CN104342426A - Novel aquilaria sinensis sesquiterpenes synthetase, encoding gene and application thereof - Google Patents
Novel aquilaria sinensis sesquiterpenes synthetase, encoding gene and application thereof Download PDFInfo
- Publication number
- CN104342426A CN104342426A CN201410612415.4A CN201410612415A CN104342426A CN 104342426 A CN104342426 A CN 104342426A CN 201410612415 A CN201410612415 A CN 201410612415A CN 104342426 A CN104342426 A CN 104342426A
- Authority
- CN
- China
- Prior art keywords
- gene
- lignum aquilariae
- aquilariae resinatum
- aquilaria sinensis
- sesquiterpenes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title abstract description 29
- 229930004725 sesquiterpene Natural products 0.000 title abstract description 8
- 241001533085 Aquilaria sinensis Species 0.000 title abstract 10
- 108090000364 Ligases Proteins 0.000 title abstract 7
- 102000003960 Ligases Human genes 0.000 title abstract 3
- 241001265525 Edgeworthia chrysantha Species 0.000 claims abstract description 39
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 101710093888 Pentalenene synthase Proteins 0.000 claims description 41
- 101710115850 Sesquiterpene synthase Proteins 0.000 claims description 41
- 150000004354 sesquiterpene derivatives Chemical class 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 2
- 241000271309 Aquilaria crassna Species 0.000 description 20
- 239000000523 sample Substances 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 16
- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 description 9
- VWFJDQUYCIWHTN-UHFFFAOYSA-N Farnesyl pyrophosphate Natural products CC(C)=CCCC(C)=CCCC(C)=CCOP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-UHFFFAOYSA-N 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 9
- 238000012797 qualification Methods 0.000 description 9
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 108010033040 Histones Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 101150044508 key gene Proteins 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- FQTLCLSUCSAZDY-UHFFFAOYSA-N (+) E(S) nerolidol Natural products CC(C)=CCCC(C)=CCCC(C)(O)C=C FQTLCLSUCSAZDY-UHFFFAOYSA-N 0.000 description 2
- 101800000263 Acidic protein Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108030003495 Delta-guaiene synthases Proteins 0.000 description 2
- 235000009414 Elaeocarpus kirtonii Nutrition 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000218922 Magnoliophyta Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 229910020820 NaAc-HAc Inorganic materials 0.000 description 2
- FQTLCLSUCSAZDY-ATGUSINASA-N Nerolidol Chemical compound CC(C)=CCC\C(C)=C\CC[C@](C)(O)C=C FQTLCLSUCSAZDY-ATGUSINASA-N 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000236151 Tabebuia pallida Species 0.000 description 2
- 235000013584 Tabebuia pallida Nutrition 0.000 description 2
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- WASNIKZYIWZQIP-AWEZNQCLSA-N nerolidol Natural products CC(=CCCC(=CCC[C@@H](O)C=C)C)C WASNIKZYIWZQIP-AWEZNQCLSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 238000013081 phylogenetic analysis Methods 0.000 description 2
- 210000001995 reticulocyte Anatomy 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000002470 solid-phase micro-extraction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108020005096 28S Ribosomal RNA Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000984061 Aquilaria Species 0.000 description 1
- 241001647745 Banksia Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000012569 chemometric method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 150000008195 galaktosides Chemical class 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 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
- 238000011534 incubation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 108010087432 terpene synthase Proteins 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
The invention discloses a novel aquilaria sinensis sesquiterpenes synthetase, an encoding gene and an application thereof. The amino acid sequence of the aquilaria sinensis sesquiterpenes synthetase is shown as SEQ ID NO.2; the nucleotide sequence of the aquilaria sinensis sesquiterpenes synthetase gene As-SesTPS1 is shown as SEQ ID NO.1; the aquilaria sinensis sesquiterpenes synthetase gene As-SesTPS1 is applied to the detection for the edgeworthia chrysantha level of the aquilaria sinensis tree. Few reports for the aquilaria sinensis sesquiterpenes synthetase gene exist at present, so that the aquilaria sinensis sesquiterpenes synthetase gene As-SesTPS1 and the expression level thereof on different parts of the aquilaria sinensis provided by the invention have important application value in detecting the edgeworthia chrysantha level of the aquilaria sinensis tree and have important practical significance in increasing the quality of the edgeworthia chrysantha.
