CN109234284A - A kind of Radix Notoginseng class sweet protein gene PnTLP5 and application - Google Patents
A kind of Radix Notoginseng class sweet protein gene PnTLP5 and application Download PDFInfo
- Publication number
- CN109234284A CN109234284A CN201811071523.XA CN201811071523A CN109234284A CN 109234284 A CN109234284 A CN 109234284A CN 201811071523 A CN201811071523 A CN 201811071523A CN 109234284 A CN109234284 A CN 109234284A
- Authority
- CN
- China
- Prior art keywords
- pntlp5
- plant
- radix notoginseng
- tobacco
- gene
- 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 42
- 241000180649 Panax notoginseng Species 0.000 title claims abstract description 26
- 235000003143 Panax notoginseng Nutrition 0.000 title claims abstract description 26
- 239000003764 sweet protein Substances 0.000 title claims abstract description 11
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 33
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 241000427940 Fusarium solani Species 0.000 claims abstract description 6
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims abstract description 6
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 6
- 235000008434 ginseng Nutrition 0.000 claims abstract description 6
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 5
- 240000006365 Vitis vinifera Species 0.000 claims abstract description 5
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 241000208340 Araliaceae Species 0.000 claims abstract 2
- 125000003729 nucleotide group Chemical group 0.000 claims abstract 2
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 241000212253 Alternaria panax Species 0.000 claims description 3
- 241000555706 Botryosphaeria dothidea Species 0.000 claims description 2
- 241000233732 Fusarium verticillioides Species 0.000 claims description 2
- 241000221696 Sclerotinia sclerotiorum Species 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 45
- 230000009261 transgenic effect Effects 0.000 abstract description 16
- 241000233866 Fungi Species 0.000 abstract description 9
- 230000000843 anti-fungal effect Effects 0.000 abstract description 7
- 239000000892 thaumatin Substances 0.000 abstract description 7
- 235000010436 thaumatin Nutrition 0.000 abstract description 7
- 230000002018 overexpression Effects 0.000 abstract description 5
- 230000001857 anti-mycotic effect Effects 0.000 abstract description 4
- 239000002543 antimycotic Substances 0.000 abstract description 4
- 239000013604 expression vector Substances 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 4
- 101710096342 Pathogenesis-related protein Proteins 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 102100027364 Cysteine-rich protein 3 Human genes 0.000 description 19
- 201000010099 disease Diseases 0.000 description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 9
- 241000589158 Agrobacterium Species 0.000 description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 244000131316 Panax pseudoginseng Species 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 210000002421 cell wall Anatomy 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 208000031888 Mycoses Diseases 0.000 description 5
- 108700019146 Transgenes Proteins 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 244000082988 Secale cereale Species 0.000 description 4
- 235000007238 Secale cereale Nutrition 0.000 description 4
- 241000607479 Yersinia pestis Species 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 108010060199 cysteinylproline Proteins 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229920001817 Agar Polymers 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
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 101710203193 Thaumatin-like protein Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 244000075850 Avena orientalis Species 0.000 description 2
- 235000007319 Avena orientalis Nutrition 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 description 2
- 241001481296 Malus spectabilis Species 0.000 description 2
- 235000003181 Panax pseudoginseng Nutrition 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 206010039509 Scab Diseases 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
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 244000098338 Triticum aestivum Species 0.000 description 2
- 102100022962 Vam6/Vps39-like protein Human genes 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
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000003967 crop rotation Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010038745 tryptophylglycine Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- ROLXPVQSRCPVGK-XDTLVQLUSA-N Ala-Glu-Tyr Chemical compound N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O ROLXPVQSRCPVGK-XDTLVQLUSA-N 0.000 description 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 1
- JJHBEVZAZXZREW-LFSVMHDDSA-N Ala-Thr-Phe Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O JJHBEVZAZXZREW-LFSVMHDDSA-N 0.000 description 1
- 241001124076 Aphididae Species 0.000 description 1
- 102000010637 Aquaporins Human genes 0.000 description 1
- 108010063290 Aquaporins Proteins 0.000 description 1
- QIWYWCYNUMJBTC-CIUDSAMLSA-N Arg-Cys-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QIWYWCYNUMJBTC-CIUDSAMLSA-N 0.000 description 1
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 1
- CFGHCPUPFHWMCM-FDARSICLSA-N Arg-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N CFGHCPUPFHWMCM-FDARSICLSA-N 0.000 description 1
- UHFUZWSZQKMDSX-DCAQKATOSA-N Arg-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UHFUZWSZQKMDSX-DCAQKATOSA-N 0.000 description 1
- MNBHKGYCLBUIBC-UFYCRDLUSA-N Arg-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCNC(N)=N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MNBHKGYCLBUIBC-UFYCRDLUSA-N 0.000 description 1
- YNSCBOUZTAGIGO-ZLUOBGJFSA-N Asn-Asn-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)C(=O)N YNSCBOUZTAGIGO-ZLUOBGJFSA-N 0.000 description 1
- NLCDVZJDEXIDDL-BIIVOSGPSA-N Asn-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N)C(=O)O NLCDVZJDEXIDDL-BIIVOSGPSA-N 0.000 description 1
- HLTLEIXYIJDFOY-ZLUOBGJFSA-N Asn-Cys-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O HLTLEIXYIJDFOY-ZLUOBGJFSA-N 0.000 description 1
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 1
- WIDVAWAQBRAKTI-YUMQZZPRSA-N Asn-Leu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O WIDVAWAQBRAKTI-YUMQZZPRSA-N 0.000 description 1
- REQUGIWGOGSOEZ-ZLUOBGJFSA-N Asn-Ser-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N REQUGIWGOGSOEZ-ZLUOBGJFSA-N 0.000 description 1
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 1
- KHGPWGKPYHPOIK-QWRGUYRKSA-N Asp-Gly-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KHGPWGKPYHPOIK-QWRGUYRKSA-N 0.000 description 1
- CYCKJEFVFNRWEZ-UGYAYLCHSA-N Asp-Ile-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O CYCKJEFVFNRWEZ-UGYAYLCHSA-N 0.000 description 1
- RPUYTJJZXQBWDT-SRVKXCTJSA-N Asp-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N RPUYTJJZXQBWDT-SRVKXCTJSA-N 0.000 description 1
- RVMXMLSYBTXCAV-VEVYYDQMSA-N Asp-Pro-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMXMLSYBTXCAV-VEVYYDQMSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000218646 Cedrus deodara Species 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- XTHUKRLJRUVVBF-WHFBIAKZSA-N Cys-Gly-Ser Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O XTHUKRLJRUVVBF-WHFBIAKZSA-N 0.000 description 1
- NITLUESFANGEIW-BQBZGAKWSA-N Cys-Pro-Gly Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O NITLUESFANGEIW-BQBZGAKWSA-N 0.000 description 1
- NRVQLLDIJJEIIZ-VZFHVOOUSA-N Cys-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N)O NRVQLLDIJJEIIZ-VZFHVOOUSA-N 0.000 description 1
- JIVJQYNNAYFXDG-LKXGYXEUSA-N Cys-Thr-Asn Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O JIVJQYNNAYFXDG-LKXGYXEUSA-N 0.000 description 1
- NAPULYCVEVVFRB-HEIBUPTGSA-N Cys-Thr-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CS NAPULYCVEVVFRB-HEIBUPTGSA-N 0.000 description 1
- 241000289763 Dasygaster padockina Species 0.000 description 1
- 241001124144 Dermaptera Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 235000014466 Douglas bleu Nutrition 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000221785 Erysiphales Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 241001451172 Fusarium pseudograminearum Species 0.000 description 1
