CN104894150A - A saussurea involucrata cell phenylalanine ammonia lyase gene SiPAL, a coding product thereof and applications of the gene - Google Patents
A saussurea involucrata cell phenylalanine ammonia lyase gene SiPAL, a coding product thereof and applications of the gene Download PDFInfo
- Publication number
- CN104894150A CN104894150A CN201510256188.0A CN201510256188A CN104894150A CN 104894150 A CN104894150 A CN 104894150A CN 201510256188 A CN201510256188 A CN 201510256188A CN 104894150 A CN104894150 A CN 104894150A
- Authority
- CN
- China
- Prior art keywords
- gene
- sipal
- cell
- saussurea involucrata
- phenylalanine ammonia
- 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 claims abstract description 51
- 241001145025 Saussurea involucrata Species 0.000 title claims abstract description 19
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 title abstract description 21
- 239000013604 expression vector Substances 0.000 claims abstract description 11
- QJVXKWHHAMZTBY-GCPOEHJPSA-N syringin Chemical compound COC1=CC(\C=C\CO)=CC(OC)=C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 QJVXKWHHAMZTBY-GCPOEHJPSA-N 0.000 claims abstract description 9
- QJVXKWHHAMZTBY-KSXIZUIISA-N syringin Natural products COc1cc(C=CCO)cc(OC)c1O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O QJVXKWHHAMZTBY-KSXIZUIISA-N 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 239000002299 complementary DNA Substances 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000003259 recombinant expression Methods 0.000 claims abstract description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 235000018102 proteins Nutrition 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 6
- 229920005610 lignin Polymers 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 235000013311 vegetables Nutrition 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 4
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 abstract description 2
- 241000133134 Saussurea Species 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 abstract 2
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 abstract 1
- 108700026244 Open Reading Frames Proteins 0.000 abstract 1
- 229930016911 cinnamic acid Natural products 0.000 abstract 1
- 235000013985 cinnamic acid Nutrition 0.000 abstract 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 abstract 1
- 229960005190 phenylalanine Drugs 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 241000588724 Escherichia coli Species 0.000 description 13
- 230000009182 swimming Effects 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 102100029989 SHC SH2 domain-binding protein 1 Human genes 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 3
- 102100034866 Kallikrein-6 Human genes 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical compound OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- NQBWNECTZUOWID-UHFFFAOYSA-N (E)-cinnamyl (E)-cinnamate Natural products C=1C=CC=CC=1C=CC(=O)OCC=CC1=CC=CC=C1 NQBWNECTZUOWID-UHFFFAOYSA-N 0.000 description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- NQBWNECTZUOWID-QSYVVUFSSA-N cinnamyl cinnamate Chemical compound C=1C=CC=CC=1\C=C/C(=O)OC\C=C\C1=CC=CC=C1 NQBWNECTZUOWID-QSYVVUFSSA-N 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 230000032459 dedifferentiation Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 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
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 108090000673 Ammonia-Lyases Proteins 0.000 description 1
- 102000004118 Ammonia-Lyases Human genes 0.000 description 1
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010020675 Hypermetropia Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 229930014669 anthocyanidin Natural products 0.000 description 1
- 150000001452 anthocyanidin derivatives Chemical class 0.000 description 1
- 235000008758 anthocyanidins Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 description 1
- 229940074393 chlorogenic acid Drugs 0.000 description 1
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 description 1
- 235000001368 chlorogenic acid Nutrition 0.000 description 1
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000007946 flavonol Chemical class 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 1
- 150000002515 isoflavone derivatives Chemical class 0.000 description 1
- 235000008696 isoflavones Nutrition 0.000 description 1
- ZOLPMMWNCLHKPX-UHFFFAOYSA-N ligustrin Natural products CC1=CCC2C1C3OC(=C)C(=O)C3C(O)CC2=C ZOLPMMWNCLHKPX-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-N trans-cinnamic acid Chemical compound OC(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A saussurea involucrata cell phenylalanine ammonia lyase gene SiPAL, a coding product thereof and applications of the gene are provided. The SiPAL gene is obtained by sequencing a transcriptome of saussurea involucrata cells and by analyzing and screening the sequencing results. The cDNA length of the gene is 2405 bp. The open reading frame is between 120 bp and 2264 bp. The nucleotide sequence of the gene is shown as SEQ ID NO:1. The phenylalanine ammonia lyase can be prepared by utilization of an SiPAL recombinant expression vector obtained by connecting the gene and a plant expression vector, and can be used for preparation of cinnamic acid by adopting L-phenylalanine as a substrate. By utilization of the gene and the coding product, the content of syringin that is a natural medicinal component can be increased by a genetic engineering technique especially in the field of cell and/or tissue culture of saussurea involucrate and plants of adjacent categories.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to saussurea involucrata cell phenylalanine ammonia lyase (SiPAL) gene and coded product thereof and application.