Description
Technical field:
The invention belongs to gene biological field, be specifically related to a kind of novel Lignum Aquilariae Resinatum sesquiterpene synthase and encoding gene thereof and application.
Background technology:
Effect of agalloch eaglewood tool promoting the circulation of QI to relieve pain, warming middle-JIAO to arrest vomiting, helping inspiration to relieve asthma, significant curative effect is had in treatment digestive system, respiratory system disease, cardiovascular and cerebrovascular diseases, nervous system disorders, antitumor, wind resistance diseases caused by dampness and anti-ageing wait for a long time in also have good effect (Liu the army and the people, Xu Honghua. Home-made occluder progress. Chinese medicinal materials, 2005,28 (7): 627-632).Lignum Aquilariae Resinatum can form agalloch eaglewood after mechanical induction, chemical damage or fungal induction, and the main component in agalloch eaglewood comprises sesquiterpenoids.The transcript profile sequencing result of Lignum Aquilariae Resinatum shows, Lignum Aquilariae Resinatum sesquiterpene synthase is the biosynthetic key enzyme (Wu Hongqing of agalloch eaglewood, Wang Lei, He Xin, Bai Ling, Gao Xiaoxia, severe cold is quiet, Zhang Weimin. the clone of Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS and information biology and expression analysis, 2014,45 (1): 94-101).Artificial agilawood is compared with natural agilawood, relatively low (the Gao X X of its main pharmacodynamics composition sesquiterpenoids, 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 is also not as natural agilawood, and sesquiterpene synthase is the key enzyme that sesquiterpenoids is formed, therefore theoretical direction can be provided for promoting artificial Edgeworthia chrysantha technology to the cloning and identification of Lignum Aquilariae Resinatum sesquiterpene synthase gene, the quality promoting artificial agilawood is of great significance.
At present, the floristic sesquiterpenoids synthase gene of multiple difference is by successful clone, but the biosynthesizing of agalloch eaglewood sesquiterpene synthase genes involved research is also few, only clone obtains sesquiterpene synthase gene (the Kumeta Y that principal product is guaiene, Ito M.Characterization of δ-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, Fan Yanping. the progress of plant terpene synthetic enzyme and metabolic regulation thereof. gardening journal, 2011,3 (2): 379-388).
Summary of the invention:
First object of the present invention is to provide a kind of novel Lignum Aquilariae Resinatum sesquiterpene synthase.
Lignum Aquilariae Resinatum sesquiterpene synthase of the present invention, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2.
The present invention's second object is to provide the Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 of above-mentioned Lignum Aquilariae Resinatum sesquiterpene synthase of encoding, and it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
3rd object of the present invention is to provide the application of Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 in the Edgeworthia chrysantha level detecting Lignum Aquilariae Resinatum tree body.
The present invention clone obtains Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1, phylogenetic analysis result shows that this gene belongs to the TPSa subtribe of angiosperm sesquiterpene synthase genomic constitution, the gene the highest with this genetic homology is δ-guaiene synthase gene, its amino acid sequence homology is also only 54%, is therefore speculated as a kind of novel Lignum Aquilariae Resinatum sesquiterpene synthase gene.This gene is analyzed in Lignum Aquilariae Resinatum Edgeworthia chrysantha position (containing a large amount of agalloch eaglewood), intermediate location (containing a small amount of agalloch eaglewood by quantitative fluorescent PCR, position between Edgeworthia chrysantha and non-Edgeworthia chrysantha) and the expression level at non-Edgeworthia chrysantha position (without agalloch eaglewood), the Edgeworthia chrysantha level detecting Lignum Aquilariae Resinatum tree body can being acted on, providing important molecular biology foundation for evaluating agalloch eaglewood quality.
Lignum Aquilariae Resinatum sesquiterpene synthase provided by the present invention has following physicochemical property: the molecular weight of this coded by said gene albumen is 64.14kD, iso-electric point is 5.14, for acidic protein, its transformation period in vitro Mammals reticulocyte be 30h, in yeast body, be greater than 20h, in intestinal bacteria body, be greater than 10h.Unstability index is 44.57, is labile protein.The proteins catalyse substrate farnesyl pyrophosphate of this coded by said gene generates sesquiterpenoids, is the key gene in agalloch eaglewood biosynthetic pathway.
