CN106244599A - 一种三七病程相关蛋白1家族基因PnPR1‑2及应用 - Google Patents
一种三七病程相关蛋白1家族基因PnPR1‑2及应用 Download PDFInfo
- Publication number
- CN106244599A CN106244599A CN201610836098.3A CN201610836098A CN106244599A CN 106244599 A CN106244599 A CN 106244599A CN 201610836098 A CN201610836098 A CN 201610836098A CN 106244599 A CN106244599 A CN 106244599A
- Authority
- CN
- China
- Prior art keywords
- pnpr1
- plant
- gene
- pathogenesis
- radix notoginseng
- 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
- 101710096342 Pathogenesis-related protein Proteins 0.000 title claims abstract description 14
- 241000180649 Panax notoginseng Species 0.000 title claims description 22
- 235000003143 Panax notoginseng Nutrition 0.000 title claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 36
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 36
- 235000018102 proteins Nutrition 0.000 claims abstract description 17
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 241000223218 Fusarium Species 0.000 claims abstract description 12
- 241000427940 Fusarium solani Species 0.000 claims abstract description 9
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims abstract description 6
- 241000208340 Araliaceae Species 0.000 claims abstract description 5
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims abstract description 5
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 5
- 235000008434 ginseng Nutrition 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000894006 Bacteria Species 0.000 claims description 14
- 241000233732 Fusarium verticillioides Species 0.000 claims description 6
- 108091064702 1 family Proteins 0.000 claims description 5
- 241000212253 Alternaria panax Species 0.000 claims description 2
- 108091026890 Coding region Proteins 0.000 claims description 2
- 244000061458 Solanum melongena Species 0.000 claims description 2
- 235000002597 Solanum melongena Nutrition 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 55
- 238000000034 method Methods 0.000 abstract description 22
- 230000009261 transgenic effect Effects 0.000 abstract description 14
- 241000233866 Fungi Species 0.000 abstract description 11
- 230000000843 anti-fungal effect Effects 0.000 abstract description 11
- 230000002018 overexpression Effects 0.000 abstract description 5
- 229940121375 antifungal agent Drugs 0.000 abstract description 4
- 239000013604 expression vector Substances 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 4
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 244000052769 pathogen Species 0.000 description 11
- 230000001717 pathogenic effect Effects 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- 108700019146 Transgenes Proteins 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 208000035240 Disease Resistance Diseases 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 5
- 229960004889 salicylic acid Drugs 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 208000031888 Mycoses Diseases 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000021918 systemic acquired resistance Effects 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000223221 Fusarium oxysporum Species 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 2