Background technology
Herba Saussureae Involueratae (Saussurea involucrata) is composite family Genus Saussurea plant, main product is in Xinjiang Tianshan, Kun Lun Mountain, growth is more than snow line, among the people with all herbal medicine, there is the effects such as promoting blood circulation to restore menstrual flow, dispelling cold and removing dampness, by force muscle are supporing yang, be used for the treatment of the illness such as rheumatic arthritis, lung cold cough, amenorrhoea, retention of placenta, impotence
[1].Because Herba Saussureae Involueratae growing environment is special, artificial culture is difficult, one of saussurea involucrata cell substitute becoming Herba Saussureae Involueratae.Main containing lignin compositions such as Syringins in saussurea involucrata cell
[2-4], first key enzyme of its biosynthetic pathway is exactly phenylalanine ammonia lyase.Phenylalanine ammonia lyase (Phenylalanin ammonia-lyase, PAL) be the enzyme of catalysis phenylalanine metabolic pathways approach the first step reaction, be key enzyme and the rate-limiting enzyme of this approach, it plays an important role in the secondary metabolite synthesis such as xylogen, tonka bean camphor, flavonoid, flavonol, isoflavones and anthocyanidin.Its function is that catalysis L-Phe generates trans-cinnamic acid, is further converted to tonka bean camphor, chlorogenic acid, also can forms CoA ester, then be further converted to the xylogen such as Syringin.The clone of Herba Saussureae Involueratae cell phenylalanine ammonia lyase (SiPAL) gene, improving saussurea involucrata cytoactive composition Syringin content for utilizing genetically engineered provides important foundation.
Before the present invention comes forth, not yet have any open or reported Herba Saussureae Involueratae Phenylalanine Ammonia-Lyase Gene mentioned in the present invention and aminoacid sequence thereof.
Reference:
[1] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China version () in 2010. Beijing: Chemical Industry Press, 2010:50-51.
[2] Li Junshan, Cai Shaoqing. the chemistry of saussurea involucrata flos materia medica and pharmacological research progress [J]. Chinese Pharmaceutical Journal, 1998,33 (8): 449-452
[3] Li Yan, Guo Shunxing, Wang Chunlan, etc. Herba Saussureae Involueratae cell culture chemical constitution study [J]. Chinese Pharmaceutical Journal, 2008,42 (23): 1768-1769.
[4] Chen Dao. the chemical composition of Herba Saussureae Involueratae culture and Syringin determination study [D]. Shenyang: Shenyang Pharmaceutical University, 2009.
Summary of the invention
The present invention is for solving the above-mentioned problems in the prior art, there is provided by Herba Saussureae Involueratae phenylalanine ammonia lyase (SiPAL) gene of cloning in Herba Saussureae Involueratae (Saussurea involucrata) cell, the length of this gene cDNA is 2405bp, opening code-reading frame is between 120-2264bp, and the nucleotide sequence of this gene is as shown in SEQ ID NO:1.
Another aspect of the present invention is the protein of SiPAL coded by said gene openly mentioned above, and its aminoacid sequence is as shown in SEQ ID NO:2.
Another aspect of the present invention is the amplimer of openly SiPAL gene mentioned above, and its sequence is as shown in SEQ ID NO:3 ~ 4.
Another aspect of the present invention is openly containing SiPAL gene recombinant vectors mentioned above.SiPAL gene provided by the present invention, is connected to and the expression vector of foreign gene in expression of plants can be guided to prepare SiPAL gene recombinant vectors.In preferred situation, described expression vector is plasmid pET-31b (+).SiPAL gene of the present invention, when being building up in expression vector, adds any one strong promoter or inducible promoter before its transcription initiation Nucleotide, simultaneously must be identical with the reading frame of encoding sequence, ensure the translation of whole sequence.For the ease of identifying transgenic plant cells or plant and screening, can process carrier when carrier construction, such as add selectable markers, usual spendable mark is to the gene of antibiotics resistance enzyme and Biosafety mark, also can be that GUS, GFP etc. can produce the enzyme of colour-change or the gene etc. of luminophor.
