CN108707610A - 三七defensin抗菌肽基因PnDEFL1及应用 - Google Patents
三七defensin抗菌肽基因PnDEFL1及应用 Download PDFInfo
- Publication number
- CN108707610A CN108707610A CN201810418737.3A CN201810418737A CN108707610A CN 108707610 A CN108707610 A CN 108707610A CN 201810418737 A CN201810418737 A CN 201810418737A CN 108707610 A CN108707610 A CN 108707610A
- Authority
- CN
- China
- Prior art keywords
- pndefl1
- plant
- tobacco
- radix notoginseng
- 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 39
- 241000180649 Panax notoginseng Species 0.000 title claims abstract description 24
- 235000003143 Panax notoginseng Nutrition 0.000 title claims abstract description 24
- 102000000541 Defensins Human genes 0.000 title claims abstract description 23
- 108010002069 Defensins Proteins 0.000 title claims abstract description 23
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 24
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 241000427940 Fusarium solani Species 0.000 claims abstract description 11
- 241000208340 Araliaceae Species 0.000 claims abstract description 6
- 241000223218 Fusarium 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
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 6
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 6
- 240000006365 Vitis vinifera Species 0.000 claims abstract description 6
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 6
- 235000008434 ginseng Nutrition 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 241000212253 Alternaria panax Species 0.000 claims description 3
- 241000555706 Botryosphaeria dothidea Species 0.000 claims description 3
- 241000233732 Fusarium verticillioides Species 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 abstract description 47
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 31
- 108700019146 Transgenes Proteins 0.000 abstract description 9
- 230000000843 anti-fungal effect Effects 0.000 abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 6
- 208000031888 Mycoses Diseases 0.000 abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 230000002018 overexpression Effects 0.000 abstract description 5
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 5
- 239000013604 expression vector Substances 0.000 abstract description 4
- 239000002543 antimycotic Substances 0.000 abstract description 2
- 241000208125 Nicotiana Species 0.000 abstract 3
- 206010017533 Fungal infection Diseases 0.000 abstract 1
- -1 SEQ ID NO:Shown in 1 Proteins 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 244000061176 Nicotiana tabacum Species 0.000 description 28
- 108020004414 DNA Proteins 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 241000233866 Fungi Species 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 239000002299 complementary DNA Substances 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 230000009261 transgenic effect Effects 0.000 description 10
- 241000589158 Agrobacterium Species 0.000 description 7
- 238000004458 analytical method 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
- 230000014509 gene expression Effects 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 238000006062 fragmentation reaction Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 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