- 101150054548 PR1 gene Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
- 241000219095 Vitis Species 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- 229960000723 ampicillin Drugs 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
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- BVLPIIBTWIYOML-ZKWXMUAHSA-N Ala-Val-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BVLPIIBTWIYOML-ZKWXMUAHSA-N 0.000 description 1
- SGYSTDWPNPKJPP-GUBZILKMSA-N Arg-Ala-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SGYSTDWPNPKJPP-GUBZILKMSA-N 0.000 description 1
- GXCSUJQOECMKPV-CIUDSAMLSA-N Arg-Ala-Gln Chemical compound C[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GXCSUJQOECMKPV-CIUDSAMLSA-N 0.000 description 1
- VYZBPPBKFCHCIS-WPRPVWTQSA-N Arg-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N VYZBPPBKFCHCIS-WPRPVWTQSA-N 0.000 description 1
- YNDLOUMBVDVALC-ZLUOBGJFSA-N Asn-Ala-Ala Chemical compound C[C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CC(=O)N)N YNDLOUMBVDVALC-ZLUOBGJFSA-N 0.000 description 1
- YYSYDIYQTUPNQQ-SXTJYALSSA-N Asn-Ile-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YYSYDIYQTUPNQQ-SXTJYALSSA-N 0.000 description 1
- HPNDKUOLNRVRAY-BIIVOSGPSA-N Asn-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N)C(=O)O HPNDKUOLNRVRAY-BIIVOSGPSA-N 0.000 description 1
- JPPLRQVZMZFOSX-UWJYBYFXSA-N Asn-Tyr-Ala Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 JPPLRQVZMZFOSX-UWJYBYFXSA-N 0.000 description 1
- USENATHVGFXRNO-SRVKXCTJSA-N Asp-Tyr-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 USENATHVGFXRNO-SRVKXCTJSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000555706 Botryosphaeria dothidea Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- MBPKYKSYUAPLMY-DCAQKATOSA-N Cys-Arg-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MBPKYKSYUAPLMY-DCAQKATOSA-N 0.000 description 1
- KIQKJXYVGSYDFS-ZLUOBGJFSA-N Cys-Asn-Asn Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O KIQKJXYVGSYDFS-ZLUOBGJFSA-N 0.000 description 1
- ZMWOJVAXTOUHAP-ZKWXMUAHSA-N Cys-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N ZMWOJVAXTOUHAP-ZKWXMUAHSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 101100117236 Drosophila melanogaster speck gene Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000221785 Erysiphales Species 0.000 description 1
- OFPWCBGRYAOLMU-AVGNSLFASA-N Gln-Asp-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O OFPWCBGRYAOLMU-AVGNSLFASA-N 0.000 description 1
- SOEXCCGNHQBFPV-DLOVCJGASA-N Gln-Val-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O SOEXCCGNHQBFPV-DLOVCJGASA-N 0.000 description 1
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 1
- CEXINUGNTZFNRY-BYPYZUCNSA-N Gly-Cys-Gly Chemical compound [NH3+]CC(=O)N[C@@H](CS)C(=O)NCC([O-])=O CEXINUGNTZFNRY-BYPYZUCNSA-N 0.000 description 1
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 1
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 1