Another aspect of the present invention is the host cell of openly SiPAL gene mentioned above.Preferably, this host cell contains the above-described recombinant expression vector containing SiPAL gene.In the inventive solutions, described host cell preferably adopts the cells such as E.coli BL21 (DE3), E.coli BL21.
Another aspect of the present invention is the openly application of SiPAL gene mentioned above in saussurea involucrata cell or tissue is cultivated, and specifically can be applied to the preparation of biological process synthesis lignin composition.Further, described lignin composition is the important effective constituent Syringin of Herba Saussureae Involueratae.
Herba Saussureae Involueratae cell of the present invention is that the method recorded in the patent of invention of 200510115902.0 obtains according to application number, choose saussurea involucrata in vitro tissue, the callus formed through dedifferentiation, as subculture kind, is given and is carried out the red-purple agglomerate of extensive succeeding transfer culture acquisition with certain condition.
Beneficial effect of the present invention: the present invention's success has cloned a kind of Herba Saussureae Involueratae phenylalanine ammonia lyase (SiPAL) gene from saussurea involucrata cell, and it is that substrate prepares styracin that this enzyme can be applied to L-Phe.Utilize the present invention can be improved the content of natural effective component Syringin by genetic engineering technique, the content of saussurea involucrata cell effective component Syringin can also be evaluated by gene.
Accompanying drawing explanation
Fig. 1 is the correlation analysis result of ligustrin content and Phenylalanine Ammonia-Lyase Gene expression level;
Fig. 2 is SiPAL Structure and function domain forecast analysis (deriving from ncbi database);
Fig. 3 is SiPAL systematic evolution tree;
Fig. 4 is the collection of illustrative plates of the recombinant expression vector pET-SiPAL expressing SiPAL gene of the present invention;
Fig. 5 is that the present invention contains the pET plasmid of SiPAL clone through digestion with restriction enzyme electroresis appraisal result, and wherein swimming lane 1 is 1kb DNA ladder, and swimming lane 2 cuts pET-SiPAL product for XhoI enzyme, and swimming lane 3 cuts pET-SiPAL product for NdeI and XhoI enzyme.
Fig. 6 is the SDS-polyacrylamide gel electrophoresis result of SiPAL protein.Wherein swimming lane 1 is Protein Ruler Marker l, and swimming lane 2 is E.coli BL21 (DE3) culture containing pET-31b (+) empty carrier; Swimming lane 3 is E.coli BL21 (DE3) culture containing pET-SiPAL.
Embodiment
Following embodiment is convenient to better understand the present invention, but does not limit the present invention.In addition, in following embodiment, in the test method that the present invention uses, if no special instructions, be the common practise of those skilled in the art, various buffered soln, detection reagent etc., if no special instructions, all can be obtained by commercial sources or be prepared by normal experiment method.
Reagent such as T4-DNA ligase enzyme, Marker, E. coli competent DH5 α and E.coli BL21 (DE3), pET-31b (+) carrier and DNA gel is used to reclaim test kit purchased from Beijing Quanshijin Biotechnology Co., Ltd in test, ExTaq enzyme and RNA Reverse Transcription box are purchased from company of TIANGEN Biotech (Beijing) Co., Ltd., the restriction enzymes such as NdeI, XhoI are purchased from Niu Yinglun (NEB) biotechnology Beijing company limited, and other common agents are purchased from precious biological (Dalian) Engineering Co., Ltd (TaKaRa) company.Primer synthesis and order-checking are completed by Beijing Bioisystech Co., Ltd of farsighted Boxing section.
Below by example, the present invention is described in further detail.
Embodiment 1 saussurea involucrata cell transcription group checks order
Utilize Trizol to extract the total serum IgE of Herba Saussureae Involueratae cell respectively, by the order-checking of Illumina Hiseq2000 platform, amount to output 14372403000nt data.Assembling result total Unigene148718, overall length 138529450nt, mean length 931nt, N50 reach 1515nt.Unigene functional annotation, annotation is 77514 to the Unigene in NR, NT, Swiss-Prot, KEGG, COG, GO storehouse respectively, and 63458,49750,45573,28939,55950, the Unigene on all annotations is 83169.Predictive coding albumen frame (CDS), comparison has 75735 to the CDS of protein pool, and the CDS doped has 5305, totally 81040.Annotated by GO, the software analysis such as Blast comparative analysis and MEGA6.0 phylogenetic tree construction, therefrom obtain Herba Saussureae Involueratae cell Phenylalanine Ammonia-Lyase Gene man family sequence.