- QBEWLBKBGXVVPD-RYUDHWBXSA-N Gln-Phe-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N QBEWLBKBGXVVPD-RYUDHWBXSA-N 0.000 description 1
- IYAUFWMUCGBFMQ-CIUDSAMLSA-N Glu-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N IYAUFWMUCGBFMQ-CIUDSAMLSA-N 0.000 description 1
- QIQABBIDHGQXGA-ZPFDUUQYSA-N Glu-Ile-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QIQABBIDHGQXGA-ZPFDUUQYSA-N 0.000 description 1
- CXRWMMRLEMVSEH-PEFMBERDSA-N Glu-Ile-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O CXRWMMRLEMVSEH-PEFMBERDSA-N 0.000 description 1
- 229920001503 Glucan Polymers 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
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 1
- VUUOMYFPWDYETE-WDSKDSINSA-N Gly-Gln-Cys Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN VUUOMYFPWDYETE-WDSKDSINSA-N 0.000 description 1
- IDOGEHIWMJMAHT-BYPYZUCNSA-N Gly-Gly-Cys Chemical compound NCC(=O)NCC(=O)N[C@@H](CS)C(O)=O IDOGEHIWMJMAHT-BYPYZUCNSA-N 0.000 description 1
- VLIJYPMATZSOLL-YUMQZZPRSA-N Gly-Lys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN VLIJYPMATZSOLL-YUMQZZPRSA-N 0.000 description 1
- QSQXZZCGPXQBPP-BQBZGAKWSA-N Gly-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)CN)C(=O)N[C@@H](CS)C(=O)O QSQXZZCGPXQBPP-BQBZGAKWSA-N 0.000 description 1
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 1
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 1
- DNAZKGFYFRGZIH-QWRGUYRKSA-N Gly-Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 DNAZKGFYFRGZIH-QWRGUYRKSA-N 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 241000721668 Juniperus ashei Species 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-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
- 241000446313 Lamella Species 0.000 description 1
- JQSXWJXBASFONF-KKUMJFAQSA-N Leu-Asp-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JQSXWJXBASFONF-KKUMJFAQSA-N 0.000 description 1
- DXYBNWJZJVSZAE-GUBZILKMSA-N Leu-Gln-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N DXYBNWJZJVSZAE-GUBZILKMSA-N 0.000 description 1
- RIHIGSWBLHSGLV-CQDKDKBSSA-N Leu-Tyr-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O RIHIGSWBLHSGLV-CQDKDKBSSA-N 0.000 description 1
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 1
- 235000010804 Maranta arundinacea Nutrition 0.000 description 1
- 206010027146 Melanoderma Diseases 0.000 description 1
- MDXAULHWGWETHF-SRVKXCTJSA-N Met-Arg-Val Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CCCNC(N)=N MDXAULHWGWETHF-SRVKXCTJSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 1
- XZQYIJALMGEUJD-OEAJRASXSA-N Phe-Lys-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XZQYIJALMGEUJD-OEAJRASXSA-N 0.000 description 1
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 1
- 241000123107 Phellinus Species 0.000 description 1
- TXPUNZXZDVJUJQ-LPEHRKFASA-N Pro-Asn-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O TXPUNZXZDVJUJQ-LPEHRKFASA-N 0.000 description 1
- UTAUEDINXUMHLG-FXQIFTODSA-N Pro-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 UTAUEDINXUMHLG-FXQIFTODSA-N 0.000 description 1
- PZSCUPVOJGKHEP-CIUDSAMLSA-N Pro-Gln-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PZSCUPVOJGKHEP-CIUDSAMLSA-N 0.000 description 1
- KTFZQPLSPLWLKN-KKUMJFAQSA-N Pro-Gln-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KTFZQPLSPLWLKN-KKUMJFAQSA-N 0.000 description 1
- STASJMBVVHNWCG-IHRRRGAJSA-N Pro-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 STASJMBVVHNWCG-IHRRRGAJSA-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
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 240000001416 Pseudotsuga menziesii Species 0.000 description 1
- 235000005386 Pseudotsuga menziesii var menziesii Nutrition 0.000 description 1
- 244000088401 Pyrus pyrifolia Species 0.000 description 1
- 235000011400 Pyrus pyrifolia Nutrition 0.000 description 1
- 239000009759 San-Chi Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- AEGUWTFAQQWVLC-BQBZGAKWSA-N Ser-Gly-Arg Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O AEGUWTFAQQWVLC-BQBZGAKWSA-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
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000145580 Thalia geniculata Species 0.000 description 1
- 235000012419 Thalia geniculata Nutrition 0.000 description 1
- 240000007326 Thaumatococcus daniellii Species 0.000 description 1
- 235000005266 Thaumatococcus daniellii Nutrition 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- NIEWSKWFURSECR-FOHZUACHSA-N Thr-Gly-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O NIEWSKWFURSECR-FOHZUACHSA-N 0.000 description 1
- YSXYEJWDHBCTDJ-DVJZZOLTSA-N Thr-Gly-Trp Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O YSXYEJWDHBCTDJ-DVJZZOLTSA-N 0.000 description 1
- DCRHJDRLCFMEBI-RHYQMDGZSA-N Thr-Lys-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(O)=O DCRHJDRLCFMEBI-RHYQMDGZSA-N 0.000 description 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 1
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 1
- BGHVVGPELPHRCI-HZTRNQAASA-N Thr-Trp-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)N)O BGHVVGPELPHRCI-HZTRNQAASA-N 0.000 description 1
- BRBCKMMXKONBAA-KWBADKCTSA-N Trp-Ala-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 BRBCKMMXKONBAA-KWBADKCTSA-N 0.000 description 1
- NXQAOORHSYJRGH-AAEUAGOBSA-N Trp-Gly-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 NXQAOORHSYJRGH-AAEUAGOBSA-N 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 1
- NLMXVDDEQFKQQU-CFMVVWHZSA-N Tyr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLMXVDDEQFKQQU-CFMVVWHZSA-N 0.000 description 1
- BYAKMYBZADCNMN-JYJNAYRXSA-N Tyr-Lys-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O BYAKMYBZADCNMN-JYJNAYRXSA-N 0.000 description 1
- MDXLPNRXCFOBTL-BZSNNMDCSA-N Tyr-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MDXLPNRXCFOBTL-BZSNNMDCSA-N 0.000 description 1
- JHDZONWZTCKTJR-KJEVXHAQSA-N Tyr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JHDZONWZTCKTJR-KJEVXHAQSA-N 0.000 description 1
- YQMILNREHKTFBS-IHRRRGAJSA-N Val-Phe-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N YQMILNREHKTFBS-IHRRRGAJSA-N 0.000 description 1
- DOFAQXCYFQKSHT-SRVKXCTJSA-N Val-Pro-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DOFAQXCYFQKSHT-SRVKXCTJSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 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 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001458 anti-acid effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 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
- 230000034303 cell budding Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 235000013399 edible fruits 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
- 239000003205 fragrance Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 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
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 230000008659 phytopathology Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 108700024897 plant thaumatin Proteins 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000000405 serological 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
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 241001478887 unidentified soil bacteria Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 1
- 238000012795 verification Methods 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)
- C12N15/8241—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)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of 5 same clan's sweet protein genes of pathogenesis-related proteins of Radix NotoginsengPnTLP5, nucleotide sequence is as described in SEQ IDNO:1, the Radix Notoginseng class thaumatin T of coding amino acid sequence as shown in SEQ IDNO:2；The present invention is confirmed by molecular biology and genetic engineering relation technological researchingPnTLP5Gene, which has, improves the antimycotic function of plant, and the present invention is antimycoticPnTLP5It is gene constructed on plant expression vector and overexpression in tobacco is transferred to, as a result transgenic tobacco plant has very strong extracorporeal antifungal activity, overexpressionPnTLP5Transgene tobacco there is significant inhibiting effect to the growth of sclerotinite, Fusarium solani, wheel branch sickle-like bacteria, ginseng rod method, five kinds of fungies such as grape seat chamber bacterium.