- QIVPRLJQQVXCIY-HGNGGELXSA-N His-Ala-Gln Chemical compound C[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](CCC(N)=O)C(O)=O QIVPRLJQQVXCIY-HGNGGELXSA-N 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- CSTDQOOBZBAJKE-BWAGICSOSA-N His-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N)O CSTDQOOBZBAJKE-BWAGICSOSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-DKIMLUQUSA-N Ile-Phe-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(O)=O XLXPYSDGMXTTNQ-DKIMLUQUSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 1
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 1
- HQBOMRTVKVKFMN-WDSOQIARSA-N Leu-Trp-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C(C)C)C(O)=O HQBOMRTVKVKFMN-WDSOQIARSA-N 0.000 description 1
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 1
- WYEXWKAWMNJKPN-UBHSHLNASA-N Met-Ala-Phe Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCSC)N WYEXWKAWMNJKPN-UBHSHLNASA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 241000208136 Nicotiana sylvestris Species 0.000 description 1
- BARPGRUZBKFJMA-SRVKXCTJSA-N Pro-Met-Arg Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@@H]1CCCN1 BARPGRUZBKFJMA-SRVKXCTJSA-N 0.000 description 1
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- QDDJNKWPTJHROJ-UFYCRDLUSA-N Pro-Tyr-Tyr Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 QDDJNKWPTJHROJ-UFYCRDLUSA-N 0.000 description 1
- FIDNSJUXESUDOV-JYJNAYRXSA-N Pro-Tyr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O FIDNSJUXESUDOV-JYJNAYRXSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 1
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 1
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- IXZHZUGGKLRHJD-DCAQKATOSA-N Ser-Leu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IXZHZUGGKLRHJD-DCAQKATOSA-N 0.000 description 1
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 1
- FLMYSKVSDVHLEW-SVSWQMSJSA-N Ser-Thr-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLMYSKVSDVHLEW-SVSWQMSJSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- UUSQVWOVUYMLJA-PPCPHDFISA-N Thr-Lys-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UUSQVWOVUYMLJA-PPCPHDFISA-N 0.000 description 1
- NKUIXQOJUAEIET-AQZXSJQPSA-N Trp-Asp-Thr Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@H](O)C)C(O)=O)=CNC2=C1 NKUIXQOJUAEIET-AQZXSJQPSA-N 0.000 description 1
- GWQUSADRQCTMHN-NWLDYVSISA-N Trp-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O GWQUSADRQCTMHN-NWLDYVSISA-N 0.000 description 1
- NOOMDULIORCDNF-IRXDYDNUSA-N Tyr-Gly-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NOOMDULIORCDNF-IRXDYDNUSA-N 0.000 description 1
- FPCIBLUVDNXPJO-XPUUQOCRSA-N Val-Cys-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O FPCIBLUVDNXPJO-XPUUQOCRSA-N 0.000 description 1
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000014005 Vitis hybrid cultivar Nutrition 0.000 description 1
- 241000972888 Vitis hybrid cultivar Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229960004261 cefotaxime Drugs 0.000 description 1
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- UCKZMPLVLCKKMO-LHLIQPBNSA-N cephamycin Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](C)[C@]21OC UCKZMPLVLCKKMO-LHLIQPBNSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000003967 crop rotation Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010582 ecthyma Diseases 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 1