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 241000223195 Fusarium graminearum Species 0.000 description 3
- 240000008415 Lactuca sativa Species 0.000 description 3
- 235000003228 Lactuca sativa Nutrition 0.000 description 3
- 235000005456 Pinus sylvestris var mongolica Nutrition 0.000 description 3
- 241000114025 Pinus sylvestris var. mongolica Species 0.000 description 3
- 241000813090 Rhizoctonia solani Species 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000001839 pinus sylvestris Substances 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 240000005020 Acaciella glauca Species 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 2
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 244000241257 Cucumis melo Species 0.000 description 2
- 244000000626 Daucus carota Species 0.000 description 2
- 235000002767 Daucus carota Nutrition 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 240000004658 Medicago sativa Species 0.000 description 2
- 244000302512 Momordica charantia Species 0.000 description 2
- 235000009811 Momordica charantia Nutrition 0.000 description 2
- 235000008582 Pinus sylvestris Nutrition 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000220259 Raphanus Species 0.000 description 2
- 235000019057 Raphanus caudatus Nutrition 0.000 description 2
- 244000088415 Raphanus sativus Species 0.000 description 2
- 235000011380 Raphanus sativus Nutrition 0.000 description 2
- 235000006140 Raphanus sativus var sativus Nutrition 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
- 108010076830 Thionins Proteins 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 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
- 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
- 230000001857 anti-mycotic effect Effects 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000009545 invasion 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
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- WJRXVTCKASUIFF-FXQIFTODSA-N Ala-Cys-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WJRXVTCKASUIFF-FXQIFTODSA-N 0.000 description 1
- PUBLUECXJRHTBK-ACZMJKKPSA-N Ala-Glu-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O PUBLUECXJRHTBK-ACZMJKKPSA-N 0.000 description 1
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 1
- 101710115746 Antifungal protein 1 Proteins 0.000 description 1
- NVWJMQNYLYWVNQ-BYULHYEWSA-N Asn-Ile-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O NVWJMQNYLYWVNQ-BYULHYEWSA-N 0.000 description 1
- GVPSCJQLUGIKAM-GUBZILKMSA-N Asp-Arg-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GVPSCJQLUGIKAM-GUBZILKMSA-N 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 235000011305 Capsella bursa pastoris Nutrition 0.000 description 1
- 240000008867 Capsella bursa-pastoris Species 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 241001290235 Ceratobasidium cereale 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
- 235000009842 Cucumis melo Nutrition 0.000 description 1
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108060002063 Cyclotide Proteins 0.000 description 1