Description
Technical field
The present invention relates to molecular biology and genetic engineering relation technological researching fields, especially a kind of with antimycotic
Active Radix Notoginseng class sweet protein genePnTLP5And application.
Background technique
Radix Notoginseng (Panax notoginseng) it is the long Chinese herbal medicine of Chinese history.Radix Notoginseng main product is in Yunnan Wenshan Prefecture inkstone
Also there is plantation on mountain county, Maguan, Xichou, Guangnan, Malipo, Funing, Qiu north etc., the ground such as another Guangxi Tianyang County, Jingxi, Tiandong County, Debao.
The Radix Notoginseng of Yunnan Wenshan Prefecture is with a long history, yield is big, high-quality, practises and claims " literary Radix Notoginseng ", " pseudo-ginseng ", is famous genunie medicinal materials.
Radix Notoginseng is grown in yin throughout the year and covers in environment, and pest and disease damage generation is more serious, according to statistics, the pest and disease damage occurred on Radix Notoginseng about 20
It is a variety of.Wherein, main to have root rot, black spot, Northern leaf spot, epidemic disease, powdery mildew, slug, cutworm, aphid, scale insect, ruler
Earwig etc., Radix Notoginseng is every year all because of extremely serious loss caused by the harm of disease pest.Therefore, reinforcing the Radix Notoginseng prevention and control of plant diseases, pest control is guarantee three
One major measure of seven yield and quality.
Disease problem in cultivation of pseudo-ginseng is than more prominent, and wherein Alternaria panax is exactly a Major Diseases in seven gardens.Three
Seven stem, leaf, spend can be aggrieved, is killed portion's initial stage oval light brown scab, after at dark brown, scab to upper and lower extension,
, finally there is stem in recess or floral disc kinking is dead.Aggrieved Radix Notoginseng less serious case yield and quality decline, 90% or more the severe one underproduction.Mesh
There are several types of modes for the preceding control to Radix Notoginseng fungal disease: using chemical bactericide；It takes crop rotation, avoid band soil bacteria and band
The propagation etc. of nosophyte material.But either chemical sterilization or crop rotation all cannot fundamentally solve asking for Radix Notoginseng disease
Topic.But with the continuous development of Molecular Biology and technology, people is made to be not only able to deeply recognize from molecular level
The mechanism of plant and pathogen interaction, but also disease-resistant crop new product can be quickly and efficiently cultivated by genetic engineering
Kind, it is the new method for improving plant disease-resistant ability.
Pathogenesis-related proteins (Pathogenesis-related proteins, PRs) earliest from tobacco (Nicotiana tabacum) find in the tobacco leaf that infects of mosaic virus (Tobacco mosaic virus, TMV), according to amino acid group
It is 17 different protein families, i.e. PR-1 ~ PR-17 by PRs points at, biochemistry and serological characteristic.Due to PR-5 family
In some protein amino acid sequence and West Africa arrowroot (Thaumatococcus daniellii ) thaumatin T
(Thaumatin) amino acid sequence very high homology, therefore the class thaumatin T that is otherwise known as (Thaumatin-like proteins,
TLPs) (Li Wenxian, Liu Diqiu wait the structure feature and Research progress on Function Chinese biological engineering magazine of class thaumatin T,
2010,30 (3): 100-104.).TLPs is widely distributed in higher plant, including pollen tube wall, fine cell wall, screen casing
End wall etc., they play a significant role in plant normal growth development.Importantly, TLPs is in the resistance mechanism of plant
Also play key player.TLPs reacts related with the degeneration-resistant of plant, when plant is by pathogen, chemokines, physical agent
When Deng coercing,TLPsGene expresses rapidly in plant privileged site and accumulates that (Jiang Xiaoling, Huang Qiuxian wait plant sweet tea
Progress Zhejiang A & F University of protein gene family journal, 2012,29 (2): 279-287.).Many research discoveries are planted
Object TLPs has significant antifungal activity, Yi XiezhuanTLPsThe plant of gene is significantly enhanced the resistance of fungal disease,
Thus in recent years, the anti-fungus function of TLPs is in Plant Pathology research by more and more extensive concern.
Known TLPs belongs to the 17th family of glycosyl hydrolase, and TLPs mostly important structure feature is its molecule
In by 16 cysteine residues match formed 8 disulfide bond, due to the presence of these disulfide bond, so that TLPs is provided with phase
It, being capable of heat resistanceheat resistant, resistance to enzymolysis and antiacid alkali to stable chemical structure.Such as carrotTLPsContain 16 cysteine residues, it is more
It counts and all has class thaumatin T family label G-X- [GF]-X-C-X-T- [GA]-D-C-X-(1,2)-G-X-(2,3)-C and 5
A residual (Liu D, He X, et al. Molecular cloning of a thaumatin- of conservative REDDD amino acid
like protein gene from pyrus pyrifolia and overexpression of this gene in
tobacco increased resistance to pathogenic fungi. Plant Cell, Tissue and
Organ Culture(PCTOC), 2012,111(1): 29-39.).Some TLPs contain N-terminal signal peptide, for guide at
Ripe albumen is to secretion path.Such as Hubei Chinese flowering crabapple class sweet protein geneMhPR5Also contain 1 by 25 amino acid residues in the end N
Composition signal peptide (Zhang Jiyu, the clone of Hubei Chinese flowering crabapple disease-resistant related gene and its functional analysis Agricultural University Of Nanjing,
2011).The tertiary structure of TLPs protein is generally made of 3 structural domains, and structural domain I is anti-by 11 β lamellas (β-sheet)
It being formed to parallel folds, structural domain II and structural domain III are mainly made of alpha helical region domain, and between structural domain I and structural domain II
Form the crack (Cleft) with strong electronegativity.However there are also special TLPs, tertiary structure only has knot
Structure domain I and structural domain II or even some TLPs only include structural domain I, and therefore, the molecular size range of plant TLPs differs, and one
As be 16 ~ 40 kDa.