In view of the current report about Lignum Aquilariae Resinatum sesquiterpene synthase gene is less, therefore Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 provided by the present invention and the Edgeworthia chrysantha level of expression level in detection Lignum Aquilariae Resinatum tree body at Lignum Aquilariae Resinatum different sites thereof have important using value, are of great practical significance to raising Edgeworthia chrysantha quality.
Accompanying drawing illustrates:
Fig. 1 extracts by Lignum Aquilariae Resinatum different sites the agarose gel electrophoresis qualification of RNA sample: 1 is non-Edgeworthia chrysantha sample; 2 is transition sample; 3 is Edgeworthia chrysantha position sample
Fig. 2 is the agarose gel electrophoresis qualification of Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1: 1 is the sequence of the Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 that the cDNA obtained using RT-PCR obtains as template amplification; 2 is the sequence obtained for template amplification with Lignum Aquilariae Resinatum genome; 3 cDNA obtained for RT-PCR; 4 is DL2000marker;
Fig. 3 is plant terpene synthase gene family system evolutionary tree;
Fig. 4 is the expression level analysis of Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 at Lignum Aquilariae Resinatum different sites: A.qPCR analyzes expression level, and wherein agalloch eaglewood sample, transition sample and whitewood's sample are respectively Edgeworthia chrysantha position sample, transition sample, non-Edgeworthia chrysantha sample; B. agarose gel electrophoresis qualification, wherein 1,2,3,4,5,6,7,8 be respectively reference gene Histone at Lignum Aquilariae Resinatum Edgeworthia chrysantha position, intermediate location, non-Edgeworthia chrysantha position, blank, As-SesTPS1 gene at Lignum Aquilariae Resinatum Edgeworthia chrysantha position, intermediate location, non-Edgeworthia chrysantha position, blank expression.
For containing sesquiterpene synthase recombinant bacterium supernatant catalytic substrate farnesyl pyrophosphate product GC-MS coded by As-SesTPS1, Fig. 5 identifies that figure: A. does not induce recombinant bacterium supernatant enzymatic reaction product B.IPTG to induce rear recombinant bacterium supernatant enzymatic reaction product.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
The extraction of Lignum Aquilariae Resinatum different Edgeworthia chrysantha component R NA, comprises the steps:
(1) sample (non-Edgeworthia chrysantha sample, transition sample or Edgeworthia chrysantha position sample) is fully ground in liquid nitrogen, the extracting solution added containing beta-mercaptoethanol is got in 500 μ L to 1.5mL centrifuge tubes, concussion mixing, the centrifugal 10min of 12000r/min, gets supernatant;
(2) isopyknic extract I extracting again, the centrifugal 5min of 12000r/min, gets supernatant; Add isopyknic extract II, concuss, the centrifugal 8min of 12000r/min, gets supernatant;
(3) add 1/2 volume dehydrated alcohol, 4mol/L LiCl isopyknic with supernatant, the beta-mercaptoethanol of final concentration 1%, puts upside down mixing repeatedly ,-30 DEG C of standing 4h or spend the night, 12000r/min 4 DEG C of centrifugal 10min;
(4) precipitation is dissolved in appropriate DEPC water, adds 1/10 volume 3mol/L NaAc-HAc, pH=5.2, after mixing, adds 3 times of volume dehydrated alcohols ,-30 DEG C of standing 30min, 12000r/min 4 DEG C of centrifugal 10min; Abandoning supernatant, 75% washing with alcohol precipitates 2 times, and precipitation is dissolved in 30 μ L DEPC water, and-80 DEG C save backup.
Above-mentioned improvement guanidinium isothiocyanate-CTAB extracting solution composition is 38% water-saturated phenol, 1mol/L guanidinium isothiocyanate, 2%CTAB, 100mmol/L NaAc-HAc pH=5.2,2mol/L NaCl, 2%PVP; Extract I composition is water-saturated phenol: chloroform: primary isoamyl alcohol=25: 24: 1; Extract II composition is chloroform: primary isoamyl alcohol=24: 1.