Described Herba Saussureae Involueratae cell is that the method recorded in the patent of invention of 200510115902.0 obtains according to application number, choose saussurea involucrata in vitro tissue, the callus formed through dedifferentiation, as subculture kind, is given and is carried out the red-purple agglomerate of extensive succeeding transfer culture acquisition with certain condition.
Embodiment 2 saussurea involucrata cell Phenylalanine Ammonia-Lyase Gene screens
UPLC-qTOF-MS (Ultra Performance Liquid Chromatography-quadrupole flight time mass spectrum) is utilized to measure liposoluble ingredient in Herba Saussureae Involueratae cell, and correlation analysis is carried out to Syringin content and Phenylalanine Ammonia-Lyase Gene family member expression level in the different cell mass of above-mentioned Herba Saussureae Involueratae clone, screening and activeconstituents accumulate closely-related Herba Saussureae Involueratae cell Phenylalanine Ammonia-Lyase Gene, called after SiPAL (as shown in Figure 1), its nucleotide sequence is as SEQ ID No:1, the aminoacid sequence of the protein of this genes encoding is as shown in SEQ ID NO:2.
Embodiment 3 saussurea involucrata cell Phenylalanine Ammonia-Lyase Gene
The clone of SiPAL utilizes forward primer P1:5 ' ACCACAACTCTCAGCTACCCTCCTC 3 ', reverse primer P2:5 ' TGGCAGGGGAGGGATAGAATTC 3 ', with Herba Saussureae Involueratae cell phenylalanine ammonia lyase SiPAL full length sequence for template carries out pcr amplification.Amplification system is as follows: 10 × Buffer 2.5 μ L, the each 1 μ L of dNTP (2.5mmolL-1) 1 μ L, primer P1 and P2, Taq archaeal dna polymerase (5UL-1) 0.2 μ L, template 1 μ L (about 20ng), all the other are supplied with aseptic double-distilled water.Reaction conditions: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 56 DEG C of annealing 30s, 72 DEG C extend 2min30s, 35 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.So far Herba Saussureae Involueratae Phenylalanine Ammonia-Lyase Gene SiPAL is obtained.
The bioinformatic analysis of embodiment 4 SiPAL gene
The length of the Herba Saussureae Involueratae Phenylalanine Ammonia-Lyase Gene SiPAL full-length cDNA that the present invention relates to is 2405bp, and detailed sequence is as the SEQ ID NO.1 in sequence table, and wherein opening code-reading frame is positioned at 120-2264bp.Blast program in SiPAL gene order ncbi database is carried out nucleotide homology retrieval in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR database.This gene has higher homology with the PAL in other species on amino acid levels, has typical PAL-HAL structural domain simultaneously.As Fig. 2 and Fig. 3.Wherein Fig. 2 is SiPAL Structure and function domain forecast analysis (deriving from ncbi database); Fig. 3 is SiPAL systematic evolution tree (adjacent method).
The structure of embodiment 5 SiPAL gene plant expression vector
With SiPAL cDNA for template, design specific upstream primer P3 and downstream primer P4, carry out pcr amplification reaction, in primer, dashed part is restriction enzyme site.
| Primer | Sequence names | Base sequence (5 ' → 3 ') |
| P3 | SEQ ID NO.3 | CATATGCCCCATTCAATCATCTC |
| P4 | SEQ ID NO.4 | CTCGAGGCAACAAAATACTAAAACGT |
PCR reaction system (25 μ L) is: containing 10 × PCR damping fluid 2.5 μ L, dNTP (2.5mmolL-1) 1 μ L, each 0.2 μ L, Taq archaeal dna polymerase (5UL-1) the 0.2 μ L and template 1 μ L (about 20ng) of diagnostic primers (10 μm of olL-1) in reaction system, supply reaction volume with aseptic double-distilled water.
PCR reaction conditions: 95 DEG C of denaturation 5min, 95 DEG C of 30s, 56 DEG C of annealing 30s, 72 DEG C extend 2min30s, 35 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.