TLPsThe expression of gene is related with plant growth environment and disease resistance of plant.Deodar (Juniperus ashei) flower
With allergen activity in powderTLPs5.0 times or more (A Zamani, RN are up in the content difference of different year
Sturrock, et al. Gene cloning and tissue expression analysis of a PR-5
thaumatin-like protein in phellinus weirii-infected douglas-fir.
Phytopathology, 2004 , 94 (11) :1235-1243).Barley (Hordeum vulgare) and oat (Avena sativa) from ovary wall development to during aleurone,TLPsThe expression of gene is changed.A kind of pepperTLPsGene
And it is expressed during fruit growth.Wheat crown rot bacterium (Fusarium pseudograminearum) infect 4 days after,
In disease-resistant wheat kindTLPsGene expression is significantly higher than susceptible variety.However, there are alsoTLPsGene pairs pathogen stress
It is insensitive, and (Jiang Xiaoling, Huang Qiu are refined to wait plant thaumatin T to great expression when plant is induced by chemically or physically factor
Gene family progress Zhejiang A & F University journal, 2012,29 (2): 279-287).
TLPs antibacterial action is mainly reflected on its unique structural domain and acid crack.Rye (Secale cereale)
In TLPs structural domain I in acidic residues and fungal cell membrane on aquaporin, ionophorous protein and infiltration sense
Interaction between receiver, so that TLPs is provided with antifungal activity (Chan Y, Griffith M, et al.
Cloning of a cDNA encoding the thaumatin-like protein of winter rye (Secale cereale L. Musketeer) and its functional characterization. Journal of
Experimental Botany, 1999,50 (339): 1627-1628).The combination of TLPs and pathogen beta-1,3-dextran
Crack area is betided, the carbohydrate of several residues and fungal cell wall in crack area forms hydrogen bond, and fragrance
Race's amino acid participates in forming the structure that accumulation force destroys fungal cell wall jointly at crack, makes TLPs and cell membrane contact,
Cause fungal cell dead.Changing cell wall structure is the prerequisite that TLPs inhibits fungal infection.TLPs in addition to cell wall
On β -1, outside 3- glucosan specificity Binding change cell wall structure, the structure that other approach change cell can also be passed through.
Summary of the invention
It is an object of the present invention to provide a kind of to clone the overall length base for obtaining the class thaumatin T with antifungal activity from Radix Notoginseng
CausePnTLP5, Radix Notoginseng class sweet protein genePnTLP5Nucleotide sequence is as shown in SEQ ID NO:1, the gene cDNA full length sequence
For 1170 bp, the open reading frame comprising 726 bp, the 5 ' non-translational regions of 186 bp, 258 bp 3 ' non-translational regions, compile
The protein of code amino acid sequence as shown in SEQ ID NO:2.
The present invention separates the global cDNA segment of an antimycotic related gene for clone Radix Notoginseng, utilizes Agrobacterium tumefaciems
(Agrobacterium tumefaciens) target gene is transferred in recipient plant and overexpression by mediated method, by into one
Whether step experimental verification gene has antimycotic activity, resists for the later-stage utilization improvement of genes tobacco and other plant
The ability of fungal disease lays the foundation, this unnamed gene is by inventorPnTLP5。
The present invention is by 5 same clan's sweet protein genes of Radix Notoginseng pathogenesis-related proteinsPnTLP5It applies and is improving tobacco to eggplant corruption sickle
Knife bacterium (Fusarium solani), wheel branch sickle-like bacteria (F. verticillioides), sclerotinite (Sclerotinia sclerotiorum), ginseng rod method (Alternaria panax), grape seat chamber bacterium (Botryosphaeria dothidea) in resistance, concrete operations are as follows:
(1) total serum IgE that Radix Notoginseng young leaflet tablet is extracted using guanidine isothiocyanate method, using the RNA of extraction as template, with oligo (dT)
15 be reverse transcription primer, passes through reverse transcriptase chain reaction (Reverse transcription-polymerase
Chain reaction, RT-PCR) it amplifiesPnTLP5Code area, be subsequently attached on pMD-18T carrier, through being sequenced
Obtain the clone with target gene；
(2) restriction enzyme is usedBamHI andEcoRI digestion pMD-18T-PnTLP5, recycle to obtain target gene piece by glue
Section, with same endonuclease digestion plant expression vector pCAMBIA2300s, carrier large fragment needed for glue recycling obtains, then by institute
It obtainsPnTLP5Genetic fragment is connect with pCAMBIA2300s segment, plant overexpression vector is constructed, later by constructed weight
Group carrier is expressed by Agrobacterium tumefaciens mediated be transferred in tobacco；
(3) regenerated tobacco plant is screened with the label (kalamycin resistance gene) having on recombinant vector T-DNA, and passed through
PCR and RT-PCR detects to obtain real transgenic plant, the inhibition that analysis rotaring gene plant blade albumen grows fungi
Activity finally filters out the transgenic plant that fungus resistant is remarkably reinforced.
The present invention provides a kind of new method to improve the resistance of plant against fungal disease, is trained by genetic engineering means
Traditional breeding method can be overcome the shortcomings of by educating disease-resistant plants, and not only breeding cycle shortens, but also easy to operate, be easy to get highly resistance material
Material；Class sweet protein gene in the present invention from Radix NotoginsengPnTLP5Plant can be enhanced to the resistance of a variety of disease fungus, by the base
Because importing in tobacco, it can produce new varieties and new material with fungus resistant.Resistance is cultivated using technique for gene engineering to plant
The importance that article kind and material have apparent advantage and do not replace.It not only can for large-scale production cereal crops,
The offers such as industrial crops, Chinese herbal medicine, ornamental plant are convenient, reduce the use of chemical pesticide, can also for agricultural production saving at
Originally, reduce environmental pollution, therefore the present invention has a vast market application prospect.