- QADHATDBZXHRCA-ACZMJKKPSA-N Cys-Gln-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CS)N QADHATDBZXHRCA-ACZMJKKPSA-N 0.000 description 1
- GUKYYUFHWYRMEU-WHFBIAKZSA-N Cys-Gly-Asp Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O GUKYYUFHWYRMEU-WHFBIAKZSA-N 0.000 description 1
- ZHCCYSDALWJITB-SRVKXCTJSA-N Cys-Phe-Cys Chemical compound N[C@@H](CS)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CS)C(O)=O ZHCCYSDALWJITB-SRVKXCTJSA-N 0.000 description 1
- 108010025905 Cystine-Knot Miniproteins Proteins 0.000 description 1
- MMCIFJWGSIWJLP-XAMPRYAMSA-N Dambonitol Chemical compound CO[C@H]1[C@H](O)C(O)[C@H](O)[C@@H](OC)C1O MMCIFJWGSIWJLP-XAMPRYAMSA-N 0.000 description 1
- 101710085792 Defensin-like protein 1 Proteins 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- UPEZCKBFRMILAV-JNEQICEOSA-N Ecdysone Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@H]([C@@H](O)CCC(O)(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 UPEZCKBFRMILAV-JNEQICEOSA-N 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- XHWLNISLUFEWNS-CIUDSAMLSA-N Glu-Gln-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O XHWLNISLUFEWNS-CIUDSAMLSA-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
- IVSWQHKONQIOHA-YUMQZZPRSA-N Gly-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN IVSWQHKONQIOHA-YUMQZZPRSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- PELCGFMHLZXWBQ-BJDJZHNGSA-N Ile-Ser-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)O)N PELCGFMHLZXWBQ-BJDJZHNGSA-N 0.000 description 1
- 244000018716 Impatiens biflora Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 1
- KWURTLAFFDOTEQ-GUBZILKMSA-N Leu-Cys-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KWURTLAFFDOTEQ-GUBZILKMSA-N 0.000 description 1
- RNYLNYTYMXACRI-VFAJRCTISA-N Leu-Thr-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O RNYLNYTYMXACRI-VFAJRCTISA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- WRODMZBHNNPRLN-SRVKXCTJSA-N Lys-Leu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O WRODMZBHNNPRLN-SRVKXCTJSA-N 0.000 description 1
- LUAJJLPHUXPQLH-KKUMJFAQSA-N Lys-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](CCCCN)N LUAJJLPHUXPQLH-KKUMJFAQSA-N 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 206010027146 Melanoderma Diseases 0.000 description 1
- QGQGAIBGTUJRBR-NAKRPEOUSA-N Met-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCSC QGQGAIBGTUJRBR-NAKRPEOUSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000009812 Momordica cochinchinensis Nutrition 0.000 description 1
- 235000018365 Momordica dioica Nutrition 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- BYAIIACBWBOJCU-URLPEUOOSA-N Phe-Ile-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BYAIIACBWBOJCU-URLPEUOOSA-N 0.000 description 1
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-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
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 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