Get 5 μ l amplified productions to add 3 μ l bromine Finland and carry out agarose gel electrophoresis, take a picture after half an hour, observe glue figure, amplified fragments is 2144bp.Cut amplified production 2 hours with NdeI and XhoI enzyme at 37 DEG C, utilize DNA gel to reclaim test kit (Takara company, China) purifying digestion products.Utilize NdeI and XhoI enzyme at 37 DEG C to cut pET-31b (+) carrier 2 hours simultaneously, add 5 μ l bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize DNA gel to reclaim test kit recovery 5300bp fragment.
The two spends the night 8 DEG C of connections through T4 ligase enzyme.Electroporated bacillus coli DH 5 alpha competent cell, the LB flat board containing penbritin screens recon.Containing the pET plasmid of SiPAL clone through PCR and digestion with restriction enzyme electroresis appraisal (as Fig. 5) and DNA sequence analysis, preserve there is correct target sequence recombinant plasmid pET-SiPAL for expressing conversion.In Fig. 4, swimming lane 1 is 1kb DNA ladder, and swimming lane 2 cuts pET-SiPAL product for XhoI enzyme, and swimming lane 3 cuts pET-SiPAL product for NdeI and XhoI enzyme, this plant expression vector called after pET-SiPAL (Fig. 4).
The abduction delivering of embodiment 6 engineering bacteria
With target plasmid pET-SiPAL electricity Transformed E .coli BL21 (DE3) competent cell, cultivate the positive strain (E.coli BL21 (DE3)-SiPAL) of screening containing pET-SiPAL plasmid.This bacterial strain contains can the restructuring t7 rna polymerase gene of abduction delivering, thus starts the high expression of the SiPAL recombination of the upper T7 promotor control of pET-SiPAL.Positive colony E.coliBL21 (DE3)-SiPAL is inoculated in the LB substratum containing 50 μ g/ml penbritins, 37 DEG C of shaking culture 12-16h, OD600=0.9-1.0, be inoculated in fresh in the substratum of antibiotics ampicillin with 1: 100 ratio, 37 DEG C of shaking culture 3 ~ 5h increase, get 1ml bacterium liquid as contrast before induction, then the isopropylthio-β-D-galactoside (IPTG) that final concentration is 1mmol/L is added, engineering bacterium expression is induced in 37 DEG C of shaking culture, 1ml is sampled after induction 3h, with E.coli BL21 (DE3) culture containing pET-31b (+) empty carrier for negative control.SDS-polyacrylamide gel electrophoresis result shows, is about 76.3kD place at molecular weight, occurs an obvious specific protein band of expression, consistent with theoretical value (as Fig. 6).Wherein swimming lane 1 is Protein Ruler Marker l, and swimming lane 2 is E.coli BL21 (DE3) culture containing pET-31b (+) empty carrier; Swimming lane 3 is the culture containing pET-SiPAL.
The preparation of embodiment 7 crude enzyme liquid
1. the preparation of thick zyme extract: obtain positive strain (E.coli BL21 (DE3)-SiPAL) containing pET-SiPAL plasmid in the LB substratum containing 50 μ g/ml penbritins in embodiment 6,37 DEG C of shaking culture, after cell density reaches OD600=1.8-2.0,12000r/min, 4 DEG C, centrifugal 1min.Collect bacterial sediment, be resuspended in PBS (pH7.4) damping fluid, wash 2 times.Add the N,O-Diacetylmuramidase of 10mg/ml, room temperature places 15min, then adds 100 μ l cell pyrolysis liquids (containing 1mmol/LDTT, 0.1mmol/L EDTA, PBS, the pH7.4 of 10% glycerine), thermal agitation 1min, ice bath 1min, repeats 6 times.12000r/min, 4 DEG C, centrifugal 20min, Aspirate supernatant, as thick zyme extract, is put for subsequent use on ice.