Detailed description of the invention
Fig. 1 is part of the present inventionPnTLP5The PCR testing result of transgene tobacco genomic DNA, in figure: Marker is
DL2000 DNA Marker (the precious biology in Dalian)；Positive control is plasmid pMD-18T-PnTLP5Product is tied for the PCR of template；
WT is the product that non-transgenic tobacco (wild type) total DNA is template PCR；
Fig. 2 is some positive of the present inventionPnTLP5In transgene tobaccoPnTLP5The expression analysis result figure of transcriptional level；In figure:
Marker is DL2000 DNA Marker (the precious biology in Dalian)；WT is that non-transgenic tobacco total serum IgE reverse transcription cDNA is template
PCR product；Positive control is plasmid pMD-18T-PnTLP5For the PCR product of template；
Fig. 3 is the present inventionPnTLP5Transgene tobacco In Vitro Bacteriostatic effect picture；In figure a, b, c, d, e be respectively sclerotinite,
Ginseng rod method, wheel branch sickle-like bacteria, Fusarium solani, grape seat chamber bacterium；WT is the total protein of wild-type tobacco；Buffer is sky
White control, i.e., without protein control (for extracting the buffer of albumen).
Specific embodiment
Below by drawings and examples, invention is further described in detail, but the scope of the present invention is not limited to
The content, method is conventional method unless otherwise specified in embodiment, and the reagent used is routine unless otherwise specified
Commercial reagent or the reagent prepared according to a conventional method.
Embodiment 1:PnTLP5 full-length genes clone and sequence analysis
It takes 3 years raw sangqi ginseng blades to extract total serum IgE, with liquid nitrogen then sanchi leaf slice lapping is transferred in centrifuge tube at powder, is adopted
Total serum IgE is extracted with guanidine isothiocyanate method, uses reverse transcriptase M-MLV (promega) using total serum IgE as templated synthesis cDNA first
Chain, reaction system and operating process are as follows: take 5 μ g Total RNA, sequentially add 50 ng oligo(dT), 2 μ L dNTP
(2.5mM each), DEPC water to reaction volume are 14.5 μ L；It is cold on ice rapidly after 70 DEG C of 5 min of heat denatured after mixing
But then 5 min sequentially adds 4 μ L 5 × First-stand buffer, 0.5 μ L RNasin(200U), 1 μ L M-MLV
(200U) is mixed and is centrifuged in short-term, 42 DEG C of 1.5 h of warm bath, 70 DEG C of 10 min of heating after taking-up, terminate reaction.The first chain of cDNA
Synthesis is placed on -20 DEG C and saves backup.
Using the first chain cDNA of synthesis as template, amplifying target genesPnTLP5, upstream and downstream primer sequence used is respectively
5 ' TTGTCAACTTAAACAATATGAGCTA3 ' and 5 ' AACCTTTTAACTTATTGGAACTGCT3 '.Using AdvantageTM
2 PCR Enzyme(Clontech) amplify target gene；PCR reaction condition: 95 DEG C of 2 min；95 DEG C of 30 s, 57 DEG C 30
S, 72 DEG C of 1 min, 30 circulations；72℃10 min；Reaction system (20 μ L) is 1 μ L cDNA, 2 μ L 10 ×
2 PCR Buffer of Advantage, 1.8 μ 50 × dNTP of L Mix (10mM each), 0.2 μ L forward primer (10 μM),
0.2 μ L reverse primer (10 μM), 0.2 μ L Advantage, 2 PCR Polymerase Mix, 14.6 μ L PCR-Grade
water；After PCR, take 5 μ L for agarose gel electrophoresis, to detect the specificity and size of amplified production.
Agarose gel electrophoresis results show that PCR product only has a DNA band, therefore directly carry out TA grams to PCR product
Grand, the kit used is the precious biology in the Dalian pMD18-T vector kit(), reaction system and operating process are as follows: take 1.5 μ L
PCR product sequentially adds 1 μ L pMD18-T vector(50 ng/ μ L) and 2.5 μ L 2 × Ligation solution I,
Mixing is placed on 16 DEG C of reaction overnights.Connection product is transferred in bacillus coli DH 5 alpha using heat-shock transformed method.Using containing ammonia
The LB solid medium screening positive clone of parasiticin (Ampicillin, Amp), selects several single colonies, uses after shaking bacterium
AmplificationPnTLP5Special primer identify multiple cloning sites insertionPnTLP5Clone, the clone identified is sequenced,
Finally obtainPnTLP5Full-length cDNA be 1170 bp, by NCBI ORF finder (http: //
Www.ncbi.nlm.nih.gov/gorf/gorf.html) analysis finds the opening code-reading frame it includes 726 bp (see sequence
List),PnTLP5 codings one contain the protein PnTLP5 of 241 amino acid, and protein is 26.4 kD, etc.
Electric point (pI) is 7.75, shows that the albumen is basic protein.Including 16 acidic amino acids (D, E), 19 basic amine groups
Sour (K, R, H), 113 hydrophobic amino acids and 89 hydrophilic amino acids.Genomic organization discovery is carried out using SMART,
PnTLP5 has a possible signal peptide.
Embodiment 2: plant overexpression vector building
Insertion is extracted using a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA (the raw work in Shanghai)PnTLP5Escherichia coli matter
Grain pMD-18T-PnTLP5And the plasmid of plant expression vector pCAMBIA2300s, take 1 μ L for agarose gel electrophoresis with
Detect the integrality and concentration level of extracted plasmid；Use restriction enzymeBamHI(TaKaRa) andEcoRI(TaKaRa) divide
It is other to plasmid pMD-18T-PnTLP5Double digestion (100 μ L system) is carried out with pCAMBIA2300s, reaction system and operating process
Are as follows: take 20 μ L pMD-18T-PnTLP5With pCAMBIA2300s plasmid, sequentially add 10 μ L 10 × K buffer, 5 μ LBamHI、5 μL EcoRI、60 μL ddH2O is centrifuged in short-term after mixing, is placed in 37 DEG C of reaction overnights；By all digestion products points
Electrophoresis is carried out in Ago-Gel, it is then rightPnTLP5Segment and pCAMBIA2300s carrier large fragment carry out glue respectively and return
It receives；It takes 1 μ L recovery product to detect the size and concentration of recycling segment by agarose gel electrophoresis, it is standby to be placed in -20 DEG C of preservations
With.
Utilize T4 DNA Ligase(TaKaRa), by recyclingPnTLP5 DNA fragmentation and pCAMBIA2300s carrier-pellet
Section connects, reaction system (20 μ L) and operating process are as follows: take 10 μ LPnTLP5 DNA fragmentation sequentially adds 2 μ L
PCAMBIA2300s carrier DNA, 2 μ L 10 × T4 DNA Ligase Buffer, 1 μ L T4 DNA Ligase, 5 μ L
ddH2O is centrifuged in short-term after mixing, then 16 DEG C of water-bath reaction overnights.Then connection product is transferred to greatly using heat-shock transformed method
In enterobacteria DH5 α, with the solid medium screening positive clone for containing 50 mg/L kanamycins (Kanamycin, Km).It selects
Single colonie shakes bacterium, expands by template of bacterium solutionPnTLP5Special primer carry out PCR, pick outPnTLP5With
The clone that pCAMBIA2300s is successfully connected, bacterial strain detected are placed in -80 DEG C and save backup if the positive, addition glycerol.