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- XOTBWOCSLMBGMF-SUSMZKCASA-N Thr-Glu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOTBWOCSLMBGMF-SUSMZKCASA-N 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- VVZDBPBZHLQPPB-XVKPBYJWSA-N Val-Glu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VVZDBPBZHLQPPB-XVKPBYJWSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- UPEZCKBFRMILAV-UHFFFAOYSA-N alpha-Ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C(O)CCC(C)(C)O)C)CCC33O)C)C3=CC(=O)C21 UPEZCKBFRMILAV-UHFFFAOYSA-N 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000002091 cationic group Chemical group 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
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- UPEZCKBFRMILAV-JMZLNJERSA-N ecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@H]([C@H](O)CCC(C)(C)O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 UPEZCKBFRMILAV-JMZLNJERSA-N 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
- 230000006353 environmental stress Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 244000037671 genetically modified crops Species 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 108010053156 lipid transfer protein Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 235000011091 sodium acetates Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003245 working effect 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)
- C12N15/8241—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
Abstract
本发明公开了一种三七抗菌肽基因PnDEFL1及应用，PnDEFL1基因的核苷酸序列如SEQ ID NO：1所示，编码defensin抗菌肽，本发明通过功能基因组学相关技术研究证实PnDEFL1基因具有提高植物抗真菌侵染的功能，将本发明抗真菌PnDEFL1基因构建到植物表达载体上并转入烟草中过量表达，转基因烟草具有很强的体外抗真菌活性；PnDEFL1超表达的转基因烟草对人参链格孢、茄腐镰刀菌、串珠状赤霉菌、葡萄座腔菌的生长均有显著的抑制作用。
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，特别是具有抗真菌活性的三七defensin抗菌肽基因PnDEFL1及应用。
背景技术
病原菌是引起植物产量和品质损失的重要影响因素，由病原菌引起的植物病害，可使作物减产20%-40%；其中真菌病害是植物病害中数量最大的一类，约占病害总数的70%-80%；传统控制植物病害的方法主要是使用化学农药、改进栽培管理措施和培育抗性新品种，这些方法虽然取得了一定成效，但传统育种周期长，栽培措施效果差，农药易导致环境安全和食品安全等问题；随着生物技术的快速发展，利用基因工程的方法来培育抗病新品种不仅能克服上述防治方法的诸多弊端，还可最大限度地降低对土壤中有益微生物的伤害，实现农业的可持续发展。
植物抗菌肽(antimicrobial protein，AMP)是植物免疫系统的一部分，对细菌、真菌和病毒等病原微生物都具有抑制或杀灭作用，在植物防卫反应中起着重要的作用(Zasloff M. Antimicrobial peptides of multicellular organisms. Nature, 2002,415(6870): 389–395.)。当植物受到生物或非生物胁迫时，能迅速产生抗菌肽以抑制或杀灭入侵的病原微生物或提高对逆境胁迫的耐受性。根据抗菌肽的氨基酸序列及二级结构，可将植物多肽抗菌肽分为9类，包括硫素(thionins)、植物防御素(plant defensins)、转脂蛋白(lipid transfer proteins, LTPs)、橡胶素(heveins)、打结素(knottins)、凤仙花素(1b-AMPs)和荠菜素(shepherdins)、蜕皮素(snakins)、环肽(cyclotides) (陈艳, 赵文明, 田颖川. 植物多肽抗生素研究进展. 生物化学与生物物理进展. 2003, 30(6): 838–843.)。
defensin抗菌肽（防御素）是富含半胱氨酸的阳离子肽，分子量大约在5 KDa左右，常由45～50个氨基酸组成(Vriens K, Cammue B P, Thevissen K. Antifungal plantdefensins: mechanisms of action and production．Molecules, 2014, 19 (8) :12280－12303)。其典型的三级结构是由一个α螺旋和三股反平行的β折叠构成的CSαβ模体结构，大部分的植物防御素以 8个保守的半胱氨酸残基形成4个分子内二硫键来稳定其三级结构(Fant F, Vranken W, Broekaert W, et al. Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1HNMR. Journal of Molecular Biology, 1998, 279(1) : 257–270)。在10-6μM浓度水平，defensin能够抑制一系列植物病原菌的生长，尤其是对丝状真菌具有强烈的活性作用，但对人、畜、植物细胞无害(廖乾生，林福呈，李德葆. 植物防御素及其研究进展. 浙江大学学报. 2003, 29(1): 113–118.)。defensin抗菌肽的结构及其功能与它们的正电荷和两性分子的性质有关，植物防御素最初通过与病原菌膜上的特殊受体相互作用，与病原菌结合后，会导致Ca2 +和 K+的流入和流出。最后，阻止 Ca2+通道，从而达到抑制病原菌的目的(AertsAM, Francois IEJA, Cammue BPA, et al. The mode of antifungal action of plant,insect and human defensins. Cell Mol Life Sci, 2008, 65 :2069–2079.)。