Extract the RNA obtained and identify through agarose gel electrophoresis, result as shown in Figure 1, obtains the RNA of non-Edgeworthia chrysantha sample, transition sample, Edgeworthia chrysantha position sample respectively.It is limpid in sight that 28S and the 18S band of RNA is carried by result display institute, and 28S RNA brightness is about 2 times of 18S RNA brightness, and two bands all trail without obvious.Through UV spectrophotometer measuring OD
260/280size is between 1.8-2.0, and RNA integrity is better, degrades, can meet subsequent experimental requirement.
Embodiment 2: the acquisition of Lignum Aquilariae Resinatum cDNA sequence, specifically comprises the steps:
To extract the RNA of the Edgeworthia chrysantha position sample obtained for template, with Oligo (dT)
15random primer carries out RT-PCR reaction, and reaction system is as follows:
Adopt RT-PCR condition as follows:
Obtain reverse transcription cDNA
To obtain cDNA and Lignum Aquilariae Resinatum Edgeworthia chrysantha position genome after reverse transcription completes as template, with primer: 5 '-atgtcagctgctcaggtctcac-3 '; 5 '-tcatatagtaattggatggaccagc-3 ' carries out pcr amplification, and reaction conditions is as follows:
Gained PCR primer 1% agarose gel electrophoresis is identified, result as shown in Figure 2, take cDNA as the fragment that template amplification obtains 1671bp, and its sequence is as shown in SEQ ID NO:1, and this coded by said gene aminoacid sequence is as shown in SEQ ID NO:2.Take genome as the band that template obtains about 2200bp.Both are delivered to order-checking company to check order, both discoveries have the homology of this part nucleotide sequence of 1671bp to reach 100%, illustrate with genome be the sequence that obtains of template amplification except open reading frame, containing intron sequences.Phylogenetic analysis result shows that this gene belongs to the TPSa subtribe (Fig. 3) of angiosperm sesquiterpene synthase genomic constitution, be Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 (its nucleotide sequence is as shown in SEQ ID NO.1) by this unnamed gene, the proteolytic enzyme called after of its coding: Lignum Aquilariae Resinatum sesquiterpene synthase (its aminoacid sequence is as shown in SEQ ID NO.2).
Lignum Aquilariae Resinatum sesquiterpene synthase has following physicochemical property: the molecular weight of this coded by said gene albumen is 64.14kD, iso-electric point is 5.14, is acidic protein, its transformation period in vitro Mammals reticulocyte be 30h, in yeast body, be greater than 20h, in intestinal bacteria body, be greater than 10h.Unstability index is 44.57, is labile protein.The proteins catalyse substrate farnesyl pyrophosphate of this coded by said gene generates sesquiterpenoids, is the key gene in agalloch eaglewood biosynthetic pathway.
Embodiment 3: Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1, in the expression level analysis of Lignum Aquilariae Resinatum different sites, comprises following steps:
With the RNA at the Lignum Aquilariae Resinatum Edgeworthia chrysantha position of extracting, intermediate location and non-Edgeworthia chrysantha position for template, carry out RT-PCR by condition described in embodiment 1 and obtain cDNA sequence;
With obtained cDNA sequence for template, with primer: 5 '-atggtggaaggatcttgatttcaaaaca-3 ', 5 '-acctctcaattgcatctgtgagtagctt-3 ' carries out qPCR amplification, using Histone gene as internal reference, reference gene primer is: 5 '-gtaccgctaccggagggaagttgaaga-3 ', 5 '-cttcttgggcgacttggtagccttggt-3 '
Reaction system is as follows:
Reaction conditions is as follows:
Calculate the expression level of the relative reference gene Histone of different sample, 1% agarose gel electrophoresis qualification.Qualification result as shown in Figure 4, Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 high expression level in Edgeworthia chrysantha position, have in intermediate location and express in a small amount, almost do not express at whitewood position (non-Edgeworthia chrysantha position), the expression level at Edgeworthia chrysantha position is 8.87 times of intermediate location, it is 27.66 times of non-Edgeworthia chrysantha position expression level, results of statistical analysis shows, the Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 expression level at Lignum Aquilariae Resinatum Edgeworthia chrysantha position is significantly higher than the expression level (p<0.01) of intermediate location and non-Edgeworthia chrysantha position As-SesTPS1.Agarose gel electrophoresis result also shows, the band of the Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 at Lignum Aquilariae Resinatum Edgeworthia chrysantha position becomes clear, intermediate location only has faint band, and non-Edgeworthia chrysantha position and blank then all do not show the band (Fig. 4 B) of goal gene.Making banksia rose sesquiterpene synthase Gene A s-SesTPS1 clear is the biosynthetic key gene of agalloch eaglewood sesquiterpenoids, for detect Edgeworthia chrysantha level in Lignum Aquilariae Resinatum tree body and evaluate produce agalloch eaglewood quality there is important using value.