It is extracted using SanPrep pillar plasmid extraction kit and purifies the pCAMBIA2300s- in above-mentioned Escherichia coliPnTLP5Plasmid.Then use frozen-thawed method by the plant expression vector pCAMBIA2300s- of above-mentioned buildingPnTLP5It is transferred to root
In cancer Agrobacterium LBA4404 competent cell.Operating procedure are as follows: take 2 μ g pCAMBIA2300s-PnTLP5Plasmid addition contains
In the centrifuge tube of 200 μ L competent cells, rear 5 min of ice bath is mixed gently, then continues in liquid nitrogen and freezes 1 min, it is then fast
Speed is placed in 37 DEG C of 5 min of water-bath, later 2 min of ice bath immediately, and 800 μ L LB liquid mediums are added in 28 DEG C of shaken cultivations 4
h.Agrobacterium after activation is applied on the LB solid medium containing 50 mg/L Km, 28 DEG C of static gas wave refrigerators.Picking individual colonies
Shake bacterium, then with amplificationPnTLP5Specific primer carry out PCR, detect pCAMBIA2300s-PnTLP5Whether Agrobacterium is transferred to
In.For positive colony, addition glycerol is placed on -80 DEG C and saves backup.
Embodiment 3: the Genetic Transformation in Higher Plants of mediated by agriculture bacillus and genetically modified plants screening
The transgene receptor of this experiment is tobacco, 75% alcohol of tobacco seed is impregnated 30s, with using after sterile water washing
0.1% HgCl2Impregnate 8 min, then again with sterile water washing several times, be seeded on 1/2 MS culture medium, 28 DEG C of dark cultures
6 d go to illumination box (25 DEG C, 16h/d illumination) after germination, later monthly primary with 1/2MS culture medium subculture.
That preservation is taken out from -80 DEG C of refrigerators contains pCAMBIA2300s-PnTLP5The Agrobacterium LBA4404 bacterium of plasmid
Kind, it is inoculated in 5 mL and contains in the LB liquid medium of 50 mg/L Km and 20 mg/L rifampins, 28 DEG C of cultures are muddy to culture medium
It is turbid.It draws on bacterium solution to the LB solid medium containing 50 mg/L Km of 1 mL muddiness, 28 DEG C of 48 h of culture；Then LB is consolidated
Agrobacterium on body culture medium scrapes to be inoculated in the MGL fluid nutrient medium for being attached with the acetosyringone of 20 mg/L in right amount, and 28
DEG C shaken cultivation 2-3 h is to activate Agrobacterium.
Tobacco aseptic seedling leaf is taken to be cut into 1 cm2The leaf dish of left and right is completely soaked in the above-mentioned MGL containing activation Agrobacterium
In fluid nutrient medium, immerged time is 15 min, and the bacterium solution on leaf dish surface is blotted with aseptic filter paper, leaf dish is placed in co-cultivation base
Upper carry out incubated at room temperature, the co-cultivation base of Transformation of tobacco be MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/L sucrose+
6 g/L agar co-culture 2 days under 22 DEG C of no light conditions.
Leaf dish after co-cultivation is gone into seedling differentiation in the MS screening and culturing medium added with antibiotic, while screening transgenic
Plant.Tobacco screening and culturing medium is+6 g/L agar+50 of MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose
Mg/L Km+200 mg/L cephalosporin (Cefotaxime sodium salt, Cef)；Culture bottle is shifted when screening and culturing
To illumination box culture (25 DEG C, 16h/d illumination, 8h/d dark), is used after the long budding of tobacco and contain 50 mg/L Km and 200
The MS culture medium squamous subculture of mg/L Cef finally selects preferable regrowth progress PCR detection of taking root.
The genomic DNA that transgenic tobacco plant blade is extracted using CTAB method, takes 1 μ L to lead to the genomic DNA of extraction
It crosses agarose gel electrophoresis and detects its integrality and concentration, expanded by template of the genomic DNA of transgenic plantPnTLP5
Special primer carry out PCR, after PCR, take 8 μ L products for agarose gel electrophoresis to detect positive transgenic plant,
The amplification of Partial Tobacco transgenic plant as shown in Figure 1,PnTLP5Transgene tobacco screens 30 plants of positive transgenics altogether
Plant.
Embodiment 4: in transgene tobaccoPnTLP5Expression analysis and transgenic plant antifungal activity analysis
The tender leaf of positive transgenic single plant and non-transgenic tobacco (wild type) is taken to extract total serum IgE, reverse transcription generates cDNA the
One chain, and expanded as templatePnTLP5Special primer carry out PCR, according in each transgenosis single plant of PCR interpretation of resultPnTLP5The method of the expression of transcriptional level, Total RNAs extraction and RT-PCR are in the same manner as in Example 1, after PCR terminates, take 5
μ L is used for agarose gel electrophoresis, and the testing result of part single plant is as shown in Figure 2.
By laboratory save several fungies be inoculated in PDA solid medium (200 g/L potatos, 15 g/L agar, 20
G/L glucose) on, 28 DEG C of dark cultures grow to when bacterium colony and add albumen when diameter is about 2 ~ 3cm, analyze transgenic plant body
Outer antifungal activity.The extracted albumen of other living contaminants in order to prevent, entire vegetable protein extraction process are sterile behaviour
Make, takes 1 g transgene tobacco single plant (number is respectively 4,11,19,26) and wild-type leaves to be put into mortar first, be added 1
ML protein extract (1 M NaCl, 0.1 M sodium acetate, 1% PVP, pH6), is fully ground；It is transferred in 1.5 mL centrifuge tubes, mixes
Stood overnight for 4 DEG C after even, 4 DEG C of centrifugations 30 min(12,000 g/min), take supernatant in 1.5 new mL centrifuge tubes, and take suitable
Amount measures total protein concentration with UV detector.The total protein concentration of transgenosis and WT lines is adjusted to 1.2 μ
Then g/ μ L takes 20 μ L drops on the aseptic filter paper of each fungi culture medium, in addition to adding not on the plate of each fungi respectively
With the total protein of transgenic tobacco plant, while the total protein of parallel addition wild-type tobacco and blank control (extract albumen institute
Solution), the case where each processing fungi grows is observed in 28 DEG C of cultures after a few days, and evaluates accordinglyPnTLP5Transgene tobacco
Extracorporeal antifungal activity, as a result as shown in figure 3,PnTLP5Transgenic tobacco leaf albumen is to sclerotinite, ginseng rod method, wheel
The growth of branch sickle-like bacteria, Fusarium solani, grape seat chamber bacterium has very strong inhibiting effect.