萝卜(Raphanus sativus) RsAFP2基因对禾谷镰刀菌(Fusarium graminearum)和立枯丝核菌(Rhizoctonia solani)有体外抗菌活性，过表达RsAFP2的小麦植株增加了对禾谷镰刀菌和立枯丝核菌的抗性(Li Z, Zhou M, Zhang Z, et al. Expression of aradish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis. Funct Integr Genomics. 2011, 11(1):63–70. )。Khairutdinov等从樟子松(Pinus sylvestris)中分离纯化了抗菌肽基因PsDef1，用灰葡萄孢、尖孢镰刀菌侵染樟子松后，樟子松中PsDef1基因的表达水平上调，表明PsDef1与真菌胁迫有关；樟子松经茉莉酸处理诱导后，与对照组相比，其PsDef1基因的表达水平明显增加 (Khairutdinov BI, Ermakova EA, Yusypovych YM, et al. NMR structure,conformational dynamics, and biological activity of PsDef1 defensin fromPinus sylvestris. Biochim Biophys Acta. 2017 ;1865(8):1085–1094. )。此外，defensin抗菌肽基因还能与其他抗病基因协同作用，起到叠加抗性的作用。将紫花苜蓿(Medicago sativa)的抗菌肽基因MsDef1与谷氨酸蛋白酶基因在番茄植株中组合表达，增加了转基因番茄植株对枯萎病的抗性(Abdallah NA, Shah D, Abbas D, et al. Stableintegration and expression of a plant defensin in tomato confers resistanceto fusarium wilt. GM Crops. 2010, 1(5):344–350. )。
本发明中defensin抗菌肽基因PnDEFL1来自三七[Panax notoginseng (Burk)F.H. Chen]。三七是我国的传统名贵中药材，其种植区域主要分布于我国云南省文山州。由于三七性喜温暖阴湿，对光敏感，要求在遮阳网下栽培，其独特的生长环境易诱发病虫害的发生，尤其是根腐病、黑斑病等真菌病害，严重影响了三七原药材的产量和品质，迫切需要加强三七与根腐病等病害的分子互作机制研究，并且通过转录组测序，发掘和克隆响应病原菌入侵的防卫相关基因，为三七的抗病育种提供理论基础。
发明内容
本发明的目的是提供一种从三七中克隆获得的defensin抗菌肽的全长基因PnDEFL1，PnDEFL1的核苷酸序列如SEQ ID NO:1所示，该基因全长为702 bp，包含一个234bp的开放阅读框、70 bp的5′非翻译区(untranslated regions，UTR)及398 bp的3′UTR，编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明所述defensin抗菌肽基因PnDEFL1的编码区是序列表SEQ ID NO:1中第71-304位所示的核苷酸序列。
本发明分离克隆三七的一个抗真菌相关基因的完整cDNA片段，通过根癌农杆菌(Agrobacterium tumefaciens)介导将目的基因转入受体植物中过量表达，并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PnDEFL1。
本发明涉及分离包含PnDEFL1的DNA片段并鉴定其功能；其中所述DNA片段如序列表所示，对该基因进行分析，表明PnDEFL1全长cDNA为702 bp，包含一个234 bp的开放阅读框、70 bp的5′UTR及398 bp的3′UTR，其中ORF编码一个具有77个氨基酸的蛋白质。BLASTn分析表明PnPDF1的部分序列与黄胡萝卜（Daucus carota）的defensin-like基因（XR_001802595.1）具有69%的相似性，与莴苣（Lactuca sativa）的DEFL （XM_023891793.1）具有69%的相似性；蛋白质同源性分析表明PnDEFL1编码的蛋白质序列与香瓜（Cucumis melo）的defensin-like protein 1（XP_008454663.1）具有58%的相似性，与苦瓜（Momordica charantia）、莴苣的DEFL1分别具有54%和53%的相似性。PnDEFL1编码的蛋白质序列具有defensin超家族的保守结构域，这表明其属于三七中的defensin类似蛋白。超表达序列表SEQ ID NO:1所示序列可以增强烟草对人参链格孢(Alternaria panax)、茄腐镰刀菌(Fusarium solani)、串珠状赤霉菌(Gibberella moniliformis)、葡萄座腔菌(Botryosphaeria dothidea)的抗性。
本发明另一目的是将三七defensin抗菌肽基因PnDEFL1应用在提高烟草对葡萄座腔菌（Botryosphaeria dothidea）、人参链格孢（Alternaria panax）、串珠状赤霉菌（Gibberella moniliformis）、茄腐镰刀菌（Fusarium solani)抗性中。
上述PnDEFL1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PnDEFL1的特异引物，从接种茄腐镰刀菌后的三七根中提取总RNA，通过逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction，RT-PCR)扩增出PnDEFL1的ORF，然后将其连接到pMD-18T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pMD18-T-PnDEFL1载体和植物表达载体pCAMBIA2300S，通过胶回收得到目的基因片段和载体大片段；再将所获得PnDEFL1基因片段与pCAMBIA2300S载体片段连接，构建植物超表达载体；然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检测得到阳性转基因植株，分析转基因植株对于病原真菌的抗性，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，不仅缩短了育种周期，而且操作简单，容易获得高抗材料。本发明中来自三七的PnDEFL1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、药材等提供方便，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是本发明中部分PnDEFL1转基因烟草基因组DNA的PCR检测结果，其中Marker：DL2000 DNA Marker (大连宝生物)，由2,000 bp、1,000 bp、750 bp、500 bp、250 bp以及100 bp六条DNA片段组成；阳性对照：质粒pMD18-T-PnDEFL1为模板的PCR反应；WT：非转基因烟草(野生型)总DNA为模板进行的PCR；
