Gene A s-SesTPS1 nucleotide sequence is as shown in SEQ ID NO.1.
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