Embodiment 4:As-SesTPS1 coded by said gene Lignum Aquilariae Resinatum sesquiterpene synthase catalytic substrate farnesyl pyrophosphate generates sesquiterpenoids nerolidol, and specific experiment scheme is as follows:
1) expression of As-SesTPS1 in intestinal bacteria
To cDNA (concrete sequence is as shown in SEQ ID NO.1) full length sequence NcoI and XhoI of the As-SesTPS1 gene obtained be cloned in 37 DEG C of double digestion 2h, PCR primer reclaims test kit and reclaims product, by pET28a NcoI and XhoI in 37 DEG C of double digestion 2h, PCR primer reclaims test kit and reclaims product, then in connecting 5h by reclaiming the digestion products obtained in 22 DEG C, transformation of E. coli DH5 α competent cell, picking mono-clonal enlarged culturing, the plasmid extracting gained bacterium liquid carries out double digestion qualification.Order-checking is sent by positive colony.
Gained positive colony is converted into e. coli bl21 (DE3) competent cell, picked clones enlarged culturing, gained positive colony (inserts in pET28a carrier by As-SesTPS1 gene, conversion enters in e. coli bl21 (DE3)) be seeded to LB liquid nutrient medium, the IPTG (thioisopropyl galactoside) that final concentration is 1mM is added when OD is about 0.5, what similarity condition was cultivated does not add IPTG containing in the LB liquid nutrient medium of As-SesTPS1 recombinant bacterium, after continuing to cultivate 3h in 37 DEG C, collected by centrifugation thalline, ultrasonication, the centrifugal 10min of 8000rpm, discard precipitation and retain supernatant.Do not add the recombinant bacterium of IPTG in contrast.PAGE production sample, with Anti-His mouse resource monoclonal antibody for primary antibodie carries out Western blot qualification, do not induce in recombinant bacterium supernatant and do not obtain any band, and the band obtaining about 65kD in recombinant bacterium supernatant, can be detected, consistent with the sesquiterpene synthase molecular weight that the As-SesTPS1 as shown in SEQ ID NO.2 of theoretical prediction encodes, restructuring sesquiterpene synthase successful expression is described.
2) the GC-MS qualification of catalytic substrate farnesyl pyrophosphate and product thereof:
Not induce the supernatant of recombinant bacterium in contrast, with farnesyl pyrophosphate (FPP) for substrate, break the enzyme activity of supernatant after bacterium after detecting induction containing As-SesTPS1 recombinant bacterium, enzymatic reaction system is as follows:
Be totally 200 μ l, containing Tris-HCl buffer (25mM, pH 7.0), 10% glycerine, 100mM MgS04,5mM DTT, 46 μMs of FPP, 50 μ l recombinant bacterium supernatants.At 30 DEG C of incubation 1h after abundant mixing.With solid-phase microextraction SPME adsorbing volatilizing material 0.5h.
Gas chromatograph-mass spectrometer is adopted to detect catalysate.Concrete testing conditions is as follows:
Instrument: Agilent7890-5975 GC-MS is analyzed, chromatographic column: 30m × 0.25mm × 0.25 μm VF-5MS quartz capillary column.
Ionization mode: EI, electron energy 70eV, carrier gas is helium, and flow velocity is 1mL/min, injector temperature 250 DEG C, and starting temperature is 80 DEG C, rises to 180 DEG C with 5 DEG C/min, then rises to 250 DEG C with 20 DEG C/min.Quality of scanning scope 30 ~ 500amu.
As shown in Figure 5, do not induce in recombinant bacterium supernatant enzymatic reaction product and agalloch eaglewood feature product do not detected, and can detect and agalloch eaglewood characteristic component nerolidol in the recombinant bacterium supernatant enzymatic reaction product (containing As-SesTPS1 genes encoding Lignum Aquilariae Resinatum sesquiterpene synthase) of IPTG induction.Illustrate that the restructuring Lignum Aquilariae Resinatum sesquiterpene synthase through abduction delivering can generate agalloch eaglewood characteristic component by catalysis farnesyl pyrophosphate, therefore As-SesTPS1 gene can be used as the characterizing gene of qualification agalloch eaglewood quality.
Claims (3)
1. a Lignum Aquilariae Resinatum sesquiterpene synthase, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2.
2. a Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 for coding Lignum Aquilariae Resinatum sesquiterpene synthase according to claim 1, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
3. Lignum Aquilariae Resinatum sesquiterpene synthase Gene A s-SesTPS1 according to claim 2 is detecting the application in Lignum Aquilariae Resinatum tree body Edgeworthia chrysantha level.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410612415.4A CN104342426B (en) | 2014-11-04 | 2014-11-04 | A kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410612415.4A CN104342426B (en) | 2014-11-04 | 2014-11-04 | A kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104342426A true CN104342426A (en) | 2015-02-11 |
| CN104342426B CN104342426B (en) | 2017-09-12 |
Family
ID=52498886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410612415.4A Active CN104342426B (en) | 2014-11-04 | 2014-11-04 | A kind of new suspension culture of Aquilaria sinensis sesquiterpene synthase and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104342426B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866019A (en) * | 2018-07-18 | 2018-11-23 | 青岛农业大学 | A kind of preparation method and applications recombinating streptococcus pneumonia dihyrofolate reductase |
| CN108977426A (en) * | 2018-06-29 | 2018-12-11 | 中国林业科学研究院热带林业研究所 | A kind of oil nanmu sesquiterpene synthase and its encoding gene and application |
| CN109679943A (en) * | 2019-01-30 | 2019-04-26 | 天津大学 | A kind of Celastrus angulatus sesquiterpene synthase CaTPS3 and its gene order |
| CN109837266A (en) * | 2019-01-24 | 2019-06-04 | 天津大学 | A kind of calyculus tongue fur sesquiterpene synthase MTc and its gene order |
| CN113234740A (en) * | 2021-04-30 | 2021-08-10 | 中国医学科学院药用植物研究所 | Aquilaria sinensis terpene synthase |
| CN114410671A (en) * | 2022-01-27 | 2022-04-29 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis sesquiterpene synthase gene ASS15, and encoding product and application thereof |
| CN117604006B (en) * | 2023-10-24 | 2024-05-17 | 中国热带农业科学院橡胶研究所 | Rubber tree Hedycaryol synthetase gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009005678A (en) * | 2007-06-01 | 2009-01-15 | Univ Kinki | Nucleic acid encoding polypeptide having sesquiterpene synthase activity |
| CN103243083A (en) * | 2012-02-06 | 2013-08-14 | 中国科学院上海生命科学研究院 | Novel sesquiterpene synthetase and application thereof |
-
2014
- 2014-11-04 CN CN201410612415.4A patent/CN104342426B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009005678A (en) * | 2007-06-01 | 2009-01-15 | Univ Kinki | Nucleic acid encoding polypeptide having sesquiterpene synthase activity |
| CN103243083A (en) * | 2012-02-06 | 2013-08-14 | 中国科学院上海生命科学研究院 | Novel sesquiterpene synthetase and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ALTANZUL KHOROLRAGCHAA ET AL.: "Isolation of Sesquiterpene Synthase Homolog from Panax ginseng C.A. Meyer", 《J. GINSENG RES.》 * |
| HE X. ET AL.: "NCBI：GenBank：KM881473.1", 《NCBI》 * |
| 吴宏清等: "白木香倍半萜合成酶基因As-SesTPS的克隆及生物信息学与表达分析", 《中草药》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977426A (en) * | 2018-06-29 | 2018-12-11 | 中国林业科学研究院热带林业研究所 | A kind of oil nanmu sesquiterpene synthase and its encoding gene and application |
| CN108977426B (en) * | 2018-06-29 | 2020-08-14 | 中国林业科学研究院热带林业研究所 | Phoebe sesquiterpene synthetase, and coding gene and application thereof |
| CN108866019A (en) * | 2018-07-18 | 2018-11-23 | 青岛农业大学 | A kind of preparation method and applications recombinating streptococcus pneumonia dihyrofolate reductase |
| CN109837266A (en) * | 2019-01-24 | 2019-06-04 | 天津大学 | A kind of calyculus tongue fur sesquiterpene synthase MTc and its gene order |
| CN109837266B (en) * | 2019-01-24 | 2021-12-31 | 天津大学 | Small sepal moss sesquiterpene synthetase MTc and gene sequence thereof |
| CN109679943A (en) * | 2019-01-30 | 2019-04-26 | 天津大学 | A kind of Celastrus angulatus sesquiterpene synthase CaTPS3 and its gene order |
| CN113234740A (en) * | 2021-04-30 | 2021-08-10 | 中国医学科学院药用植物研究所 | Aquilaria sinensis terpene synthase |
| CN114410671A (en) * | 2022-01-27 | 2022-04-29 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis sesquiterpene synthase gene ASS15, and encoding product and application thereof |
| CN114410671B (en) * | 2022-01-27 | 2023-06-27 | 中国医学科学院药用植物研究所海南分所 | Aquilaria sinensis sesquiterpene synthase gene ASS15, and encoding product and application thereof |
| CN117604006B (en) * | 2023-10-24 | 2024-05-17 | 中国热带农业科学院橡胶研究所 | Rubber tree Hedycaryol synthetase gene and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104342426B (en) | 2017-09-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104342426A (en) | Novel aquilaria sinensis sesquiterpenes synthetase, encoding gene and application thereof | |
| CN104560927B (en) | A kind of arginine deiminase of mutation and its preparation method and application | |
| EA201001435A1 (en) | CELL, ENZYME SUGAR-PENTOSE | |
| CN104946575A (en) | Escherichia coli expression strain for high production of tyrosol and/or salidroside and icarisid D2 and application of escherichia coli expression strain | |
| CN103232986B (en) | Method for producing isoprene | |
| CN113234740B (en) | Aquilaria sinensis terpene synthase | |
| CN101631864A (en) | Method for preparing butanol through butyryl-coa as an intermediate using yeast | |
| CN107164471B (en) | Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm | |
| Zhou et al. | Purification and characterization of a urethanase from Penicillium variabile | |
| CN109929828A (en) | A kind of Celastrus angulatus sesquiterpene synthase CaTPS1 and its gene order | |
| CN103710367A (en) | Recombinant homo sapiens kallikrein 1 as well as coding agene and preparation method thereof | |
| CN104480127A (en) | Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK | |
| CN106929438A (en) | One plant height produces the saccharomyces cerevisiae and its construction method of Tetramethylpyrazine | |
| CN109706138A (en) | A kind of Celastrus angulatus sesquiterpene synthase CaTPS2 and its gene order | |
| CN109679943A (en) | A kind of Celastrus angulatus sesquiterpene synthase CaTPS3 and its gene order | |
| CN104560900A (en) | Cinnamate-4-hydroxylase derived from ornithogalum saundersiae, and nucleotide sequence and application thereof | |
| CN109852600B (en) | Small sepal moss sesquiterpene synthetase MTb and gene sequence thereof | |
| CN107739733B (en) | Aspartate aminotransferase and preparation method thereof | |
| CN112342148A (en) | Construction and application of ureidoamidohydrolase display strain | |
| CN114480450B (en) | Polygonum multiflorum resveratrol synthase gene FmRS2 and encoding product and application thereof | |
| CN114410671B (en) | Aquilaria sinensis sesquiterpene synthase gene ASS15, and encoding product and application thereof | |
| CN103898149B (en) | A kind of method utilizing albumen support to improve isoprene synthesis in intestinal bacteria | |
| CN104745618B (en) | The saccharomyces cerevisiae and its construction method of a kind of high-yield ethanol and application | |
| CN1600858A (en) | SiRNA for restraining melanin, preparation method and application | |
| CN103571857B (en) | Chinese herbaceous peony acetyl-CoA acyltransferase (PLAACT) gene and coded product thereof and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Guangzhou City, Guangdong province 510070 martyrs Road No. 100 Patentee after: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) Address before: Guangzhou City, Guangdong province 510070 martyrs Road No. 100 Patentee before: Guangdong Institute of Microbiology |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: No. 56, courtyard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong 510070 Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Address before: No. 56, courtyard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong 510070 Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: No. 56, courtyard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong 510070 Patentee after: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) Address before: 510070 No. 100 martyrs Middle Road, Guangdong, Guangzhou Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |
|
| CP02 | Change in the address of a patent holder |