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000003032 phytopathogenic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 101150108007 prs gene Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 108010029895 rubimetide Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种三七的病程相关蛋白1家族基因PnPR1‑2，其核苷酸序列如SEQ ID NO：1所述，编码如SEQ ID NO：2所示氨基酸序列的蛋白质，本发明通过功能基因组学相关技术研究证实PnPR1‑2基因具有提高植物抗真菌的功能，将本发明抗真菌PnPR1‑2基因构建到植物表达载体上并转入烟草中过量表达，结果转基因烟草植株具有很强的体外抗真菌活性，超表达PnPR1‑2的转基因烟草对茄腐镰刀菌、轮枝镰刀菌、串珠状赤霉菌、人参链格孢等多种真菌的生长具有明显的抑制作用。
Description
技术领域
本发明涉及分子生物学以及基因工程相关技术研究领域，特别是一种具有抗真菌活性的三七病程相关蛋白1家族基因PnPR1-2及应用。
背景技术
植物病害是农业生产中一个非常棘手的问题，尤其是真菌病害，约占到植物总病害的80％，严重影响农作物的产量和品质。传统的病害防治方法取得了一定成效，一是依靠传统育种方法培育抗性品种，二是化学农药的使用，三是采取轮作等耕作制度。然而，这些方法都存在或多或少的弊端，如抗性品种培育的周期长、化学农药残留高且极易造成环境污染、耕作制度调整则费时费力，所以传统控制植物病害的方法均不能彻底解决问题。随着重组DNA技术的创立和发展，利用基因工程技术来培育抗病植物新品种已取得初步成效，且有望从根本上解决真菌病害问题。
植物具有保护自身免受各种病原体侵害的防卫机制。在植物体受到病原体入侵时，植物通过迅速改变抗病相关基因表达来对病原菌和外部胁迫做出反应，诱导一些特殊蛋白的重新合成，如病程相关蛋白（pathogenesis-related proteins，PRs）。植物细胞在病原菌攻击后使病原的侵染局部化，并在侵染的部位形成坏死斑限制病原的扩展（Loon LCV,Strien EAV. The families of pathogenesis-related proteins, their activities,and comparative analysis of PR-1 type proteins. Physiological and MolecularPlant Pathology, 1999, 55(2): 85-97）。PRs基因是植物防卫系统的重要组成部分，与超敏反应（hypersensitive response，HR）及系统获得性抗性（systematic acquireresistance，SAR）密切相关，它的诱导表达常作为SAR建立的标志，同时其基因编码产物已成为近年来抗性研究的热点之一。
PR1首先在被TMV侵染的烟草叶片中检测到，它与超敏反应及系统获得抗性密切相关。植物获得性系统抗性是指在同一植物上未感染部位出现的抗性，这种抗性在植物受到再次侵染时，不仅对与初次感染相同的病原物表现出抗性，而且往往对其他类型的病原物也起作用(Carr JP, Beachy RN, Klessig DF. Are the PR1 proteins of tobaccoinvolved in genetically engineered resistance to TMV?. Virology, 1989, 169(2): 470-473)。经TMV感染后诱导的烟草系统获得抗性，不仅对TMV本身有作用，而且对其他的病原物如烟草枯斑病毒、真菌和细菌也有抵抗作用（郭金芳, 潘俊松, 王琛, 赵智燕,何亚丽. 病程相关蛋白与植物抗病性关系的研究及其在草坪草抗病育种中的应用. 草业学报, 2008, 17(6): 156-163）。植物的SAR可能包括多种机制，但是，烟草对真菌、细菌或病毒的共同反应都是产生PR1，并且在具有系统获得抗性的未感染的叶片上也出现PR1蛋白，这说明PR1和植物的SAR之间有紧密的联系。
水杨酸（salicylic acid，SA）能通过诱导植物体中PR1基因的表达，提高植物的抗病性。在HR发生前，有一个短暂的氧爆发阶段，使细胞中活性氧的浓度明显提高。而活性氧可以直接杀伤病原物、参与膜脂过氧化、导致HR，而且能促进寄主细胞壁木质化，增强细胞壁的结构，诱导木质素的产生。同时H2O2是一种能自由扩散的小分子，可以跨过细胞膜进入病原物侵染以外的组织中，作为第二信使来激活防卫基因的表达，提高植物抗病性。SA是将病害和创伤的信息传递到植物其他部分的信号分子之一（Métraux JP. Recentbreakthroughs in the study of salicylic acid biosynthesis. Trends in PlantScience, 2002, 7(8): 332-334）。SA是诱发PR1的信号分子，病菌侵染植物体后，活性氧瞬时达到高峰，寡糖素转化为水杨酸，通过胞内信号传递，PR1基因表达开始表达，产生菌类细胞壁水解酶（吴建丽, 郝建军. 水杨酸与植物诱导抗病性. 辽宁林业科技, 2005, (1):33-35）。
本发明的病程相关蛋白1家族基因PnPR1-2来自三七（Panax notoginseng）。三七主产于云南文山州砚山县、马关、西畴、广南、麻栗坡、富宁、邱北等，另广西田阳、靖西、田东、德保等地也有种植。云南文山州三七历史悠久、产量大、质量好，为著名的道地药材。三七常年生长在阴蔽环境中，病害发生较为严重。三七上发生的病害主要的有根腐病、黑斑病、圆斑病、疫病、白粉病等，三七每年都因病害的大面积发生造成极为严重的损失。因此，加强三七抗病机制研究以及抗性基因克隆迫在眉睫。
发明内容
本发明的目的是提供一种从三七中克隆获得编码具有抗真菌活性的病程相关蛋白1家族基因PnPR1-2，三七病程相关蛋白1家族基因PnPR1-2核苷酸序列如SEQ ID NO：1所示，该基因cDNA全长序列为852bp，包含一个501bp的开放阅读框、104bp的5’非翻译区、247bp的3’非翻译区，编码如SEQ ID NO：2所示氨基酸序列的蛋白质。
本发明中PnPR1-2基因的编码区是序列表SEQ ID NO：1中第105-605位所示的核苷酸序列。
本发明分离三七的一个抗真菌相关基因的完整cDNA片段，利用根癌农杆菌(Agrobacterium tumefaciens)介导将目的基因转入受体植物中并过量表达，通过进一步实验验证该基因是否具有抗真菌的活性，为后期利用该基因改良烟草及其他植物抵御真菌病害的能力奠定基础，发明人将这个基因命名为PnPR1-2。
PR1蛋白具有与体外抑菌活性，是植物受病原物侵染或非生物因子刺激后产生的一类水溶性蛋白。PR1蛋白通过攻击病原物、降解细胞壁大分子、降解病原物毒素等途径来提高植物对病原菌的抗性。