图2是本发明中部分阳性PnDEFL1转基因烟草中PnDEFL1转录水平的表达分析结果图，其中Marker：DL2000 DNA Marker(大连宝生物)；WT：非转基因烟草总RNA逆转录cDNA为模板的PCR产物；阳性对照：质粒pMD18-T-PnDEFL1为模板的PCR产物；
图3是本发明中PnDEFL1转基因烟草体外抗真菌活性的抑菌效果图；其中a、b、c、d图示中的真菌分别是葡萄座腔菌、人参链格孢、串珠状赤霉菌、茄腐镰刀菌；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进一步说明，但本发明保护范围不局限于所述内容，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例1：PnDEFL1全长cDNA克隆以及序列分析
用茄腐镰刀菌接种三七根，用接种后4 h的根提取总RNA，用液氮将接种后的三七根研磨成粉末，然后转入离心管中，采用异硫氰酸胍法提取总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 total RNA，依次加入50 ng oligo (dT)，2 μL dNTP Mix (2.5 mM each)，用DEPC水将反应体积补齐至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-stand buffer、0.5 μL RNasin (200U)、1 μL M-MLV (200U)，混匀并简短离心，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PnDEFL1，所用上下游引物序列分别为5’GAAAGTGGTTTAGTTCAATTCAAGA3’及5’GTGACCCATGAAATTGCTACTTAG3’。采用AdvantageTM 2PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件：94℃ 5min；94℃ 30s，56℃ 30s，72℃ 30s，32个循环；72℃ 5min。反应体系(20 μL)为1 μL cDNA、2 μL 10×Advantage 2PCR Buffer、1.8 μL dNTP Mix (10mM each)、0.2 μL 正向引物(10 μM)、0.2 μL 反向引物(10 μM)、0.2 μL Advantage 2 PCR Polymerase Mix、14.6 μL PCR-Grade water。PCR结束后，取8 μL进行琼脂糖凝胶电泳，用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，直接对PCR产物进行TA克隆，使用的试剂盒为pMD18-T vector kit (大连宝生物)，反应体系和操作过程为：取1.5 μL PCR产物，依次加入1μL pMD18-T vector (50 ng/μL)和2.5 μL 2×Ligation solution I，混匀后置于16℃过夜反应。通过热激转化法将连接产物转入大肠杆菌DH5α感受态中。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PnDEFL1的特异引物检测多克隆位点插入PnDEFL1的克隆。将得到的阳性克隆进行测序，最终获得的PnDEFL1全长cDNA为702 bp，通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个 234bp的开放读码框(见序列表)。PnDEFL1编码一个含77个氨基酸的蛋白质PnDEFL1，其分子量约为 8.63 KDa，等电点为7.51。借助生物信息学软件SignalP 4.1分析PnDEFL1编码的蛋白序列，检测其是否具有N端信号肽。结果显示在PnDEFL1的N端存在信号肽，因此推测该蛋白是分泌蛋白。
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PnDEFL1的大肠杆菌质粒pMD18-T-PnDEFL1以及植物表达载体pCAMBIA2300S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pMD18-T-PnDEFL1和pCAMBIA2300S进行双酶切(100 μL体系)，反应体系和操作过程为：分别取20 μL pMD18-T-PnPDF1和pCAMBIA2300S质粒、依次加入10 μL 10×Kbuffer、4.5 μL BamHI、5.5 μL EcoRI、60 μL ddH2O，混匀后短时离心，置于37℃过夜反应。将所有酶切产物进行琼脂糖凝胶电泳，然后使用SanPrep柱式DNA胶回收试剂盒 (上海生工)对PnDEFL1片段和pCAMBIA2300S载体大片段分别进行胶回收，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，置于-20℃保存备用。
利用T4 DNA Ligase (TaKaRa)，将回收的PnDEFL1DNA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 PnDEFL1DNA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PnDEFL1的特异引物进行PCR，挑选出PnDEFL1与pCAMBIA2300S成功连接的克隆，并向检测得到的阳性菌株中加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌DH5α中的pCAMBIA2300S-PnDEFL1质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PnDEFL1转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为：取2 μg pCAMBIA2300S-PnDEFL1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1 min，然后迅速置于37℃水浴5min，再冰浴2 min，之后加入500 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50 mg/L Km的LB固体培养基上，28℃倒置培养；挑选单菌落摇菌，再用扩增PnDEFL1的特异性引物进行PCR反应，检测pCAMBIA2300S-PnDEFL1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(Nicotiana tabacum)。将烟草种子用75%的酒精浸泡30s，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5-8 d，发芽后转至光照培养箱(25℃，16 h/d光照)，以后每月用MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300S-PnDEFL1质粒的农杆菌LBA4404菌种，取20 μL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50 mg/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5-8 h以活化农杆菌。
取烟草无菌烟草幼嫩叶片切成约1 cm2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，25℃浸染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，22℃黑暗条件下共培养2天。烟草转化的共培养基为MS+0.02 mg/L 6-BA+2.1 mg/LNAA+30 g/L蔗糖+6 g/L琼脂。
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+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L蔗糖+6 g/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℃，16 h/d光照，8 h/d黑暗)。待烟草分化出芽后用含有50 mg/L Km和200 mg/L Cef的MS培养基继代培养。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幼苗进行PCR分析。
采用CTAB法提取转基因烟草植株叶片的基因组DNA，取1 μL所得基因组DNA进行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PnDEFL1的特异引物进行PCR反应。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，PnDEFL1转基因烟草共筛选到37株阳性转基因植株。
实施例4：转基因烟草中PnDEFL1的表达分析以及转基因植株抗真菌活性分析
分别取阳性转基因植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PnDEFL1的特异引物进行PCR，根据PCR结果分析各转基因植株中PnDEFL1转录水平的表达量；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。
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，20 g/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。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0.5 g转基因烟草单株(编号分别为4、6、10、11)及野生型叶片放入研钵中，加入1 mL蛋白提取液(1 M NaCl，0.1 M 乙酸钠，1% PVP，pH6.0)，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)，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(蛋白提取液)，28℃培养几天后观察各处理真菌生长的情况，并据此来评价PnDEFL1转基因烟草的体外抗真菌活性。结果如图3所示，PnDEFL1转基因烟草蛋白对人参链格孢、茄腐镰刀菌、串珠状赤霉菌以及葡萄座腔菌的生长具有明显的抑制作用。
序列表
<110> 昆明理工大学
<120> 三七defensin抗菌肽基因PnDEFL1及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 702
<212> DNA
<213> 三七(Panax notoginseng)
<400> 1
atccaagaat cttttactat aaattcccca caaaatgaaa gtggtttagt tcaattcaag 60
aagattaaac atggcaataa aatttagccc aactaccttc attacaatct ctctaatttt 120
tttcctcctt gctaacacag aaacaaatat aggtgttgag ggaaaattat gtgaaaaact 180
aagcttgaca tggtccggga ggtgcggaga ctcaggccac tgtgaacagc aatgccagaa 240
cgcggaatct gcaaaacgtg gagcatgtcg cgatcggaga tgcttctgtt actttgattg 300
ctgatgatct aattaacggc gccaagaaat aaatatatat cgatcagaat cttcttaaat 360
caaatttctt cttatcgtgc taaataaata aataaaatga aatgcttctt atcgtgctaa 420
gtagcaattt catgggtcac tatagctagg caggcatatg ataactaaag ttgtcatgaa 480
agtgtctttc atatatatta tatatgtatt atgtgtgtgt gtgtgtgtct gtacgtttcg 540
atcaatttat gtcttatgaa aaaatttaag tgtttctttg attctataaa ttttctttga 600
aaatacttca atataattat ggcgtgtaat acttctgaaa aattaatcta tattgacgct 660
tatgatcatc atcgtaaaaa aaaaaaaaaa aaaaaaaaaa aa 702
<210> 2
<211> 77
<212> PRT
<213> 三七(Panax notoginseng)
<400> 2
Met Ala Ile Lys Phe Ser Pro Thr Thr Phe Ile Thr Ile Ser Leu Ile
1 5 10 15
Phe Phe Leu Leu Ala Asn Thr Glu Thr Asn Ile Gly Val Glu Gly Lys
20 25 30
Leu Cys Glu Lys Leu Ser Leu Thr Trp Ser Gly Arg Cys Gly Asp Ser
35 40 45
Gly His Cys Glu Gln Gln Cys Gln Asn Ala Glu Ser Ala Lys Arg Gly
50 55 60
Ala Cys Arg Asp Arg Arg Cys Phe Cys Tyr Phe Asp Cys
65 70 75
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 3
gaaagtggtt tagttcaatt caaga 25
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial)
<400> 4
gtgacccatg aaattgctac ttag 24
Claims (2)
1.一种三七defensin抗菌肽基因PnDEFL1，其特征在于：其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的三七defensin抗菌肽基因PnDEFL1在提高烟草对葡萄座腔菌（Botryosphaeria dothidea）、人参链格孢（Alternaria panax）、串珠状赤霉菌（Gibberella moniliformis）、茄腐镰刀菌（Fusarium solani)抗性中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810418737.3A CN108707610B (zh) | 2018-05-04 | 2018-05-04 | 三七defensin抗菌肽基因PnDEFL1及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810418737.3A CN108707610B (zh) | 2018-05-04 | 2018-05-04 | 三七defensin抗菌肽基因PnDEFL1及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108707610A true CN108707610A (zh) | 2018-10-26 |
| CN108707610B CN108707610B (zh) | 2021-01-05 |
Family
ID=63867749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810418737.3A Active CN108707610B (zh) | 2018-05-04 | 2018-05-04 | 三七defensin抗菌肽基因PnDEFL1及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108707610B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604477A (zh) * | 2021-08-20 | 2021-11-05 | 昆明理工大学 | 一种岷江百合defensin抗菌肽基因LrDef1及应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102295689A (zh) * | 2010-06-28 | 2011-12-28 | 中国农业大学 | 植物耐旱相关蛋白AtDi19及其编码基因与应用 |
| CN103194457A (zh) * | 2013-04-24 | 2013-07-10 | 昆明理工大学 | 岷江百合类萌发素蛋白基因LrGLP2及其应用 |
| CN104878019A (zh) * | 2015-05-14 | 2015-09-02 | 昆明理工大学 | 漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用 |
-
2018
- 2018-05-04 CN CN201810418737.3A patent/CN108707610B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102295689A (zh) * | 2010-06-28 | 2011-12-28 | 中国农业大学 | 植物耐旱相关蛋白AtDi19及其编码基因与应用 |
| CN103194457A (zh) * | 2013-04-24 | 2013-07-10 | 昆明理工大学 | 岷江百合类萌发素蛋白基因LrGLP2及其应用 |
| CN104878019A (zh) * | 2015-05-14 | 2015-09-02 | 昆明理工大学 | 漾濞大泡核桃类萌发素蛋白基因JsGLP1及应用 |
Non-Patent Citations (1)
| Title |
|---|
| 杨丹等: "三七病程相关蛋白PR10-2 基因的克隆、表达及功能初步分析", 《中国中药杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604477A (zh) * | 2021-08-20 | 2021-11-05 | 昆明理工大学 | 一种岷江百合defensin抗菌肽基因LrDef1及应用 |
| CN113604477B (zh) * | 2021-08-20 | 2023-03-24 | 昆明理工大学 | 一种岷江百合defensin抗菌肽基因LrDef1及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108707610B (zh) | 2021-01-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105441460B (zh) | 一种岷江百合WRKY转录因子基因LrWRKY1及应用 | |
| CN105861517B (zh) | 一种三七抗菌肽基因PnSN1及其应用 | |
| CN106244599B (zh) | 一种三七病程相关蛋白1家族基因PnPR1-2及应用 | |
| CN108251432B (zh) | 三七类病程相关蛋白基因PnPRlike及应用 | |
| CN104488945B (zh) | 杀虫蛋白的用途 | |
| CN111235165B (zh) | 一种百合的易感真菌基因LrWRKY-S1及其应用 | |
| CN107267526B (zh) | 三七MYB转录因子基因PnMYB2及其应用 | |
| CN104131015B (zh) | 一种岷江百合病程相关蛋白10基因LrPR10-5的应用 | |
| CN103194456B (zh) | 岷江百合抗真菌基因Lr14-3-3及其应用 | |
| CN103194457B (zh) | 岷江百合类萌发素蛋白基因LrGLP2及其应用 | |
| CN105755020B (zh) | 三七丝裂原活化蛋白激酶激酶基因PnMAPKK1及其应用 | |
| 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 | |
| CN106244598B (zh) | 三七Dirigent类似蛋白基因PnDIR1及应用 | |
| CN102732532A (zh) | 一种提高水稻抗逆性的小分子热激蛋白基因的应用 | |
| CN103320448B (zh) | 一种岷江百合bZIP转录因子基因LrbZIP1及应用 | |
| CN104878041B (zh) | 漾濞大泡核桃转录因子基因JsWRKY1的应用 | |
| CN104744577B (zh) | 球孢白僵菌抗菌蛋白Bbafp及其表达载体、含有其基因的转基因植物的制备方法和应用 | |
| CN108707610A (zh) | 三七defensin抗菌肽基因PnDEFL1及应用 | |
| CN107267525B (zh) | 三七多聚半乳糖醛酸酶抑制蛋白基因PnPGIP的应用 | |
| CN107365794A (zh) | 三七几丁质酶基因PnCHI1的应用 | |
| CN104878027B (zh) | 漾濞大泡核桃核糖核酸酶基因JsRNase及应用 | |
| CN107964547B (zh) | 一种三七病程相关蛋白10基因PnPR10-3及应用 | |
| CN109295068A (zh) | 一种三七类甜蛋白基因PnTLP2及应用 | |
| CN104131014A (zh) | 岷江百合类萌发素蛋白基因LrGLP1的应用 |
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 |