2.PAL determination of activity: determination of activity is undertaken by following condition: reaction system is 0.1mol/L Tris-HCl (pH8.8) damping fluid 600 μ l, 0.01mol/L L-Phe (L-Phe) 150 μ l, thick zyme extract 30 μ l; Blank is with 30 μ l ddH
2o substitutes crude enzyme liquid.37 DEG C of water bath heat preservation reaction 1h.With the OD before and after ultraviolet spectrophotometer assaying reaction during 0h and 1h
290, with the actual vigor of the two mathematic interpolation cell crude enzyme liquid PAL.Enzyme activity unit adopts international unit of enzyme (IU), both generates 1 μm of ol styracin for 1IU with per minute.The unit of activity of formula specific activity of enzyme=enzyme/crude enzyme liquid protein content is utilized to calculate the Rate activity of restructuring PAL enzyme, find that SiPAL transgenic engineering bacterial strain (E.coli BL21 (D32)-SiPAL) is 237.3 μm of ol/ming pro, namely 237.3U/g, SiPAL protein (phenylalanine ammonia lyase) expression amount reaches about the 9wt% of total protein content.
Claims (9)
1. the SiPAL gene of Herba Saussureae Involueratae (Saussurea involucrata) cell, it is characterized in that, the length of this gene cDNA is 2405bp, and opening code-reading frame is between 120-2264bp, and the nucleotide sequence of this gene is as shown in SEQ ID NO:1.
2. the protein of SiPAL genes encoding according to claim 1, its aminoacid sequence is as shown in SEQ ID NO:2.
3. the primer of amplification SiPAL gene according to claim 1, its sequence is as shown in SEQ ID NO:3 ~ 4.
4. the recombinant expression vector containing gene according to claim 1.
5. the host cell containing gene according to claim 1.
6. host cell according to claim 5, is characterized in that, this host cell contains recombinant expression vector according to claim 4.
7. the application of SiPAL gene according to claim 1 in saussurea involucrata and close kind vegetable cell and/or tissue culture thereof.
8. SiPAL gene according to claim 1 is utilizing the application in Biological preparation lignin composition.
9. application according to claim 8, is characterized in that, described lignin composition is Syringin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510256188.0A CN104894150B (en) | 2015-05-18 | 2015-05-18 | Saussurea involucrate cell phenylalanine ammonia lyase gene SiPAL and encoding product and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510256188.0A CN104894150B (en) | 2015-05-18 | 2015-05-18 | Saussurea involucrate cell phenylalanine ammonia lyase gene SiPAL and encoding product and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104894150A true CN104894150A (en) | 2015-09-09 |
| CN104894150B CN104894150B (en) | 2020-06-23 |
Family
ID=54027109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510256188.0A Active CN104894150B (en) | 2015-05-18 | 2015-05-18 | Saussurea involucrate cell phenylalanine ammonia lyase gene SiPAL and encoding product and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104894150B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647943A (en) * | 2016-03-14 | 2016-06-08 | 大连普瑞康生物技术有限公司 | Saussurea involucrata cell squalene synthase (SiSQS) gene as well as products coded by same and application thereof |
| CN116004673A (en) * | 2022-12-19 | 2023-04-25 | 中国科学院青岛生物能源与过程研究所 | Saussurea involucrata syringin SiUGT72E gene and encoding product and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305454A (en) * | 2012-03-14 | 2013-09-18 | 中国医学科学院药物研究所 | Method for producing Syringin, chlorogenic acid and 1, 5-dicaffeoylquinic acid by cell culture of herba saussureae involucratae |
| CN103374586A (en) * | 2012-04-16 | 2013-10-30 | 中国中医科学院中药研究所 | Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene |
-
2015
- 2015-05-18 CN CN201510256188.0A patent/CN104894150B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305454A (en) * | 2012-03-14 | 2013-09-18 | 中国医学科学院药物研究所 | Method for producing Syringin, chlorogenic acid and 1, 5-dicaffeoylquinic acid by cell culture of herba saussureae involucratae |
| CN103374586A (en) * | 2012-04-16 | 2013-10-30 | 中国中医科学院中药研究所 | Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene |
Non-Patent Citations (3)
| Title |
|---|
| UNIPROT: "UniProtKB-B1GS31", 《UNIPROT》 * |
| 刘永刚: "雪莲细胞培养及其次生代谢产物黄酮的代谢调控", 《中国优秀硕士学位论文全文数据库》 * |
| 赵康: "桑葚中白藜芦醇及其苷的含量和光稳定性研究及MaPAL基因的克隆分析", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647943A (en) * | 2016-03-14 | 2016-06-08 | 大连普瑞康生物技术有限公司 | Saussurea involucrata cell squalene synthase (SiSQS) gene as well as products coded by same and application thereof |
| CN105647943B (en) * | 2016-03-14 | 2020-05-05 | 大连普瑞康生物技术有限公司 | Saussurea involucrate cell squalene synthase gene SiSQS and coded product and application thereof |