本发明涉及分离包含PnPR1-2的DNA片段并鉴定其功能，其中所述DNA片段如序列表SEQ ID NO：1所示，对该基因进行序列分析，发现PnPR1-2全长cDNA为852 bp，包含一个501 bp的开放阅读框(ORF)、104bp的5’非翻译区(untranslated region，UTR)、247 bp的3’UTR，其中ORF编码一个具有166个氨基酸的蛋白质。通过NCBI/BIastp进行同源性分析表明，PnPR1-2基因编码蛋白与葡萄（Vitis hybrid cultivar）、烟草（Nicotiana sylvestris）PR1蛋白相似性分别为68%、63%。PnPR1-2基因编码一个由166个氨基酸残基组成的蛋白质，生物信息学分析结果显示PnPR1-2蛋白质分子质量约为18.1KD，等电点为9.16。超表达序列表SEQ ID NO：1所示序列可以增强烟草对茄腐镰刀菌（Fusarium solani）、轮枝镰刀菌（F. verticillioides）、串珠状赤霉菌（Gibberella moniliformis）、人参链格孢菌（Alternaria panax）的抗性。
上述PnPR1-2基因可以应用于提高烟草的抗真菌特性，具体操作如下：
（1）采用扩增PnPR1-2的特异引物，从接种茄腐镰刀菌后的三七根中提取总RNA，以oligo(dT)18为反转录引物，通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction，RT-PCR)扩增出PnPR1-2编码区，然后将其连接到pGEM-T载体上，经测序获得具有目的基因的克隆；
（2）用限制性内切酶BamHI和EcoRI酶切pGEM-T-PnPR1-2，通过胶回收得到目的基因片段，用同样的内切酶酶切植物表达载体pCAMBIA2300s，胶回收获得所需载体大片段，再将所获得PnPR1-2基因片段与pCAMBIA2300s片段连接，构建植物超表达载体，之后将所构建的重组载体通过根癌农杆菌介导转入烟草中表达；
（3）以重组载体T-DNA上具有的抗性标记筛选转化子，并通过PCR以及RT-PCR检测得到真正的转基因植株，分析转基因植物蛋白对真菌生长的抑制活性，最后筛选出对真菌抗性明显增强的转基因植株。
本发明为提高植物对真菌病害的抗性提供了一种新的方法，通过基因工程手段培育抗病植物可以克服传统育种的不足，不仅育种周期缩短，而且操作简单，容易获得高抗材料。本发明中来自三七的PnPR1-2基因能增强植物对几种病原真菌的抗性，将该基因导入烟草中，可以产生具有真菌抗性的新品种和新材料。利用基因工程技术培育抗性植物品种和材料具有明显的优势和不可取代的重要性。它不仅可以为大规模生产作物、花卉、药用植物等提供方便，减少化学农药的使用，还可以为农业生产节约成本、减少环境污染，因此本发明具有广阔的市场应用前景。
附图说明
图1是本发明PnPR1-2转基因烟草基因组DNA的PCR检测结果示意图，图中：Marker为DL2000 DNA Marker (大连宝生物)；阳性对照为质粒pGEM-T-PnPR1-2为模板的PCR结产物；WT为非转基因烟草(野生型)总DNA为模板PCR的产物；
图2是本发明阳性PnPR1-2转基因烟草中PnPR1-2转录水平的表达分析结果图；图中：Marker是DL2000 DNA Marker (大连宝生物)；WT是非转基因烟草总RNA逆转录cDNA为模板的PCR产物；阳性对照是质粒pGEM-T-PnPR1-2为模板的PCR产物；
图3是本发明PnPR1-2转基因烟草体外抑菌活性效果图；图中a、b、c、d分别是对茄腐镰刀菌、轮枝镰刀菌、串珠状赤霉菌、人参链格孢；WT为野生型烟草的总蛋白；CK为空白对照，即无蛋白对照(用于提取蛋白的缓冲液)。
具体实施方式
下面通过附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：PnPR1-2全长基因克隆以及序列分析
用茄腐镰刀菌接种一年生三七的根部，取接种后12 h的根提取总RNA。采用逆转录酶M-MLV (promega)以总RNA为模板合成cDNA第一链，反应体系和操作过程为：取5 μg TotalRNA，依次加入50 ng oligo（dT）、2 μL dNTP（2.5mM each）、DEPC水至反应体积为14.5 μL；混匀后，70℃加热变性5 min后迅速在冰上冷却5 min，然后依次加入4 μL 5×First-standbuffer、0.5μL RNasin（200U）、1μL M-MLV（200U），混匀并短时离心，42℃温浴1.5 h，取出后70℃加热10 min，终止反应。cDNA第一链合成后置于-20℃保存备用。
以合成的第一链cDNA为模板，扩增目的基因PnPR1-2，所用上下游引物序列分别为5’CTTGTTCTATAAATAATCTTCATTGC3’及5’ATATGGATGAGAATTGTGTAAAAAG3’。采用AdvantageTM2 PCR Enzyme（Clontech）扩增出目的基因；PCR反应条件：95℃ 1 min；95℃ 30 s，55℃ 30s，72℃ 40s，30个循环；72℃ 5 min；反应体系（10 μL）为1 μL cDNA、1 μL 10×Advantage2 PCR Buffer、0.5 μL 50×dNTP Mix （10mM each）、0.2 μL 正向引物（10 μM）、0.2μL 反向引物（10 μM）、0.2 μL Advantage 2 PCR Polymerase Mix、6.9 μL PCR-Grade water；PCR结束后，取5 μL用于琼脂糖凝胶电泳，以检测扩增产物的特异性以及大小。
所得到PCR产物只有一条DNA带，故直接对PCR产物进行TA克隆，使用的试剂盒为pGEM-T vector（Promega），反应体系和操作过程为：取1.5 μL PCR产物，依次加入1 μLpGEM-T vector（50 ng/μL）和2.5 μL 2×Ligation solution I，混匀后置于16 ℃过夜反应；采用热激转化法将连接产物转入大肠杆菌DH5α中；使用含有氨苄青霉素（ampicillin，Amp）的LB固体培养基筛选阳性克隆，挑选若干个单菌落，摇菌后用扩增PnPR1-2的特异引物鉴定出多克隆位点插入PnPR1-2的克隆，将所鉴定的克隆进行测序，最终获得的PnPR1-2全长cDNA为852bp，通过NCBI ORF finder （http://www.ncbi.nlm.nih.gov/gorf/gorf.html）分析发现其包含一个501bp的开放读码框（见序列表），PnPR1-2编码一个含166个氨基酸的蛋白质PnPR1-2，其蛋白质分子质量约为18.1 k D，等电点(pI)约为9.16。其中包括7个酸性氨基酸(D，E)、19个碱性氨基酸(K，R，H)、72个疏水氨基酸和89个极性氨基酸。信号肽预测结果显示PnPR1-2蛋白第1至第22个氨基酸为信号肽。
实施例2：植物超表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒（上海生工）提取插入PnPR1-2的大肠杆菌质粒pGEM-T- PnPR1-2以及植物表达载体pCAMBIA2300s的质粒，取1 μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性及浓度高低；用限制性内切酶BamHI（TaKaRa）和EcoRI（TaKaRa）分别对质粒pGEM-T- PnPR1-2和pCAMBIA2300s进行双酶切（100 μL体系），反应体系和操作过程为：取20 μL pGEM-T- PnPR1-2和pCAMBIA2300s质粒，分别依次加入10 μL 10×Kbuffer、5μL BamHI、5 μL EcoRI、60 μL ddH2O，混匀后短时离心，置于37℃过夜反应；将所有酶切产物点于琼脂糖凝胶中进行电泳，然后分别对PnPR1-2片段和pCAMBIA2300s载体大片段进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒（上海生工）；取1 μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度，其余产物置于-20℃保存备用。
利用T4 DNA Ligase（TaKaRa），将回收的PnPR1-2 DNA片段和pCAMBIA2300s载体片段连接起来，反应体系（20 μL）和操作过程为：取10 μL PnPR1-2DNA片段依次加入2 μLpCAMBIA2300s载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μLddH2O，混匀后短时离心，然后16℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌DH5α中，用含有50 mg/L卡那霉素（kanamycin，Km）的固体培养基筛选阳性克隆。挑选单菌落摇菌，以菌液为模板用扩增PnPR1-2的特异引物进行PCR，挑选出PnPR1-2与pCAMBIA2300s成功连接的克隆，所检测的菌株若为阳性，加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌中的pCAMBIA2300s-PnPR1-2质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-PnPR1-2转入根癌农杆菌LBA4404感受态细胞中。操作步骤为：取2μg pCAMBIA2300s-PnPR1-2质粒加入含有200 μL感受态细胞的离心管中，轻轻混匀后冰浴5 min，随后转入液氮中冷冻1min，然后迅速置于37℃水浴5 min，之后立即冰浴2 min，加入800 μL LB液体培养基于28℃振荡培养4 h。将活化后的农杆菌涂于含有50mg/L Km的LB固体培养基上，28℃静止培养。挑选单菌落摇菌，再用扩增PnPR1-2的特异性引物进行PCR，检测pCAMBIA2300s-PnPR1-2是否转入农杆菌中，对于阳性克隆，加入甘油后置于-80℃保存备用。
实施例3：农杆菌介导的植物遗传转化以及转基因植物筛选
本实验的转基因受体是烟草，将烟草种子用75%的酒精浸泡30s，用无菌水洗涤后用0.1%的HgCl2浸泡8 min，然后再用无菌水洗涤若干次，播种于1/2 MS培养基上，28℃暗培养6d，发芽后转至光照培养箱（25℃，16h/d光照），以后每月用1/2MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300s-PnPR1-2质粒的农杆菌LBA4404菌种，接种于5 mL含有50 mg/L Km和20 mg/L利福平的LB液体培养基中，28℃培养至培养基浑浊。吸取1 mL浑浊的菌液至含有50mg/L Km的LB固体培养基上，28℃培养48 h；随后将LB固体培养基上的农杆菌刮下适量接种于附加有20 mg/L的乙酰丁香酮的MGL液体培养基中，28℃振荡培养2-3 h以活化农杆菌。
取烟草无菌苗叶片切成1 cm2左右的叶盘，完全浸泡于上述含有活化农杆菌的MGL液体培养基中，浸染时间为15 min，用无菌滤纸吸干叶盘表面的菌液，将叶盘置于共培养基上进行培养，烟草转化的共培养基为MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/L sucrose+6 g/L琼脂，22℃无光条件下共培养2天。
将共培养后的叶盘转到加有抗生素的MS筛选培养基中分化成苗，同时筛选转基因植株。烟草筛选培养基为MS+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+6g/L琼脂+50mg/L Km+200 mg/L 头孢霉素（cefotaxime sodium salt，Cef）；筛选培养时将培养瓶转移至光照培养箱培养（25℃，16h/d光照，8h/d黑暗），待烟草长出芽后用含有50 mg/L Km和200 mg/L Cef的MS培养基继代培养，因烟草愈伤分化率较高，故需要对再生植株进行进一步筛选，将烟草再生苗移至含有50 mg/L Km的MS培养基上使其生根，最后选用生根较好的再生苗进行PCR检测。
采用CTAB法提取转基因烟草植株叶片的基因组DNA，将提取的基因组DNA取1 μL通过琼脂糖凝胶电泳检测其完整性和浓度，以转基因植株的基因组DNA为模板用扩增PnPR1-2的特异引物进行PCR，PCR结束后，取8 μL产物用于琼脂糖凝胶电泳以检测阳性转基因植株，部分烟草转基因植株的扩增结果如图1所示，PnPR1-2转基因烟草共筛选到41株阳性转基因植株。
实施例4：转基因烟草中PnPR1-2的表达分析以及转基因植株抗真菌活性分析
取阳性转基因单株以及非转基因烟草（野生型）的嫩叶提取总RNA，逆转录生成cDNA第一链，并以此为模板用扩增PnPR1-2的特异引物进行PCR，根据PCR结果分析各转基因单株中PnPR1-2转录水平的表达，总RNA提取以及RT-PCR的方法与实施例1中相同，PCR结束之后，取8 μL用于琼脂糖凝胶电泳，部分单株的检测结果如图2所示，共检测到29个转基因单株中PnPR1-2在转录水平大量表达，这些单株的编号为1～29。
将实验室保存的几种真菌接种于PDA固体培养基（200 g/L马铃薯，15 g/L琼脂，20g/L葡萄糖）上，28℃暗培养，待菌落生长至直径约为2~3cm时添加蛋白，分析转基因植株体外抗真菌活性，供试真菌共有6种：茄腐镰刀菌（Fusarium solani）、轮枝镰刀菌（F. verticillioides）、串珠状赤霉菌（Gibberella moniliformis）、尖孢镰刀菌（F. oxysporum）、核盘菌（Sclerotinia sclerotiorum）、葡萄座腔菌（Botryosphaeria dothidea）。
为了防止其它杂菌污染所提取的蛋白，整个植物蛋白提取过程均是无菌操作，首先取1 g转基因烟草单株（编号分别为3、5、9、15）及野生型叶片放入研钵中，加入1 mL蛋白提取液（1M NaCl，0.1M 乙酸钠，1% PVP，pH6），充分研磨；转入1.5 mL离心管中，混匀后4℃静置过夜，4℃离心30 min（12,000 g/min），取上清于新的1.5 mL离心管中，并取适量用紫外分光光度仪测定总蛋白浓度。将转基因和野生型植株的总蛋白浓度调整至0.2 μg/μL，然后分别取20 μL滴于各真菌培养基的无菌滤纸上，在每个真菌的平板上除了添加不同转基因烟草植株的总蛋白，同时平行添加野生型烟草的总蛋白和空白对照（提取蛋白所用的溶液），28℃培养几天后观察各处理真菌生长的情况，并据此来评价PnPR1-2转基因烟草的体外抗真菌活性，结果如图3所示，PnPR1-2转基因烟草蛋白对茄腐镰刀菌、轮枝镰刀菌、串珠状赤霉菌以及人参链格孢的生长具有很强的抑制作用。
序列表
<110> 昆明理工大学
<120> 一种三七病程相关蛋白1家族基因PnPR1-2及应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 852
<212> DNA
<213> Panax notoginseng
<220>
<221> mRNA
<222> (1)..(852)
<220>
<221> 5'UTR
<222> (1)..(104)
<220>
<221> CDS
<222> (105)..(605)
<220>
<221> 3'UTR
<222> (606)..(852)
<400> 1
gaccatctat tttttctctt tcttgttcta taaataatct tcattgcaac ttagtccaat 60
aactcatcaa taccaccttt ctccaatacc aaaaacccca caaaatggca ttcaccaaaa 120
tttcactact cattttctta agtaccattt tccattcgtc ccatgctcaa aactccccac 180
aagactacct caacgcccac aacgccgccc gtgcccaagt ccgcgttgga ccaatgagat 240
gggacaccac cgtcgccgcc tacgcacaaa actacgccaa cacattgatt tccagctgcc 300
gacttgtcca ctcgagcggc agcggctacg gcgagaacct tgcctatggc ttccctactt 360
taaccggcac ggcggccgtt gatttgtggg taaaggaaaa gccttattac gactatgact 420
caaactcttg tataggggga gtgtgtgggc attataccca agtggtttgg cagacctcga 480
accggctcgg ctgcggtagg gcgaggtgta acaatggtgg gtatattgtt tcttgcaatt 540
atgcaccgcc gggtaacatt attggaaggc gcccttatgt tagatcattg gtttcatcca 600
agtaattaat tatatatata tatatgggtg cggatcaata acactaagtt ttatacaaaa 660
ctttttacac aattctcatc catatttatg gtgcattcca ttccatgcaa taaaaaatgt 720
aatttttttt atatgtgatt tgatacgtaa gatgtatttg ctatacaaat acaagtgtaa 780
gatttccggc ttattgataa agtggtttta atatttatgg ttgtggaaaa aaaaaaaaaa 840
aaaaaaaaaa aa 852
<210> 2
<211> 166
<212> PRT
<213> Panax notoginseng
<400> 2
Met Ala Phe Thr Lys Ile Ser Leu Leu Ile Phe Leu Ser Thr Ile Phe
1 5 10 15
His Ser Ser His Ala Gln Asn Ser Pro Gln Asp Tyr Leu Asn Ala His
20 25 30
Asn Ala Ala Arg Ala Gln Val Arg Val Gly Pro Met Arg Trp Asp Thr
35 40 45
Thr Val Ala Ala Tyr Ala Gln Asn Tyr Ala Asn Thr Leu Ile Ser Ser
50 55 60
Cys Arg Leu Val His Ser Ser Gly Ser Gly Tyr Gly Glu Asn Leu Ala
65 70 75 80
Tyr Gly Phe Pro Thr Leu Thr Gly Thr Ala Ala Val Asp Leu Trp Val
85 90 95
Lys Glu Lys Pro Tyr Tyr Asp Tyr Asp Ser Asn Ser Cys Ile Gly Gly
100 105 110
Val Cys Gly His Tyr Thr Gln Val Val Trp Gln Thr Ser Asn Arg Leu
115 120 125
Gly Cys Gly Arg Ala Arg Cys Asn Asn Gly Gly Tyr Ile Val Ser Cys
130 135 140
Asn Tyr Ala Pro Pro Gly Asn Ile Ile Gly Arg Arg Pro Tyr Val Arg
145 150 155 160
Ser Leu Val Ser Ser Lys
165
<210> 3
<211> 26
<212> DNA
<213> 人工序列
<400> 3
cttgttctat aaataatctt cattgc 26
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
atatggatga gaattgtgta aaaag 25
Claims (3)
1.一种三七病程相关蛋白1家族基因PnPR1-2，其特征在于：其核苷酸序列如SEQ IDNO：1所示，编码如SEQ ID NO：2所示氨基酸序列的蛋白质。
2.根据权利要求1所述三七病程相关蛋白1家族基因PnPR1-2，其特征在于：PnPR1-2基因的编码区是序列表SEQ ID NO：1中第105-605位所示的核苷酸序列。
3.权利要求1和2所述的三七病程相关蛋白1家族基因PnPR1-2在提高烟草对茄腐镰刀菌（Fusarium solani）、轮枝镰刀菌（F. verticillioides）、串珠状赤霉菌（Gibberella moniliformis）、人参链格孢菌（Alternaria panax）抗性中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610836098.3A CN106244599B (zh) | 2016-09-21 | 2016-09-21 | 一种三七病程相关蛋白1家族基因PnPR1-2及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610836098.3A CN106244599B (zh) | 2016-09-21 | 2016-09-21 | 一种三七病程相关蛋白1家族基因PnPR1-2及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106244599A true CN106244599A (zh) | 2016-12-21 |
| CN106244599B CN106244599B (zh) | 2019-07-16 |
Family
ID=57599962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610836098.3A Active CN106244599B (zh) | 2016-09-21 | 2016-09-21 | 一种三七病程相关蛋白1家族基因PnPR1-2及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106244599B (zh) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018294A (zh) * | 2018-01-02 | 2018-05-11 | 昆明理工大学 | 一种三七植物激素结合蛋白基因PnPhBP1及应用 |
| CN108341861A (zh) * | 2018-05-14 | 2018-07-31 | 南京农业大学 | 一种来源于杜梨的抗病性相关基因及其编码蛋白与应用 |
| CN109182587A (zh) * | 2018-10-29 | 2019-01-11 | 中国农业科学院特产研究所 | 人参病程相关基因及其检测引物、试剂盒及应用与检测方法 |
| CN109280719A (zh) * | 2018-10-29 | 2019-01-29 | 中国农业科学院特产研究所 | 人参病程相关基因及其检测引物、试剂盒及应用与检测方法 |
| CN116103315A (zh) * | 2023-02-07 | 2023-05-12 | 云南农业大学 | 一种三七病程蛋白基因PnPR4及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131015A (zh) * | 2014-08-13 | 2014-11-05 | 昆明理工大学 | 一种岷江百合病程相关蛋白10基因LrPR10-5的应用 |
| CN104152470A (zh) * | 2014-08-13 | 2014-11-19 | 昆明理工大学 | 一种岷江百合病程相关蛋白10基因LrPR10-6的应用 |
| CN105861517A (zh) * | 2016-04-20 | 2016-08-17 | 昆明理工大学 | 一种三七抗菌肽基因PnSN1及其应用 |
-
2016
- 2016-09-21 CN CN201610836098.3A patent/CN106244599B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131015A (zh) * | 2014-08-13 | 2014-11-05 | 昆明理工大学 | 一种岷江百合病程相关蛋白10基因LrPR10-5的应用 |
| CN104152470A (zh) * | 2014-08-13 | 2014-11-19 | 昆明理工大学 | 一种岷江百合病程相关蛋白10基因LrPR10-6的应用 |
| CN105861517A (zh) * | 2016-04-20 | 2016-08-17 | 昆明理工大学 | 一种三七抗菌肽基因PnSN1及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| 李瑞博 等: "三七病程相关蛋白1 基因的克隆与表达分析", 《药学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018294A (zh) * | 2018-01-02 | 2018-05-11 | 昆明理工大学 | 一种三七植物激素结合蛋白基因PnPhBP1及应用 |
| CN108341861A (zh) * | 2018-05-14 | 2018-07-31 | 南京农业大学 | 一种来源于杜梨的抗病性相关基因及其编码蛋白与应用 |
| CN109182587A (zh) * | 2018-10-29 | 2019-01-11 | 中国农业科学院特产研究所 | 人参病程相关基因及其检测引物、试剂盒及应用与检测方法 |
| CN109280719A (zh) * | 2018-10-29 | 2019-01-29 | 中国农业科学院特产研究所 | 人参病程相关基因及其检测引物、试剂盒及应用与检测方法 |
| CN116103315A (zh) * | 2023-02-07 | 2023-05-12 | 云南农业大学 | 一种三七病程蛋白基因PnPR4及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106244599B (zh) | 2019-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106244599B (zh) | 一种三七病程相关蛋白1家族基因PnPR1-2及应用 | |
| CN105861517B (zh) | 一种三七抗菌肽基因PnSN1及其应用 | |
| CN110734482B (zh) | 一种岷江百合WRKY转录因子基因LrWRKY4及应用 | |
| CN106868023B (zh) | 黄瓜CsERF004基因及其编码蛋白和应用 | |
| CN105755020B (zh) | 三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用 | |
| CN103194456B (zh) | 岷江百合抗真菌基因Lr14-3-3及其应用 | |
| CN107267526B (zh) | 三七MYB转录因子基因PnMYB2及其应用 | |
| CN105861501B (zh) | 水稻褐飞虱为害诱导型启动子区域的分离及表达模式鉴定 | |
| CN104131015A (zh) | 一种岷江百合病程相关蛋白10基因LrPR10-5的应用 | |
| CN104878019B (zh) | 漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用 | |
| CN104878028B (zh) | 漾濞大泡核桃几丁质酶基因JsCHI1及应用 | |
| CN112359049B (zh) | 一种岷江百合几丁质酶基因LrCHI2及其应用 | |
| CN106244598B (zh) | 三七Dirigent类似蛋白基因PnDIR1及应用 | |
| CN104878041B (zh) | 漾濞大泡核桃转录因子基因JsWRKY1的应用 | |
| CN107267525B (zh) | 三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用 | |
| CN103320448B (zh) | 一种岷江百合bZIP转录因子基因LrbZIP1及应用 | |
| CN104878027B (zh) | 漾濞大泡核桃核糖核酸酶基因JsRNase及应用 | |
| CN108707610B (zh) | 三七defensin抗菌肽基因PnDEFL1及应用 | |
| CN104774847B (zh) | 漾濞大泡核桃富含脯氨酸蛋白基因JsPRP1及应用 | |
| CN107365794A (zh) | 三七几丁质酶基因PnCHI1的应用 | |
| CN109295068B (zh) | 一种三七类甜蛋白基因PnTLP2及应用 | |
| CN104152465B (zh) | 一种岷江百合细胞色素b5基因LrCyt-b5及其应用 | |
| CN108707611B (zh) | 一种三七类逆渗透蛋白基因PnOLP1及应用 | |
| CN107964547B (zh) | 一种三七病程相关蛋白10基因PnPR10-3及应用 | |
| CN104774855B (zh) | 漾濞大泡核桃病程相关蛋白10基因JsPR10‑1的应用 |
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 |