Sequence table
<110>Kunming University of Science and Technology
<120>a kind of Radix Notoginseng class sweet protein gene PnTLP5 and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1170
<212> DNA
<213> Panax notoginseng
<400> 1
ggccgcctct agttttagta caatatagtg gcggtattct ttaactaaaa taatgacaag 60
tcagaagagt ggcttatcta ggtgaagtta aaagccgccc ttaccctata taaatccttc 120
atttgctcac aataaaaaac caagctcaaa ttatacaaat attaatttgt tgtcaactta 180
aacaatatga gctacttggc cattgtccca ttcctcctcc tcctccctag cctcttcacc 240
atcaccagtg ccgcgacttt tgaaatccgc aacaactgcc cttacaccgt gtgggctgca 300
gcttcgccag gtggtggtcg tcgacttaac cgaggcgaaa cttggtggtt atatgcacct 360
cctggcacca aaatggcacg catttggggt cgaaccaatt gcaactttga tggttccggt 420
aggggccgct gccaaacagg agattgtggg tccctccagt gcactggctg gggagtccca 480
ccgaataccc tagccgaata tgccctgaac caattcggta acctagattt ttatgacatt 540
tcactagtag atggattcaa tattcctatg gatttcagcc ccaccactaa tcttggaggg 600
aaatgcaaac aacttctgtg tacggcggat atcaacgggc aatgcccgaa cccaatgcgg 660
gttgcgggtg ggtgtaataa cccatgcacg acgttcaaga ctccacaata ctgttgcacc 720
aatggaccat gtggccccac aggttactca aggtttttca aggaaaggtg ccctgatgcc 780
tatagttatc ctcaggatga tccaactagt acttttactt gccccggaaa tagtaattat 840
agggttgtgt tttgcccttg gggatctcct catctggaga taaatggaag tgattataag 900
cagttccaat aagttaaaag gttcaaaatt tgcacgcatg cgtgtgtgaa ggttccacca 960
ctaccgtaca aataagtttt ttgttaaaac caaataacag tgagaaagtg agggaaaaga 1020
gtgtgctagt aattaagtgg tggttgtttt aatgtaattt gttatcttct tgttgttttg 1080
atgtaatttg ttagctaact tcttgttagc agatggaaac atgatcagta gtactatata 1140
tgactaaaaa aaaaaaaaaa aaaaaaaaaa 1170
<210> 2
<211> 241
<212> PRT
<213> Panax notoginseng
<400> 2
Met Ser Tyr Leu Ala Ile Val Pro Phe Leu Leu Leu Leu Pro Ser Leu
1 5 10 15
Phe Thr Ile Thr Ser Ala Ala Thr Phe Glu Ile Arg Asn Asn Cys Pro
20 25 30
Tyr Thr Val Trp Ala Ala Ala Ser Pro Gly Gly Gly Arg Arg Leu Asn
35 40 45
Arg Gly Glu Thr Trp Trp Leu Tyr Ala Pro Pro Gly Thr Lys Met Ala
50 55 60
Arg Ile Trp Gly Arg Thr Asn Cys Asn Phe Asp Gly Ser Gly Arg Gly
65 70 75 80
Arg Cys Gln Thr Gly Asp Cys Gly Ser Leu Gln Cys Thr Gly Trp Gly
85 90 95
Val Pro Pro Asn Thr Leu Ala Glu Tyr Ala Leu Asn Gln Phe Gly Asn
100 105 110
Leu Asp Phe Tyr Asp Ile Ser Leu Val Asp Gly Phe Asn Ile Pro Met
115 120 125
Asp Phe Ser Pro Thr Thr Asn Leu Gly Gly Lys Cys Lys Gln Leu Leu
130 135 140
Cys Thr Ala Asp Ile Asn Gly Gln Cys Pro Asn Pro Met Arg Val Ala
145 150 155 160
Gly Gly Cys Asn Asn Pro Cys Thr Thr Phe Lys Thr Pro Gln Tyr Cys
165 170 175
Cys Thr Asn Gly Pro Cys Gly Pro Thr Gly Tyr Ser Arg Phe Phe Lys
180 185 190
Glu Arg Cys Pro Asp Ala Tyr Ser Tyr Pro Gln Asp Asp Pro Thr Ser
195 200 205
Thr Phe Thr Cys Pro Gly Asn Ser Asn Tyr Arg Val Val Phe Cys Pro
210 215 220
Trp Gly Ser Pro His Leu Glu Ile Asn Gly Ser Asp Tyr Lys Gln Phe
225 230 235 240
Gln
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
ttgtcaactt aaacaatatg agcta 25
<210> 4
<211> 25
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
aaccttttaa cttattggaa ctgct 25
Claims (2)
1. a kind of Radix Notoginseng class sweet protein genePnTLP5, it is characterised in that: its nucleotide sequence is as shown in SEQ IDNO:1, coding
The protein of the amino acid sequence as shown in SEQ IDNO:2.