| CN116004673A (en) * | 2022-12-19 | 2023-04-25 | 中国科学院青岛生物能源与过程研究所 | Saussurea involucrata syringin SiUGT72E gene and encoding product and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104894150B (en) | 2020-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2460789C2 (en) | New hydrogenase seq id no:5 purified from thermococcus onnurienus na1 with help of carbon monoxide, genes coding it, and methods for producing hydrogen with use of microorganism having said genes | |
| CN105647943A (en) | Saussurea involucrata cell squalene synthase (SiSQS) gene as well as products coded by same and application thereof | |
| CN106947766A (en) | A kind of bacillus subtilis has DNA fragmentation and its application of promoter function | |
| Lovell et al. | Community‐level analysis: key genes of CO2‐reductive acetogenesis | |
| CN104894150A (en) | A saussurea involucrata cell phenylalanine ammonia lyase gene SiPAL, a coding product thereof and applications of the gene | |
| CN112410356B (en) | Resveratrol synthase gene RS from radix tetrastigme and application thereof | |
| CN103087998B (en) | Enzyme for synthesizing cetyl-coenzyme A through cordyceps sinensis, gene and application thereof | |
| CN107384893A (en) | A kind of thermostable lipase and its preparation and application | |
| CN107365755A (en) | A kind of novel lipase and its gene, engineering bacteria and preparation method | |
| CN101812434B (en) | Invertase and application of encoding gene thereof | |
| CN103374586B (en) | Lonicera japonica thunb phenylalanine ammonia-lyase (LJPAL) gene as well as product coded by same and application of gene | |
| Shi et al. | Characterization of Three Genes Encoding the Subunits of Light‐Independent Protochlorophyllide Reductase in Chlorella protothecoides CS‐41 | |
| CN107937359A (en) | The clonal expression and purposes of Chinese toon cinnamoyl_CoA reductase gene | |
| CN111763250A (en) | Application of gene in improving salt tolerance of plant | |
| CN105924512A (en) | GhLPAAT5-like protein relevant with grease content as well as coding gene and application of GhLPAAT5-like protein | |
| CN112410352A (en) | 4-coumaric acid-coenzyme A ligase gene Th4CL and application thereof | |
| CN103937810B (en) | Super enriching plant black nightshade metallothionein gene（MT‑L3）Sequence and its cloning process | |
| CN110616206A (en) | Tiger skin fish PtScd1 gene and application thereof | |
| CN104120112B (en) | Cordyceps sinensis 3-Isopropylmalate dehydrogenase B, encoding gene and application thereof | |
| CN103374582B (en) | Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene and coded product thereof and application | |
| KR20090060124A (en) | Novel hydrogenases purified from thermococcus spp., genes encoding them, and methods for producing hydrogen using microorganism having the genes | |
| Recipon et al. | ATP synthase subunit c/III/9 gene sequences as a tool for interkingdom and metaphytes molecular phylogenies | |
| CN117448350B (en) | Application of Phellinus linteus Pb-bHLH9 gene in improving multiple stress resistance of Saccharomyces cerevisiae | |
| Keum et al. | Complete genome sequence of Flavisolibacter ginsenosidimutans Gsoil 636 T, a ginsenoside-converting bacterium, isolated from soil used for cultivating ginseng | |
| CN104120114B (en) | Cordyceps 3-Isopropylmalate dehydrogenase A, encoding gene and application thereof |
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 | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Phenylalanine ammonia lyase gene sipal from Saussurea involucrata and its coding products and Application Effective date of registration: 20200930 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020210000054 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210929 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020210000054 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Phenylalanine ammonia lyase gene sipal in Saussurea involucrata cells and its coding product and Application Effective date of registration: 20210930 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021210000067 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20221213 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021210000067 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Phenylalanine ammonia lyase gene SiPAL from Saussurea involucrata cells and its coding products and application Effective date of registration: 20221214 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2022210000215 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231214 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2022210000215 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: The phenylalanine ammonia lyase gene SiPAL in Tianshan Snow Lotus cells and its encoded products and applications Effective date of registration: 20231218 Granted publication date: 20200623 Pledgee: Dalian Xigang District Enterprise Credit Financing Guarantee Co.,Ltd. Pledgor: DALIAN PRACTICAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023980072352 |