2. Radix Notoginseng class sweet protein gene described in claim 1PnTLP5Improve tobacco to Fusarium solani (Fusarium solani), wheel branch sickle-like bacteria (F. verticillioides), sclerotinite (Sclerotinia sclerotiorum), ginseng
Rod method (Alternaria panax), grape seat chamber bacterium (Botryosphaeria dothidea) in application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811071523.XA CN109234284B (en) | 2018-09-14 | 2018-09-14 | Pseudo-ginseng sweet protein gene PnTLP5 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811071523.XA CN109234284B (en) | 2018-09-14 | 2018-09-14 | Pseudo-ginseng sweet protein gene PnTLP5 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109234284A true CN109234284A (en) | 2019-01-18 |
| CN109234284B CN109234284B (en) | 2021-04-09 |
Family
ID=65059463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811071523.XA Active CN109234284B (en) | 2018-09-14 | 2018-09-14 | Pseudo-ginseng sweet protein gene PnTLP5 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109234284B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000065039A2 (en) * | 1999-04-28 | 2000-11-02 | Syngenta Participations Ag | Identification of dna control elements responsive to specific stimuli |
| WO2001066146A1 (en) * | 2000-03-08 | 2001-09-13 | Vdf Futureceuticals | Methods and compositions for dietary supplements |
| US20010051195A1 (en) * | 2000-03-08 | 2001-12-13 | Miljkovic Dusan A. | Methods and compositions for increasing fermentation of a microorganism |
| EP1539966A1 (en) * | 2002-09-12 | 2005-06-15 | Greenovation Biotech GmbH | Protein production method |
| KR20060111205A (en) * | 2005-04-22 | 2006-10-26 | 고려대학교 산학협력단 | An antiviral agent comprising nttlp1 isolated from nicotiana tabacum to cmv infection |
| CN101117639A (en) * | 2007-06-28 | 2008-02-06 | 贵州省生物技术研究所 | Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap |
| CN101736024A (en) * | 2010-01-22 | 2010-06-16 | 昆明理工大学 | Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application |
| CN101857869A (en) * | 2010-04-23 | 2010-10-13 | 南京农业大学 | Disease-resistant gene engineering application of soybean TLP (Trypsin-Like Protease) protein coding gene GmTLP1 |
| CN104862307A (en) * | 2014-02-26 | 2015-08-26 | 河北农业大学 | Thaumatin-like protein related to wheat leaf rust resistance and encoding gene and application thereof |
| CN106636091A (en) * | 2016-11-21 | 2017-05-10 | 中国热带农业科学院环境与植物保护研究所 | Litchi leaf specific expression gene LcGRX promoter and application thereof |
| US20170252417A1 (en) * | 2016-03-07 | 2017-09-07 | Massachusetts Institute Of Technology | Protein-chaperoned t-cell vaccines |
| CN108251432A (en) * | 2018-01-02 | 2018-07-06 | 昆明理工大学 | Radix Notoginseng class PR gene PnPRlike and application |
-
2018
- 2018-09-14 CN CN201811071523.XA patent/CN109234284B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000065039A2 (en) * | 1999-04-28 | 2000-11-02 | Syngenta Participations Ag | Identification of dna control elements responsive to specific stimuli |
| WO2001066146A1 (en) * | 2000-03-08 | 2001-09-13 | Vdf Futureceuticals | Methods and compositions for dietary supplements |
| US20010051195A1 (en) * | 2000-03-08 | 2001-12-13 | Miljkovic Dusan A. | Methods and compositions for increasing fermentation of a microorganism |
| EP1539966A1 (en) * | 2002-09-12 | 2005-06-15 | Greenovation Biotech GmbH | Protein production method |
| KR20060111205A (en) * | 2005-04-22 | 2006-10-26 | 고려대학교 산학협력단 | An antiviral agent comprising nttlp1 isolated from nicotiana tabacum to cmv infection |
| CN101117639A (en) * | 2007-06-28 | 2008-02-06 | 贵州省生物技术研究所 | Method for acquiring disease-resistance expression of agrobacterium-mediated potato transgenic hrap |
| CN101736024A (en) * | 2010-01-22 | 2010-06-16 | 昆明理工大学 | Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application |
| CN101857869A (en) * | 2010-04-23 | 2010-10-13 | 南京农业大学 | Disease-resistant gene engineering application of soybean TLP (Trypsin-Like Protease) protein coding gene GmTLP1 |
| CN104862307A (en) * | 2014-02-26 | 2015-08-26 | 河北农业大学 | Thaumatin-like protein related to wheat leaf rust resistance and encoding gene and application thereof |
| US20170252417A1 (en) * | 2016-03-07 | 2017-09-07 | Massachusetts Institute Of Technology | Protein-chaperoned t-cell vaccines |
| WO2017155981A1 (en) * | 2016-03-07 | 2017-09-14 | Massachusetts Institute Of Technology | Protein-chaperoned t-cell vaccines |
| CN106636091A (en) * | 2016-11-21 | 2017-05-10 | 中国热带农业科学院环境与植物保护研究所 | Litchi leaf specific expression gene LcGRX promoter and application thereof |
| CN108251432A (en) * | 2018-01-02 | 2018-07-06 | 昆明理工大学 | Radix Notoginseng class PR gene PnPRlike and application |
Non-Patent Citations (7)
| Title |
|---|
| K. DATTA 等: ""Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease"", 《THEOR APPL GENET》 * |
| LIU,D.等: ""Panax notoginseng thaumatin-like protein (TLP5) mRNA, complete cds"", 《GENBANK DATABASE》 * |
| MADDUMAGE,R.等: ""thaumatin-like protein [Actinidia deliciosa]"", 《GENBANK DATABASE》 * |
| XIN LI等: ""Thaumatin-like protein genes of Panax notoginseng confers resistance to Alternaria panax"", 《PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY》 * |
| 姜晓玲等: ""植物类甜蛋白基因家族研究进展"", 《浙江农林大学学报》 * |
| 李文娴等: ""类甜蛋白的结构特征以及功能研究进展"", 《中国生物工程杂志》 * |
| 金红等: ""利用类甜蛋白基因诱导表达提高马铃薯对晚疫病的抗性研究"", 《华北农学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109234284B (en) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105441460B (en) | A kind of Ming River lily WRKY transcription factor genes LrWRKY1 and application | |
| CN105861517B (en) | A kind of Radix Notoginseng antibacterial peptide gene PnSN1 and its application | |
| CN106244599B (en) | A kind of 1 family gene PnPR1-2 of Radix Notoginseng pathogenesis-related proteins and application | |
| CN103194456B (en) | Lilium regale antifungal gene Lr14-3-3 and application thereof | |
| CN108251432A (en) | Radix Notoginseng class PR gene PnPRlike and application | |
| CN107267526B (en) | Radix Notoginseng myb transcription factor gene PnMYB2 and its application | |
| CN104131015A (en) | Application of lilium regale wilson pathogenesis-related protein 10 gene LrPR10-5 | |
| CN105755020B (en) | Radix Notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 and its application | |
| CN104878028B (en) | Yangbi bulla walnut chitinase gene JsCHI1 and application | |
| CN104878019B (en) | Yangbi bulla walnut class sprouts fibroin gene JsGLP1 and application | |
| CN106244598B (en) | Radix Notoginseng Dirigent albuminoid gene PnDIR1 and application | |
| CN104878041B (en) | Yangbi bulla walnut transcription factor gene JsWRKY1 application | |
| CN103320448B (en) | Lilium regle bZIP transcription factor LrbZIP1 and application | |
| CN107365794A (en) | Pseudo-ginseng chitinase gene PnCHI1 application | |
| CN107267525B (en) | Application of panax notoginseng polygalacturonase inhibitor protein gene PnPGIP | |
| CN102174547B (en) | Beta-1,3-glucanase gene (i)PpGlu(/i) of Pyrus pyrifolia Nakai and application thereof | |
| CN104878027B (en) | Yangbi bulla walnut ribonuclease gene JsRNase and application | |
| CN103937819A (en) | Glutathione S-transferase gene LrGSTL1 of lilium regale and application thereof | |
| CN109234284A (en) | A kind of Radix Notoginseng class sweet protein gene PnTLP5 and application | |
| CN109295068B (en) | Pseudo-ginseng sweet protein gene PnTLP2 and application | |
| CN108707610B (en) | Notoginseng defensein antibacterial peptide genePnDEFL1And applications | |
| CN107964547B (en) | A kind of 10 gene PnPR10-3 of Radix Notoginseng pathogenesis-related proteins and application | |
| CN104152465A (en) | Lilium regale cytochrome b5 gene LrCyt-b5 and application thereof | |
| CN104774847B (en) | Yangbi bulla walnut Pro-rich GFP JsPRP1 and application | |
| CN104131014A (en) | Application of lilium regale germin protein gene LrGLP1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |