CN112029766A - 一种双链rna的应用 - Google Patents
一种双链rna的应用 Download PDFInfo
- Publication number
- CN112029766A CN112029766A CN202010754468.5A CN202010754468A CN112029766A CN 112029766 A CN112029766 A CN 112029766A CN 202010754468 A CN202010754468 A CN 202010754468A CN 112029766 A CN112029766 A CN 112029766A
- Authority
- CN
- China
- Prior art keywords
- seq
- double
- vector
- rna
- enzyme
- 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
- 108091032973 (ribonucleotides)n+m Proteins 0.000 title claims abstract description 122
- 102000040650 (ribonucleotides)n+m Human genes 0.000 title claims abstract description 62
- 239000013598 vector Substances 0.000 claims abstract description 74
- 241000238631 Hexapoda Species 0.000 claims abstract description 34
- 230000009261 transgenic effect Effects 0.000 claims abstract description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 6
- 239000002299 complementary DNA Substances 0.000 claims description 49
- 238000001976 enzyme digestion Methods 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 30
- 102000004190 Enzymes Human genes 0.000 claims description 28
- 108090000790 Enzymes Proteins 0.000 claims description 28
- 241000196324 Embryophyta Species 0.000 claims description 27
- 108091030071 RNAI Proteins 0.000 claims description 27
- 230000009368 gene silencing by RNA Effects 0.000 claims description 27
- 230000003321 amplification Effects 0.000 claims description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 23
- 229920000742 Cotton Polymers 0.000 claims description 22
- 239000012634 fragment Substances 0.000 claims description 22
- 239000013612 plasmid Substances 0.000 claims description 22
- 241000255967 Helicoverpa zea Species 0.000 claims description 16
- 238000003776 cleavage reaction Methods 0.000 claims description 16
- 230000007017 scission Effects 0.000 claims description 16
- 101150036126 sNPF gene Proteins 0.000 claims description 15
- 238000010839 reverse transcription Methods 0.000 claims description 12
- 238000005520 cutting process Methods 0.000 claims description 10
- 230000000968 intestinal effect Effects 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 241000209510 Liliopsida Species 0.000 claims description 5
- 210000004556 brain Anatomy 0.000 claims description 5
- 241001233957 eudicotyledons Species 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 230000008685 targeting Effects 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 17
- 241000607479 Yersinia pestis Species 0.000 abstract description 9
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 241001147381 Helicoverpa armigera Species 0.000 description 56
- 108020004414 DNA Proteins 0.000 description 49
- 238000006243 chemical reaction Methods 0.000 description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- 238000012360 testing method Methods 0.000 description 25
- 239000000203 mixture Substances 0.000 description 23
- 101710101888 Short neuropeptide F Proteins 0.000 description 22
- 241000219146 Gossypium Species 0.000 description 21
- 239000000047 product Substances 0.000 description 21
- 238000002347 injection Methods 0.000 description 16
- 239000007924 injection Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- 238000002156 mixing Methods 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 238000003786 synthesis reaction Methods 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 12
- 235000004341 Gossypium herbaceum Nutrition 0.000 description 11
- 240000002024 Gossypium herbaceum Species 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 230000035611 feeding Effects 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 102000006382 Ribonucleases Human genes 0.000 description 6
- 108010083644 Ribonucleases Proteins 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 108090000189 Neuropeptides Proteins 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 235000021050 feed intake Nutrition 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 3
- 229920002527 Glycogen Polymers 0.000 description 3
- 241000255777 Lepidoptera Species 0.000 description 3
- 241000233855 Orchidaceae Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 3
- 229940096919 glycogen Drugs 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000012536 storage buffer Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 2
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 2
- CVGNCMIULZNYES-WHFBIAKZSA-N Ala-Asn-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CVGNCMIULZNYES-WHFBIAKZSA-N 0.000 description 2
- 240000007185 Albizia julibrissin Species 0.000 description 2
- 235000011468 Albizia julibrissin Nutrition 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 2
- UISQLSIBJKEJSS-GUBZILKMSA-N Arg-Arg-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(O)=O UISQLSIBJKEJSS-GUBZILKMSA-N 0.000 description 2
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 2
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 2
- ICRHGPYYXMWHIE-LPEHRKFASA-N Arg-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ICRHGPYYXMWHIE-LPEHRKFASA-N 0.000 description 2
- 241000758795 Aristolochiaceae Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- GURLOFOJBHRPJN-AAEUAGOBSA-N Asn-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N GURLOFOJBHRPJN-AAEUAGOBSA-N 0.000 description 2
- RLHANKIRBONJBK-IHRRRGAJSA-N Asn-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)N)N RLHANKIRBONJBK-IHRRRGAJSA-N 0.000 description 2
- OERMIMJQPQUIPK-FXQIFTODSA-N Asp-Arg-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O OERMIMJQPQUIPK-FXQIFTODSA-N 0.000 description 2
- JGDBHIVECJGXJA-FXQIFTODSA-N Asp-Asp-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JGDBHIVECJGXJA-FXQIFTODSA-N 0.000 description 2
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 2
- ZVGRHIRJLWBWGJ-ACZMJKKPSA-N Asp-Ser-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVGRHIRJLWBWGJ-ACZMJKKPSA-N 0.000 description 2
- 240000007124 Brassica oleracea Species 0.000 description 2
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 2
- 235000009046 Convallaria majalis Nutrition 0.000 description 2
- 244000068485 Convallaria majalis Species 0.000 description 2
- 240000001980 Cucurbita pepo Species 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 2
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- KVYVOGYEMPEXBT-GUBZILKMSA-N Gln-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O KVYVOGYEMPEXBT-GUBZILKMSA-N 0.000 description 2
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 2
- OWOFCNWTMWOOJJ-WDSKDSINSA-N Gln-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O OWOFCNWTMWOOJJ-WDSKDSINSA-N 0.000 description 2
- AVZHGSCDKIQZPQ-CIUDSAMLSA-N Glu-Arg-Ala Chemical compound C[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AVZHGSCDKIQZPQ-CIUDSAMLSA-N 0.000 description 2
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 2
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 description 2
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 2
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 2
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 2
- AFWYPMDMDYCKMD-KBPBESRZSA-N Gly-Leu-Tyr Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AFWYPMDMDYCKMD-KBPBESRZSA-N 0.000 description 2
- VOEGKUNRHYKYSU-XVYDVKMFSA-N His-Asp-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O VOEGKUNRHYKYSU-XVYDVKMFSA-N 0.000 description 2
- DURWCDDDAWVPOP-JBDRJPRFSA-N Ile-Cys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N DURWCDDDAWVPOP-JBDRJPRFSA-N 0.000 description 2
- FHPZJWJWTWZKNA-LLLHUVSDSA-N Ile-Phe-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N FHPZJWJWTWZKNA-LLLHUVSDSA-N 0.000 description 2
- 241001113425 Iridaceae Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 2
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 2
- VKOAHIRLIUESLU-ULQDDVLXSA-N Leu-Arg-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VKOAHIRLIUESLU-ULQDDVLXSA-N 0.000 description 2
- ILJREDZFPHTUIE-GUBZILKMSA-N Leu-Asp-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ILJREDZFPHTUIE-GUBZILKMSA-N 0.000 description 2
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 2
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 2
- XWEVVRRSIOBJOO-SRVKXCTJSA-N Leu-Pro-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O XWEVVRRSIOBJOO-SRVKXCTJSA-N 0.000 description 2
- JDBQSGMJBMPNFT-AVGNSLFASA-N Leu-Pro-Val Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O JDBQSGMJBMPNFT-AVGNSLFASA-N 0.000 description 2
- MEQLGHAMAUPOSJ-DCAQKATOSA-N Lys-Ser-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O MEQLGHAMAUPOSJ-DCAQKATOSA-N 0.000 description 2
- XIGAHPDZLAYQOS-SRVKXCTJSA-N Met-Pro-Pro Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 XIGAHPDZLAYQOS-SRVKXCTJSA-N 0.000 description 2
- 102000003797 Neuropeptides Human genes 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 241000256259 Noctuidae Species 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- RFEXGCASCQGGHZ-STQMWFEESA-N Phe-Gly-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O RFEXGCASCQGGHZ-STQMWFEESA-N 0.000 description 2
- ITUDDXVFGFEKPD-NAKRPEOUSA-N Pro-Ser-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ITUDDXVFGFEKPD-NAKRPEOUSA-N 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- NLQUOHDCLSFABG-GUBZILKMSA-N Ser-Arg-Arg Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NLQUOHDCLSFABG-GUBZILKMSA-N 0.000 description 2
- OHKLFYXEOGGGCK-ZLUOBGJFSA-N Ser-Asp-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OHKLFYXEOGGGCK-ZLUOBGJFSA-N 0.000 description 2
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 2
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 2
- 241000233945 Typhaceae Species 0.000 description 2
- ZRPLVTZTKPPSBT-AVGNSLFASA-N Tyr-Glu-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZRPLVTZTKPPSBT-AVGNSLFASA-N 0.000 description 2
- VDPRBUOZLIFUIM-GUBZILKMSA-N Val-Arg-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C(C)C)N VDPRBUOZLIFUIM-GUBZILKMSA-N 0.000 description 2
- VSCIANXXVZOYOC-AVGNSLFASA-N Val-Pro-His Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N VSCIANXXVZOYOC-AVGNSLFASA-N 0.000 description 2
- GBIUHAYJGWVNLN-AEJSXWLSSA-N Val-Ser-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N GBIUHAYJGWVNLN-AEJSXWLSSA-N 0.000 description 2
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 108010068380 arginylarginine Proteins 0.000 description 2
- 108010062796 arginyllysine Proteins 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 235000012631 food intake Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 108010040030 histidinoalanine Proteins 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229930014550 juvenile hormone Natural products 0.000 description 2
- 239000002949 juvenile hormone Substances 0.000 description 2
- 150000003633 juvenile hormone derivatives Chemical class 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 230000001418 larval effect Effects 0.000 description 2
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000219144 Abutilon Species 0.000 description 1
- 241000499929 Agapanthus Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 235000017334 Alcea rosea Nutrition 0.000 description 1
- 240000000530 Alcea rosea Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 244000295724 Allium chinense Species 0.000 description 1
- 235000016790 Allium chinense Nutrition 0.000 description 1
- 240000002234 Allium sativum Species 0.000 description 1
- 241000572565 Alpinia oxyphylla Species 0.000 description 1
- 241000223600 Alternaria Species 0.000 description 1
- 235000017303 Althaea rosea Nutrition 0.000 description 1
- 241000219317 Amaranthaceae Species 0.000 description 1
- 241000234270 Amaryllidaceae Species 0.000 description 1
- 241000209524 Araceae Species 0.000 description 1
- 241000722824 Ardisia crenata Species 0.000 description 1
- 241000233788 Arecaceae Species 0.000 description 1
- 241000726094 Aristolochia Species 0.000 description 1
- 241000432824 Asparagus densiflorus Species 0.000 description 1
- 241001532036 Aspidistra Species 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- 235000011274 Benincasa cerifera Nutrition 0.000 description 1
- 244000036905 Benincasa cerifera Species 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 241001478750 Chlorophytum comosum Species 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241001672694 Citrus reticulata Species 0.000 description 1
- 240000000560 Citrus x paradisi Species 0.000 description 1
- 235000008738 Clausena lansium Nutrition 0.000 description 1
- 244000089795 Clausena lansium Species 0.000 description 1
- 235000007354 Coix lacryma jobi Nutrition 0.000 description 1
- 244000077995 Coix lacryma jobi Species 0.000 description 1
- 241000233833 Commelinaceae Species 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 235000009852 Cucurbita pepo Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 240000007371 Cuscuta campestris Species 0.000 description 1
- 241000234646 Cyperaceae Species 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 241000244987 Daiswa polyphylla Species 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 241000234272 Dioscoreaceae Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000508725 Elymus repens Species 0.000 description 1
- 235000009008 Eriobotrya japonica Nutrition 0.000 description 1
- 244000061508 Eriobotrya japonica Species 0.000 description 1
- 241000735527 Eupatorium Species 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- 241000605372 Fritillaria Species 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 241000131505 Gloriosa Species 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 241000756137 Hemerocallis Species 0.000 description 1
- 235000002941 Hemerocallis fulva Nutrition 0.000 description 1
- 240000009206 Hemerocallis fulva Species 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 235000003935 Hippophae Nutrition 0.000 description 1
- 241000229143 Hippophae Species 0.000 description 1
- 241001482471 Hosta plantaginea Species 0.000 description 1
- 241001632576 Hyacinthus Species 0.000 description 1
- 235000017846 Ipomoea alba Nutrition 0.000 description 1
- 240000008436 Ipomoea aquatica Species 0.000 description 1
- 235000019004 Ipomoea aquatica Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 240000008285 Ipomoea violacea Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 240000003915 Lophatherum gracile Species 0.000 description 1
- 244000241838 Lycium barbarum Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 235000015468 Lycium chinense Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241001633628 Lycoris Species 0.000 description 1
- 241000218394 Magnolia liliiflora Species 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 235000009811 Momordica charantia Nutrition 0.000 description 1
- 235000009812 Momordica cochinchinensis Nutrition 0.000 description 1
- 240000001740 Momordica dioica Species 0.000 description 1
- 235000018365 Momordica dioica Nutrition 0.000 description 1
- 241000234615 Musaceae Species 0.000 description 1
- 241000124079 Mylabris Species 0.000 description 1
- 241000234479 Narcissus Species 0.000 description 1
- 235000017879 Nasturtium officinale Nutrition 0.000 description 1
- 240000005407 Nasturtium officinale Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 244000248557 Ophiopogon japonicus Species 0.000 description 1
- 240000000654 Ornithogalum longebracteatum Species 0.000 description 1
- 241000531207 Oxera splendida Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 241000218688 Podocarpaceae Species 0.000 description 1
- 235000016067 Polianthes tuberosa Nutrition 0.000 description 1
- 244000014047 Polianthes tuberosa Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101710130181 Protochlorophyllide reductase A, chloroplastic Proteins 0.000 description 1
- 240000002292 Psychopsis papilio Species 0.000 description 1
- 241000736301 Pteridaceae Species 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 240000001987 Pyrus communis Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001426527 Rhaponticum Species 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241001448530 Rohdea japonica Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241001278833 Rosa laevigata Species 0.000 description 1
- 235000000661 Rosa laevigata Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 240000000411 Sansevieria trifasciata Species 0.000 description 1
- 244000057114 Sapium sebiferum Species 0.000 description 1
- 235000005128 Sapium sebiferum Nutrition 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 241000244978 Stemonaceae Species 0.000 description 1
- 241000244155 Taenia Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000722921 Tulipa gesneriana Species 0.000 description 1
- 244000153888 Tung Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 244000015069 Zephyranthes candida Species 0.000 description 1
- 244000128884 Zier Kohl Species 0.000 description 1
- 241000234299 Zingiberaceae Species 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000005058 diapause Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 108010063039 endodeoxyribonuclease SfiI Proteins 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 230000003031 feeding effect Effects 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000004611 garlic Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000013016 learning Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000001719 neurosecretory cell Anatomy 0.000 description 1
- 101150046050 npf gene Proteins 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008058 pain sensation Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000007958 sleep Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- 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
- C12N15/8286—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 insect resistance
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Plant Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Pest Control & Pesticides (AREA)
- Cell Biology (AREA)
- Insects & Arthropods (AREA)
- Virology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种双链RNA在制备抗虫剂或培育抗虫的转基因植物中的应用。本发明还提供了一种包含双链RNA的载体或重组菌株。本发明提供的双链RNA、包含双链RNA的载体或重组菌株,可以明显干扰害虫的取食能力,抑制其危害植物,有效的培育出抗虫的转基因植物。同时,所述的双链RNA、载体或重组菌株无污染,也不会将有害物质传递到害虫的天敌体内,与环境相容性好。
Description
技术领域
本发明涉及一种基因工程技术领域,具体涉及一种双链RNA及其在制备抗虫剂或转基因植物培育中的应用。
背景技术
神经肽是一类小分子活性多肽,主要由神经分泌细胞(Enteroendocrine cells,EEs)或神经元(Neuron)产生,某种程度上具有较高的相似性:基因转录翻译后首先形成前导前体蛋白(Pre-pro-proteins),N端16-30氨基酸残基组成前体蛋白(Precursors),形成具有疏水结构的信号肽,前体蛋白(Precursors)在其识别与引导下运送至内质网进行正确折叠(Endoplasmic Reticulum,ER),前体多肽在内质网内经历一系列翻译后修饰(糖基化、羟基化、酰基化、二硫键形成),接着进入高尔基体后,氨基酸单个或者相邻的两个位点被蛋白酶切割,C-末端碱性残基由羧肽酶转化酶切除,形成具有生物活性的成熟多肽,随后通过各种代谢途径分泌到胞外,达到靶位点,与蛋白受体特别是G蛋白偶联受体(G Protein-coupled Receptor,GPCR),参与各种生理活动。
短神经肽(short NPF),也称sNPF就是其中众多神经肽的一种,在昆虫中主要参与多种重要的生理和行为活动,如痛觉、睡眠、取食、代谢、学习与记忆乃至神经系统本身的分化和发育,在不同发育期还可调节保幼激素的合成,对保幼激素合成具有一定的抑制作用。
棉铃虫属于鳞翅目、夜蛾科,主要以幼虫取食幼嫩组织为害作物,以蛹越冬。此外,棉铃虫具有显著的生理生态及行为特点,例如杂食性、滞育、迁飞等,还是研究鳞翅目昆虫的代表物种。
为防治作物的虫害,专利CN110679571A公开了一种棉花种植用高效灭虫灯,该灭虫灯通过改变高压电网的结构,并利用电动马达带动旋转,网栅和网罩主动性靠近接触灯管周围的昆虫,增加害虫触网概率,提高灭杀效率。
专利CN110558193A公开了一种高产低害的棉花种植方法,包括选种育苗、苗床管理、移栽、田间管理、收获,该种植方法通过对棉花种子的合理处理强化以及对棉花植株的有效抗病防害,大大提高了棉花苗期的存活率和抗病性,虫害率降低了25%以上。
专利CN105967896A公开了一种棉花种植用的抗病虫营养液,该营养液能够增强棉花的抗病虫能力,提高棉花的免疫能力,促进棉花植株的根系发达等等。
但是,上述现有技术中,并没有解决针对特定害虫的防治措施,有些方法的使用还会导致环境不相容。
发明内容
为解决现有技术中的问题,本发明的目的是提供一种RNAi干扰载体或双链RNA,并将其应用于培育抗虫的转基因植物。具体的:
本发明的第一方面,提供了一种双链RNA在制备抗虫剂或培育抗虫的转基因植物中的应用,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
本发明的第二方面,提供了一种包含双链RNA的载体或重组菌株,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
优选的,所述的载体包括但不限于pDNR-LIB载体。
优选的,所述的载体包含SEQ ID NO:20所示的核苷酸序列。
优选的,所述的重组菌株包括但不限于农杆菌或大肠杆菌。
在本发明的一个具体实施方式中,所述的菌株为大肠杆菌DH5α或C58农杆菌。
本发明的第三方面,提供了一种包含双链RNA、载体或重组菌株的试剂盒、细胞系、植株或其叶片。
优选的,所述的植株为单子叶植物或双子叶植物。
在本发明的一个具体实施方式中为棉花植株。
本发明的第四方面,提供了一种双链RNA的制备方法,所述的制备方法包括采用靶向靶序列SEQ ID NO:3和/或SEQ ID NO:4的引物,扩增棉铃虫sNPF基因的cDNA序列,合成双链RNA。
优选的,先采用引物SEQ ID NO:14、SEQ ID NO:15,再采用引物SEQ ID NO:12和SEQ ID NO:13扩增棉铃虫sNPF基因的cDNA序列,合成双链RNA。
在本发明的一个具体实施方式中,所述的制备方法包括:
获取棉铃虫sNPF基因的总RNA,反转录获得cDNA;优选为棉铃虫肠总RNA或脑总RNA,进一步优选为肠总RNA;
扩增所述的cDNA;优选扩增引物为SEQ ID NO:10、11;
将扩增获得的cDNA连接至载体后,转入细胞,提取质粒;
采用靶向靶序列SEQ ID NO:3和/或SEQ ID NO:4的引物,扩增提取的质粒,合成双链RNA;优选先采用引物SEQ ID NO:14和SEQ ID NO:15,然后采用引物SEQ ID NO:12和SEQID NO:13扩增提取的质粒,合成双链RNA。
本发明的第五方面,提供了一种包含双链RNA核苷酸序列的RNAi干扰载体的制备方法,所述的制备方法包括采用上游引物:包含酶切位点I、酶切位点II和SEQ ID NO:10,下游引物:包含酶切位点III、酶切位点IV和SEQ ID NO:11,扩增棉铃虫sNPF基因的cDNA,然后经酶切与载体连接获得RNAi干扰载体。
在本发明的一个具体实施方式中,所述的制备方法包括:
获取棉铃虫sNPF基因的总RNA,反转录获得cDNA;优选为棉铃虫肠总RNA或脑总RNA,进一步优选为肠总RNA;
采用上游引物:包含酶切位点I、酶切位点II和SEQ ID NO:10,下游引物:包含酶切位点III、酶切位点IV和SEQ ID NO:11,扩增cDNA,获得扩增产物;优选酶切位点I为SmaI识别位点、酶切位点II为EcoRI识别位点、酶切位点III为BamHI识别位点、酶切位点IV为HindIII识别位点;进一步优选采用引物SEQ ID NO:18、19扩增cDNA;
用酶切位点I对应的酶与酶切位点III对应的酶双酶切获得的扩增产物,获得目的片段1。
用酶切位点I对应的酶与酶切位点III对应的酶双酶切载体,获得载体骨架1;优选载体为pGreen-HY104。
将获得的目的片段1连接至载体骨架1上,获得重组质粒1。
用酶切位点II对应的酶与酶切位点IV对应的酶双酶切获得的扩增产物,获得目的片段2。
用酶切位点II对应的酶与酶切位点IV对应的酶双酶切获得的重组质粒1,获得载体骨架2。
将获得的目的片段2连接至载体骨架2上,获得RNAi干扰载体。
优选的,所述的RNAi干扰载体或包含双链RNA的载体,其具有特异DNA片段。所述的特异DNA片段包括DNA片段甲、DNA片段乙以及DNA片段甲与DNA片段乙之间的间隔序列,其中,DNA片段甲与DNA片段乙的序列反向互补。
进一步优选的,所述的RNAi干扰载体或包含双链RNA的载体,为采用骨架载体连接的DNA片段甲和DNA片段乙。
本发明的第六方面,提供了一种棉铃虫sNPF基因的cDNA文库或获取棉铃虫sNPF基因的cDNA的构建方法,包括:
1)取棉铃虫sNPF基因的总RNA,反转录获得cDNA;
优选为棉铃虫肠总RNA或脑总RNA,进一步优选为肠总RNA。
2)扩增cDNA
5'RACE扩增引物为SEQ ID NO:5、6;
优选扩增条件为:94℃3min;94℃30S,60℃→45℃,其中每个循环降低1℃,45s、72℃2min,15个循环;94℃30S、45℃45S、72℃2min,30个循环;72℃10min。
3'RACE扩增引物为SEQ ID NO:26、27;
优选扩增条件为:94℃3min;94℃30S、56℃30S、72℃30S,10个循环;4℃温浴,加入下游接头引物3AP;94℃30S、56℃30S、72℃30S,25个循环;72℃7min。
全长扩增引物为SEQ ID NO:8、9。
优选扩增条件为:94℃3min;94℃30s、65℃→55℃(每个循环降低1℃)30s、72℃1min,10个循环;94℃30s、54℃30s、72℃1min,30个循环;72℃10min。
优选的,所述的抗虫为抗鳞翅目昆虫。更优选为抗夜蛾科昆虫。
在本发明的一个具体实施方式中,所述的抗虫为抗棉铃虫。
优选的,所述的转基因植物为单子叶植物或双子叶植物。进一步优选为棉花植物。
本发明的第七方面,提供了一种抗虫的转基因植物的培育方法,所述的培育方法包括:
将本发明所述的双链RNA、本发明所述的载体或重组菌株,导入出发植物,得到抗虫的转基因植物。
优选的,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
优选的,所述的抗虫为抗鳞翅目昆虫。
在本发明的一个具体实施方式中,所述的鳞翅目昆虫为棉铃虫。
优选的,所述的出发植物为单子叶植物或双子叶植物。进一步优选为棉花出发植物。
优选的,所述的培育方法还包括育苗、苗床管理、移栽和/或田间管理等步骤。
本发明的第八方面,提供了一种抗虫剂,所述的抗虫剂包含本发明所述的双链RNA、本发明所述的载体或重组菌株。
本发明的第九方面,提供了一种试剂盒,所述的试剂盒包括本发明所述的双链RNA、本发明所述的载体或重组菌株。
本发明提供的RNAi干扰载体或双链RNA,可以明显干扰害虫的取食能力,抑制其危害植物,有效的培育出抗虫的转基因植物。同时,RNAi干扰载体或双链RNA无污染,也不会将有害物质传递到害虫的天敌体内,与环境相容性好。
同时,与现有技术(例如CN201210336702中序列9、11的NPF基因)相比,首先属于不同的基因(参见Spit J,Badisco L,H.Verlinden,P.Van Wielendaele,S.Zels,S.Dillen,J.VandenBroeck.Peptidergic control of food intake and digestion in insects 1[J].Canadian Journal of Zoology,2012,90(4):489-506);其次,本申请所述的双链RNA是在一个编码框里同时可以产生3-4个8个氨基酸的成熟肽一起起作用。而现有技术中的NPF是在一个编码框中编码长度在36个氨基酸的成熟肽在起作用。
本发明所述的“包含”在本申请中用于描述蛋白质或核酸的序列时,所述蛋白质或核酸可以是由所述序列组成,或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸,但仍然具有本发明所述的活性。
本发明所述的“鳞翅目昆虫”包括但不限于蛾、蝶两类昆虫。属有翅亚纲、全变态类。在本发明的一个具体实施方式中,所述的鳞翅目昆虫为棉铃虫。
本发明所述的“单子叶植物”包括但不限于香蒲科、露兜树科、黑三棱科、水蕹科、眼子菜科、茨藻科、冰沼草科、泽泻科、花蔺科、水鳖科、霉草科、禾本科、莎草科、棕榈科(槟榔科)、天南星科、浮萍科、须叶藤科、帚灯草科、刺鳞草科、黄眼草科、谷精草科、凤梨科、鸭跖草科、雨久花科、田葱科、灯心草科、百部科、百合科、石蒜科、蒟蒻薯科(箭根薯科)、薯蓣科、鸢尾科、芭蕉科、姜科、美人蕉科、竹芋科、水玉簪科或兰科等等。优选包括郁金香、风信子、萱草,玉簪、铃兰、吊兰、文竹、天门冬、麦冬、沿阶草、贝母、嘉兰、虎眼万年青、虎尾兰、七叶一枝花、蜘蛛抱蛋、黄精、黄花菜、大蒜、蕌头、洋葱、葱、铃兰花、百子莲、君子兰、网球花、朱顶兰、石蒜、水仙、晚香玉、玉帘、风雨花、金色葱莲、薏苡、芦根、茅根、淡竹叶、竹茹、麦芽、谷芽、龟背竹、马蹄莲、花叶芋、白掌、红掌、万年青、天南星、半夏、菖蒲、千年健、海芋、水浮莲、沙姜、草果、豆蔻、砂仁、郁金、莪术、益智、姜花、卡特兰、蝴蝶兰、石斛兰、万代兰、文心兰、兜兰、天麻、石斛、白芨、手参、石仙桃、香子兰等等。
本发明所述的“双子叶植物”包括但不限于白兰、荷花玉兰、菜心、萝卜、白菜、芥菜、西洋菜、南瓜、冬瓜、西瓜、黄瓜、苦瓜、丝瓜、棉花、烟草、山茶花、苘麻、大红花、蜀葵、蓖麻、木薯、橡胶树、乌桕、油桐、木油树、玫瑰、月季、蔷薇、金樱子、苹果、梨、枇杷、含羞草、合欢、柑、橙、柚、柠檬、黄皮、向日葵、莴苣、茼蒿、苦荬菜、菊花、烟草、马铃薯、番茄、茄、辣椒、枸杞、曼陀罗、番薯、蕹菜、五爪金龙、月光花、茑萝或菟丝子等。
附图说明
以下,结合附图来详细说明本发明的实施例,其中:
图1:棉铃虫中肠组织cDNA建库电泳图,其中,泳道M1、M2为Marker,泳道1-17分别为依次收集17个管中样品电泳结果,代表不同时间、不同片段的DNA文库。
图2:pDNR-LIB载体的结构及部分序列示意图。
图3:棉铃虫中肠组织cDNA文库的菌落PCR随机鉴定结果,M为Marker,泳道1-20分别为随机挑取的单克隆。
图4:棉铃虫神经肽sNPF基因的全长扩增产物的电泳图,其中,M为Marker,泳道1、2为两个随机样本。
图5:pGreen-HY104载体的结构示意图。
图6:HindIII和BamHI双酶切鉴定棉铃虫RNAi干扰载体,其中,M为Marker,泳道1对应pGreen-HY104载体,泳道2对应转ds EGFP对照,泳道3对应转dsNPF对应酶切后的棉铃虫RNAi干扰载体。
图7:棉铃虫sNPF dsRNA琼脂糖凝胶电泳图,其中,M为Marker,泳道1对应含有dsEGFP片段,泳道2对应棉铃虫sNPF目的基因dsRNA。
图8:棉铃虫注射sNPF dsRNA后,棉铃虫的取食量变化,其中图8-1为注射ds EGFP的对照组,图8-2为注射含有ds sNPF的试验组。
图9:注射dsRNA后,棉铃虫的取食量变化。
图10:饲喂含有转基因sNPF双链RNA的棉花叶片后棉铃虫取食效果(图10-1,10-2为含有双链RNA的转基因棉花叶片,图10-3和10-4为转入空载体的棉花叶片)。
图11:饲喂含有转基因sNPF双链RNA的棉花叶片后,棉铃虫取食叶片效果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:棉铃虫sNPF蛋白及其编码基因的获得
一、cDNA文库构建
文库构建使用SMART cDNA Library Construction Kit试剂盒。以下均参照该建库试剂盒操作说明,具体构建流程如下:取2μL棉铃虫幼虫中肠总RNA(约2μg),依次加入1μLSMART IV Oligoncleotide(5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGG-3(SEQ IDNO:23)),1μL CDS/3'PCR primer(ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N(SEQ IDNO:24)),然后加入2μL无RNase去离子水。轻轻混匀后离心,72℃孵育2min,冰上冷却2min后离心,每管中加入2μL 5x第一链合成Buffer,1μL DTT(20mM),lμL dNTP混合物(10mM),1μLMMLV反转录酶,至总体积为10μL。同时做一个阳性对照。轻轻混匀后、离心后在PCR仪中42℃孵育1h,在冰上终止第一链的合成,至此第一链合成完成。
在合成第二链的反应中选用LD-PCR法,加入第一链cDNA 2μL,去离子水80μL,10xAdvantage 2PCR Buffer 10μL,50x dNTP Mix 2μL,5'PCR引物(5'-AAGCAGTGGTATCAACGCAGAGT-3'(SEQ ID NO:25))2μL,CDS III/3'PCR引物2μL,50xAdvantage 2Polymerase Mix 2μL,总体积100μL。轻轻混匀后离心,使用以下程序进行反应:95℃1min;95℃15sec、68℃6min,22个循环。
反应结束后,取5μL在1.1%含有EtBr的胶上进行电泳检测。第二链产物合成后需用蛋白酶K进行消化。方法如下:在0.5mL的离心管中加入50μL ds cDNA和2μL蛋白酶K(20μg/μL),混匀后离心,45℃孵育20min,反应结束后加入50μL去离子水,然后加入100μL酚:氯仿:异戊醇(25:24:1,体积比)进行抽提,14000rpm离心5min后取上清。在上清中加入10μLNaAC(醋酸钠,3M,pH4.8),1.3μL Glycogen(糖原,20μg/μL)和260μL 95%ETOH(乙醇),室温下迅速14000rpm离心20min,小心弃去上清,加100μL含80%的乙醇洗涤沉淀,沉淀干燥约10min以除去残留的乙醇。加入79μL的去离子水,充分溶解沉淀。
在上述79μL ds cDNA溶液中加入10μL 10x Sfi Buffer,10μL SfiI内切酶和1μL100x BSA,混匀后50℃孵育2h进行酶切。反应结束后加入2μL 1%二甲苯青染色,然后准备进行ds cDNA片段分离。ds cDNA片段分离使用CHROMA SPIN-400柱子进行,步骤如下:
1:在离心管铁架台上放置16个1.5mL离心管;
2:将CHROMA SPIN-400柱从4℃冰箱中取出,按以下步骤处理柱
2.1室温放置lh,颠倒数次彻底悬浮胶体,或者打开上盖子用微量枪头轻轻混匀;
2.2轻轻悬浮胶体(勿产生气泡),拔掉底盖;
2.3将柱子水平固定在铁架台上,打开柱子下面封口,使贮存缓冲液自然流下,待流尽后看凝胶体积是否达到1mL,如果没有1mL,在其它柱子中取凝胶补充至1mL;
2.4柱子流速大约为1滴/40-60s,1滴约为40μL,若流速太低(<1滴/100s)或每滴体积太小(<25μL)必须重复上述步骤进行重新悬浮。
3:贮存缓冲液流完后,在柱的中心处加入700μL柱缓冲液,让其流出;
4:待柱缓冲液流干后,小心将约100μL经SfiI-酶切的cDNA和二甲苯青混合物加到胶顶部中心部位;
5:当样品充分进入胶内时,向柱内加入100μL的柱缓冲液,待柱内凝胶表面液体消失且没有液滴滴下时进行下一步;
6:准备好17个1.5mL离心管的架子置于柱子下面,第一个管子置于柱子出口下部;加入600μL的柱缓冲液,立即开始收集,每管35μL(约1滴),收集一个管子后,放入下一个管子,直至收集够全部17管;
7:制备1.1%的琼脂糖凝胶,电泳检测17管的收集物,每管用3μL连续在点样孔加样,150V电泳10min,电泳结束后检测电泳结果(如图1所示,不同时间DNA片段均匀分布于各泳道中,表明建库质量较高);收集6-11管含有cDNA的组分到一个新1.5mL的离心管中;
8:依次加入1/10体积NaAC(3M;pH=4.8),1.3μL Glycogen(20mg/mL),2.5倍体积95%ETOH(-20)℃,充分混匀,-20℃过夜沉淀;
9:室温14000rpm离心20min;
10:小心弃上清,轻离心,用移液器小心吸去剩余的上清,干燥约10min;11:以7μL无离子水溶解沉淀,轻微混匀,即制备好酶切完成的PCR片段。
将酶切完成、收集好的PCR片段与经SfiI酶切的pDNR-LIB载体(结构示意图见图2)连接。连接体系如下:dscDNA 1.0μL、pDNR-LIB载体1.0μL、10x ligation buffer 0.5μL、ATP(10mM)0.5μL、T4 DNA连接酶0.5μL、去离子水1.5μL。
将上述反应后的重组质粒,通过电击转化仪导入大肠杆菌DH5α菌株感受态细胞中。取3份25μL感受态细胞,冰上融化,分别加入上述连接产物,混匀后加入到电转杯中进行电转转化。转化后迅速取出电转杯,加入970μL 37℃预热的SOC培养基,转移到10mL离心管中,37℃震荡培养1h(200rpm/min)。取培养物涂布在含有30mg/mL氯霉素的平板上,37℃倒置培养过夜。根据菌落生长情况确定适合的连接体系,以此作为原始文库,4℃保存。棉铃虫中肠组织cDNA文库的菌落PCR鉴定结果见图3。
二、5'RACE PCR扩增
5'RACE上游引物采用引物SMART cDNA Library Construction Kit中的5'引物(5'-AAGCAGTGGTATCAACGCAGAGT-3'(SEQ ID NO:5)),下游引物采用设计的简并引物(sNPF:5'-AANCKNARNCKDATNSWNGGNGC-3'(SEQ ID NO:6))。
按照试剂盒说明书操作,反应体系(25μL):LA PCR Buffer(Mg2+)2.5μL、dNTP mix(each 10mM)4μL、LA taq 0.25μL、Primer 1(10μmol/L)1μL、Primer 2(10μmol/L)1μL、质粒cDNA文库0.5μL、ddH2O 11.25μL。
在PCR管中将其混合完毕后,放入PCR仪扩增,反应条件为:94℃3min;94℃30S、60℃→45℃(每个循环降低1℃)45s、72℃2min,15个循环;94℃30S、45℃45S、72℃2min,30个循环;72℃10min。
反应结束后经2%琼脂糖凝胶电泳,回收连接克隆后转入大肠杆菌培养,挑菌送样测序。
三、3'RACE快速扩增3'末端
1、cDNA合成
1)将模板RNA在冰上解冻;引物、10×RT mix(其中包含RNasin和DTT)、超纯dNTP混合液、RNase-free ddH2O在室温(15-25℃)解冻,解冻后迅速置于冰上。使用前将每种溶液涡旋振荡混匀,离心以收集残留在管壁的液体。
2)配制逆转录体系混合液,彻底混匀,涡旋振荡时间不超过5s。简短离心,并置于冰上,RNA模板于第4)步加入。
3)若做多个逆转录反应,可以将配制好的混合液后分装在单个反应管中,置于冰上。
4)首先取适量的RNA 1μg进行定量,冰浴条件下按照Tiangen(Quant One StepRT-PCR Kit)说明进行以下配置反转录体系:10x RT mix、dNTP混合液(2.5mM each)、dT3AP、Quant Reverse Transcriptase、RNAse-free H2O、模板RNA(在第4步加入),总体积20μL。
5)将模板RNA(50ng-2μg)加入到混合液中,彻底混匀,涡旋振荡时间不超过5s,离心15s。
6)37℃孵育60min,4℃保存备用。
2、3'RACE扩增
根据cDNA反转录时候3'-dT3AP引物结构,设计3'外引物:3Ap(5'-ATTCTAGAGGCCGAGGCGGCCGACATG-3’(SEQ ID NO:26));根据5'RACE扩增并测出的序列信息,设计出一条上游特异性引物SPF(sNPF:5'-ATGAATCATCAGCAGCGTCGGAC-3'(SEQ ID NO:27))。
3'RACE扩增采用两步PCR的方法,即第一步PCR只需在反应体系中加入上游引物,在PCR管中将其混合完毕后,放入PCR仪扩增,通过10个循环的单引物反应,增加目的基因的初始拷贝数,然后再加入下游3AP引物,进行25个左右循环的扩增。
按照试剂盒说明书操作,反应体系(25μL):LA PCR Buffer(Mg2+)2.5μL、dNTP mix(each 10mM)4μL、LA taq 0.25μL、Primer 1(SPF)(10μmol/L)1μL、ddH20 11.75μL。
反应条件:94℃3min;94℃30S、56℃30S、72℃30S,10个循环;4℃温浴,加入下游接头引物3AP;94℃30S、56℃30S、72℃30S,25个循环;72℃7min。
反应结束后进行2%琼脂糖凝胶电泳,回收连接克隆后转入大肠杆菌培养,挑菌送样测序。
四、cDNA全长的扩增
根据NPF5'和3'RACE测序结果设计扩增棉铃虫NPF全长基因序列,上游命名为P1F,下游命名为P1R。
按照试剂盒说明书操作,反应体系(25μL):LA PCR Buffer(Mg2+)2.5μL、dNTP mix(each 10mM)4μL、LA taq 0.25μL、引物P1F(10μmol/L)1μL、引物P1R(10μmol/L)1μL、棉铃虫质粒cDNA文库0.5μL、ddH20 11.25μL。
引物P1F:5'-ATGTCTCCGAGCTTCGTTTGCGCGCT-3'(SEQ ID NO:8);
引物P1R:5'-TTATTCCTGTTGTCTGTCGTCTTCCAC-3'(SEQ ID NO:9)。
反应条件为:94℃3min;94℃30s、65℃→55℃(每个循环降低1℃)30s、72℃1min,10个循环;94℃30s、54℃30s、72℃1min,30个循环;72℃10min。
反应结束进行2%琼脂糖凝胶电泳,回收连接克隆后转入大肠杆菌培养,挑菌送样测序。棉铃虫神经肽sNPF基因的全长扩增产物的电泳图见图4。
通过上述试验,获得棉铃虫sNPF基因的其中一种剪辑形式,其cDNA序列如SEQ IDNO:2所示,可以编码得到SEQ ID NO:1所示的蛋白质。
实施例2:棉铃虫sNPF双链RNA的合成
1、总RNA抽提
(1)试验准备:无RNA酶枪头、离心管,研磨棒高温灭菌等;
(2)将棉铃虫中肠组织充分研磨,使组织与Trizol试剂中完全接触,并加入1mL的Trizol试剂,于室温静置5min,加入200μL氯仿试剂,充分混匀后,室温放置5-8min;
(3)离心15min(12000r/min,4℃),将上清液转至新的无RNA酶的离心管中,并加入等体积的异丙醇试剂,充分混匀后,室温静置10min;
(4)离心10min(12000r/min,4℃),弃去上清液,将沉淀物干燥后,加75%乙醇试剂洗涤沉淀;
(5)离心5min(12000r/min,4℃),弃去上清液,将沉淀物简单干燥后,加入DEPC水,充分溶解并混匀,于紫外分光光度计测量OD值(1.8-2.0之间),保证RNA的高质量,后置于-80℃冰箱保存以便备用。
2、cDNA模板的合成
将所抽提的各组织总RNA,按照反转录试剂盒说明书操作,以便合成cDNA:2μLgDNA Eraser Buffer,1μL gDNA Eraser,1μg不同样品总RNA,适量RNase free dH2O补足至10μL体系,42℃反应2min,冰上加入2μL 5×Buffer,1μL RT Primer Mix,1μLRT Enzyme MixⅠ,4μL RNase free H2O。将以上试剂充分混合均匀后,37℃孵育15min,后于85℃孵育5s。样品保存在-80℃冰箱,以备后续试验进行。
模板扩增引物:
sNPF-F2:ATGAATCATCAGCAGCGTCGGAC(SEQ ID NO:10)
sNPF-R2:GGGCAGTAAGAACATGTTGTTGT(SEQ ID NO:11)
dsRNA合成引物:
sNPF-F:ATGAATCATCAGCAGCGTCGGAC(SEQ ID NO:14)
sNPF-R:GGGCAGTAAGAACATGTTGTTGT(SEQ ID NO:15)
T7-sNPF-F:
GATCACTAATACGACTCACTATAGGGAGAATGAATCATCAGCAGCGTCGGAC(SEQ ID NO:12)
T7-sNPF-R:
GATCACTAATACGACTCACTATAGGGAGAGGGCAGTAAGAACATGTTGTTGT(SEQ ID NO:13)
EGFPdsRNA的引物:
EGFP-F:TGGTCCCAATTCTCGTGGAAC(SEQ ID NO:28)
EGFP–R:CTTGAAGTTGACCTTGATGCC(SEQ ID NO:29)
T7 EGFP-F:GATCACTAATACGACTCACTATAGGGAGATGGTCCCAATTCTCGTGGAAC(SEQ IDNO:16)
T7 EGFP–R:GATCACTAATACGACTCACTATAGGGAGACTTGAAGTTGACCTTGATGCC(SEQ IDNO:17)
3、目的序列的PCR扩增
以反转录获得的cDNA为模版,sNPF F2/R2为引物,经PCR反应获得目的序列。反应程序为:94℃,3min(预变性);94℃,1min(变性);58℃,20s(退火);72℃,30s(延伸);35个循环;72℃,10min(延伸),获得dsTK目的序列。PCR产物经电泳45min后,于紫外凝胶成像系统中观测目的条带,以期望获得清晰的目标条带。
4、PCR产物纯化
目标条带大小正确后,使用PCR产物纯化试剂盒(上海生工生物)进行DNA回收,按照试剂盒说明书进行操作,具体步骤如下:
(1)将PCR反应液转入新的1.5mL离心管,并加入3倍反应液体积的Buffer PCR-A,混合均匀。
(2)将PCR制备管置于2mL离心管,并将步骤(1)中的混合试剂转至PCR制备管中,室温放置1-2min。
(3)室温条件下,离心机内离心1min(12,000r/min),弃去滤液。
(4)向PCR制备管中加入700μL的Buffer W2试剂,离心机内离心1min(12,000r/min),弃去滤液。此次步骤重复两次(注意:进行第二次重复时,PCR制备管中加入400μL的Buffer W2试剂。
(5)将PCR制备管放回2mL离心管,再离心(12,000r/min)。
(6)将PCR制备管放置于新的离心管(试剂盒内提供),加入25μL的Elution Buffer(60℃),37℃温育5min。
(7)室温条件下,离心机内离心1min(12,000r/min),洗脱DNA。
5、PCR纯化产物与克隆载体连接
反应体系:5μL Solution I,0.5μL T载体,100ng-200ng PCR产物,用ddH2O补足至10μL。反应条件:16℃,30min-60min(PCR仪中)。
6、感受态细胞的制备
感受态细胞制备的具体步骤如下:
(1)试验准备:超净工作台、已灭菌试管、已灭菌离心管、已灭菌LB液体培养基、大肠杆菌母液(需提前一晚挑斑摇12h左右)、0.1Mol/L冰CaCl、酒精灯、火柴、摇床、离心机、冰。
(2)向已灭菌的试管中倒入5mL的无抗LB液体培养基(超净工作台内,酒精灯前操作)。
(3)吸取100μL的大肠杆菌母液至试管中(超净工作台内,酒精灯前操作)。
(4)将试管放置摇床内(37℃,200rpm),培养1h-2h(至略有絮状即可)。
(5)将(4)倒入已灭菌的1.5mL离心管中,4℃离心(6000rpm/min,2min),舍弃上清液。重复两次。
(6)加入750μL冰CaCl2(0.1Mol/L),充分混匀,使沉淀的菌体悬浮。
(7)冰浴30min,4℃离心(4000rpm/min,5min),舍弃上清。
(8)加入200μL冰CaCl2,充分混匀,使沉淀的菌体悬浮,即为感受态细胞。4℃保存以备用。
7、载体转化至感受态细胞
载体转化至感受态细胞的具体步骤如下:
(1)试验准备:已灭菌离心管、已灭菌LB液体培养基、含Amp的LB固体平板(需提前配置LB固体培养基,并倒板)、水浴锅、摇床、培养箱。
(2)将10μL连接载体的反应液加入至200μL的感受态细胞中,轻轻吸打混匀。
(3)将离心管放置冰上,冰浴30min。
(4)水浴锅中热冲击(42℃,120s)后,立即冰浴(3-5min)。
(5)加入800μL的无抗生素的LB液体培养基,放入摇床培养1h(37℃,130rpm/min)。
(6)取50μL均匀涂布到LB固体平板(Amp)上,放入培养箱,37℃过夜培养。第二天查看菌落生长情况。
8、重组质粒的提取
以菌落为模板,扩PCR。通过琼脂糖电泳跑胶检测有无目的条带进行阳性斑筛选,如有目的条带可进行摇菌获得大量质粒。质粒抽提可使用上海莱枫生物技术有限公司试剂盒,具体操作如下:
(1)试验准备:已灭菌离心管、已灭菌枪头、离心机。
(2)将菌液收集至1.5mL离心管,离心30s(12000g/min),舍弃上清。
(3)加入250μL Buffer P1(含RNase A1),充分悬浮细菌。
(4)加入250μL Buffer P2,缓慢翻转离心管(5-10次)。
(5)加入350μL Buffer P3,缓慢翻转离心管(约10-20次),离心5min(12000g/min)。
(6)将DNA吸附柱置于收集管中,并将(5)的上清液转入DNA吸附柱中,离心1min(12000g/min),弃去废液,将DNA吸附柱放回收集管中。
(7)加入500μL Buffer WB,离心1min(12000g/min),弃去废液,将DNA吸附柱放回收集管中。此次步骤重复两次。
(8)离心2min(12000g/min)。
(9)将PCR制备管放置于新的离心管(试剂盒内提供),加入50-200μL去离子水(pH≥7.0),室温放置5min。
(10)离心机内离心1min(12,000g/min),洗脱质粒。
9、dsRNA的合成
以测序正确的质粒为模版,不含T7的sNPF F/R为引物,经第一轮PCR反应获得不含T7的目的序列,并将PCR产物纯化(具体操作参照3、4)。以纯化的PCR产物为模板,T7 sNPFF/R为引物,经第二轮PCR反应获得含有T7序列的目的片段(见图5),并将其纯化,获得dsRNA模板(具体操作步骤同3、4)。
使用MEGA script RNAi Kit(ABI公司)试剂盒进行体外合成dsRNA。反应体系为:4μL T7 promoter template,4μL 10×T7 Reaction Buffer,4μL ATP Solution,4μL GTPSolution,4μL CTP Solution,4μL UTP Solution,4μL T7 Enzyme Mix,DEPC水补足至40μL。反应条件:37℃温育4h。
核酸酶消化去除DNA和单链RNA。dsRNA合成后,反应液中加入1μL TURBO DNase,混合均匀后,37℃孵育15min。
10、dsRNA的纯化
dsRNA合成后,将反应液吸入1.5mL离心管,加入等体积的酚氯仿,充分混合均匀后,离心2min(12000r/min);转移上清液至无RNA酶的1.5mL离心管中,加入2.5倍体积的无水乙醇,充分混合均匀,-20℃静置至少4h后,离心10min(12000r/min);倒掉上清,白色粘稠状沉淀物即为dsRNA;加入适量DEPC进行溶解,并计算浓度,于-20℃低温保存以便使用。其中,紫外凝胶成像系统中观测目的条带结果见图7所示。
最终获得的dsRNA的核苷酸序列如SEQ ID NO:22所示,具体如下:
atgtctccgagcttcgtttgcgcgctggctgtctgcggtttcgcggcgatctgctcattgcccgtgtccaccgcgcaggcgcttgtttctccttatgaatcatcagcagcgtcggacagtcaaaacggttggggcgcgttaggcgggctgtacgctctactagcgcagcacgacgcactgggcggccacgcgctcgcccgcaagtcggtccgctcgccctcccgccgtctgcgcttcggacgccgatccgaccccgacatgcctcctcaagctcccctggacgagatgaatgagctgctctctctgcgcgaggtccgcacgcccgtgcgtctgcgattcggccgccgctcggaggaacgtgccgtgccccacatcttcccccaggaggagcaggaccgtgccgtgcgtgccccgtccatccgtctccggttcggacgccggtccgacaacaacatgttcttactgccctacgaatcggctctgccccaggaagtgaaagccaacggctccgtggaagacgacagacaacaggaataa
其编码的氨基酸序列如下:
MetSerProSerPheValCysAlaLeuAlaValCysGlyPhe
AlaAlaIleCysSerLeuProValSerThrAlaGlnAlaLeu
ValSerProTyrGluSerSerAlaAlaSerAspSerGlnAsn
GlyTrpGlyAlaLeuGlyGlyLeuTyrAlaLeuLeuAlaGln
HisAspAlaLeuGlyGlyHisAlaLeuAlaArgLysSerVal
ArgSerProSerArgArgLeuArgPheGlyArgArgSerAsp
ProAspMetProProGlnAlaProLeuAspGluMetAsnGlu
LeuLeuSerLeuArgGluValArgThrProValArgLeuArg
PheGlyArgArgSerGluGluArgAlaValProHisIlePhe
ProGlnGluGluGlnAspArgAlaValArgAlaProSerIle
ArgLeuArgPheGlyArgArgSerAspAsnAsnMetPheLeu
LeuProTyrGluSerAlaLeuProGlnGluValLysAlaAsn
GlySerValGluAspAspArgGlnGlnGlu(SEQ ID NO:21)。
实施例3:注射dsRNA对幼虫取食的影响
1、试验组别及棉铃虫饲料配方
1)试验组别
试验组:实施例2制备的sNPF dsRNA,溶于DEPC水。
对照组:按照实施例2的方法制备ds EGFP,溶于DEPC水。其中,合成ds EGFP采用的引物如下:
合成EGFP dsRNA的引物:
EGFP-F:TGGTCCCAATTCTCGTGGAAC(SEQ ID NO:28)
EGFP–R:CTTGAAGTTGACCTTGATGCC(SEQ ID NO:29)
T7 EGFP-F:GATCACTAATACGACTCACTATAGGGAGATGGTCCCAATTCTCGTGGAAC(SEQ IDNO:16)
T7 EGFP–R:GATCACTAATACGACTCACTATAGGGAGACTTGAAGTTGACCTTGATGCC(SEQ IDNO:17)
2)饲料
玉米粉300克、大豆粉100g、酵母粉100g、琼脂25g、维生素C 2.5g、维生素B1.5g、山梨酸2g、柠檬酸2.5g、红霉素半片、水1300mL和丙酸5mL。
2、试验方法
取蜕皮当天的棉铃虫5龄幼虫,应用10μL微量进样器通过体腔注射的方法(先将棉铃虫在冰上麻醉,然后从腹足进行注射)将实施例2制备的棉铃虫sNPF dsRNA(每头棉铃虫注射10μg棉铃虫dsRNA)导入棉铃虫体内,对照组注射等量的ds EGFP。
处理方法:注射后将棉铃虫放入装有新鲜饲料的养虫管中进行饲养(每支养虫管加入长方形饼状的饲料),每24小时取出饲料,去除饲料中的粪便(粪便为颗粒状,可以较为容易的从饼状饲料中去除,见图8,其中饼状的为饲料,颗粒状的为粪便),然后将饲料称重后放回养虫管。
具体试验方法如下:
1、体重测定
每天定点用电子天平称量并记录每只虫子的体重,记为Ln(以注射当天为第一天,n为注射后的天数),L(n-1)-L(n),作为一天的体重增长量。
2、幼虫取食量测定
测量试验期间饲料因含水量改变而发生的重量变化,因此设对照组,取相同大小的饲料3块分别放到3个相同的养虫管里,放在与试验中的养虫管相同的环境下;对照组放置和取出饲料的时间与试验组相同;首先称量对照组最初的饲料重量,每24小时后取出饲料并称取重量。
幼虫取食量的计算公式如下:
W为试验组试验开始时加入的饲料的初始重量;
L为试验组某一阶段(如24小时)结束时的饲料重量;
3、试验结果
试验第1天(即试验24小时后)棉铃虫的取食量结果见图8(单位为g)。试验第24h、48h及72h时,棉铃虫取食结果见图9,图9明显看出,注射ds sNPF比注射ds EGFP,对棉铃虫取食量的影响差距显著。结果表明,注射棉铃虫dsRNA后,棉铃虫取食量显著减少。
实施例4:sNPF RNAi干扰载体的构建
1、提取5龄棉铃虫幼虫中肠总RNA并反转录为cDNA,以cDNA为模板,采用sNPF-F2和NPF-R2为模板,采用LA taq作为DNA聚合酶,进行PCR扩增,回收PCR扩增产物。sNPF-F2:5'-(SEQ ID NO:18);sNPF-R2:5'-(SEQ ID NO:19)
sNPF-F2中,下划线标注限制性内切酶SmaI的酶切识别序列,方框标注限制性内切酶EcoRI的酶切识别序列。sNPF-R2下划线标注限制性内切酶BamHI的酶切识别序列,方框标注限制性内切酶HindⅢ的酶切识别序列。
PCR扩增体系:LA PCR Buffer(Mg2+)2.5μL、dNTP mix(each 10mM)4μL、LA taq0.25μL、sNPF-F2(10μmol/L)1μL、sNPF-R2(10μmol/L)1μL、cDNA 1.0μL、ddH20 10.75μL。
PCR扩增条件:94℃3min;94℃30s、56℃30s、72℃30s,35个循环;72℃7min;4℃保存。
2、用限制性内切酶SmaI和BamHI双酶切步骤1得到的PCR扩增产物,回收酶切产物。
3、用限制性内切酶SmaI和BamHI双酶切pGreen-HY104载体,回收载体骨架(约6kb)。
4、将步骤2的酶切产物和步骤3的载体骨架连接,得到重组质粒甲。
5、用限制性内切酶HindⅢ和EcoRI双酶切步骤1得到的的PCR扩增产物,得到酶切产物。
6、用限制性内切酶HindⅢ和EcoRI双酶切重组质粒甲,回收载体骨架(约6.4kb)。
7、将步骤5的酶切产物和步骤6的载体骨架连接,得到重组质粒乙(又称棉铃虫RNAi干扰载体)。鉴定结果见图6所示。根据测序结果,对重组质粒乙进行结构描述如下:骨架载体为pGreen-HY104载体,在骨架载体的HindⅢ和EcoRI酶切位点之间插入了DNA片段甲,在骨架载体的SmaI和BamHI酶切位点之间插入了DNA片段乙。其RNAi干扰载体的具体序列如SEQ ID NO:20所示。
实施例5:棉铃虫sNPF RNAi干扰载体在棉花体内瞬时表达
一、棉铃虫RNAi干扰载体在棉花体内瞬时表达
1、将实施例4构建的棉铃虫sNPF RNAi干扰载体导入农杆菌C58,得到重组农杆菌。
2、棉铃虫sNPF RNAi干扰载体在棉花体内瞬时表达
(1)将棉花2047B种子脱绒后播种在灭菌MS基本培养基上,7天后将无菌苗移栽到营养钵中,培养至4-5叶期(培养条件:16小时光照、25℃,8小时黑暗、20℃,培养时间约1个月)。
(2)挑取单个农杆菌菌落,接种至3-5mL YEB液体培养基(含50mg/l卡那霉素),于摇床中28℃、200rpm振荡培养过夜。
(3)取2mL步骤(2)得到的菌液,接种至50mL YEB液体培养基(含50mg/l卡那霉素、50mg/l利福平、10mmol/L MES和20μmol/L乙酰丁香酮),于摇床中28℃、200rpm振荡培养过夜。
(4)取步骤(3)得到的菌液,4℃、4000g离心6min,收集菌体细胞。
(5)用渗透培养液(含10mmol/L MES、200μmol/L乙酰丁香酮、10mM MgCl2,溶剂为水)重新悬浮步骤(4)得到的菌体细胞,使菌体浓度为OD600=0.5-1.0。
(6)将步骤(5)得到的菌液25℃静置培养3h,即为重组农杆菌菌悬液。
(7)取步骤(1)得到的植株,对顶部叶片以下的两片叶片进行注射,每片叶片的注射方法为:用针头在叶片上背面非常轻微的扎一细小的浅微孔,然后用无针头的注射器吸取2-3mL步骤(6)得到的重组农杆菌菌悬液,从针孔处轻微的注入叶片内。
(8)将完成步骤(7)的植株放回温室,继续进行常规培养(培养条件:16小时光照、25℃,8小时黑暗、20℃)。
二、棉铃虫RNAi干扰载体在棉花体内瞬时表达效果的验证
步骤一的(8)中,于刚完成注射后取用于注射的原叶片部分组织,于完成注射1周后取新长出的叶片,分别提取总RNA并反转录为cDNA,将cDNA作为模板进行PCR鉴定(引物:5'-ATGAATCATCAGCAGCGTCGGAC-3'(SEQ ID NO:30);5'-GGGCAGTAAGAACATGTTGTTGT-3'(SEQID NO:7))。结果表明,步骤(8)的植株均为导入棉铃虫RNAi干扰载体的转基因植株。
三、导入棉铃虫RNAi干扰载体的棉花对棉铃虫的抗性
试验组:在每个培养皿中放置1头蜕皮当天的棉铃虫5龄幼虫,每24小时更换放入一片刚采摘的转基因棉花植株的叶片(其中,第一次放入培养皿中的叶片命名为叶片1,24小时后更换的叶片命名为叶片2,依次类推);转基因棉花植株即步骤一的(8)中,完成注射7天至30天的棉花植株;
对照组:在每个培养皿中放置1头蜕皮当天的棉铃虫5龄幼虫,每24小时更换放入一片刚采摘的野生型棉花植株的叶片(其中,第一次放入培养皿中的叶片命名为叶片1,24小时后更换的叶片命名为叶片2,依次类推)。
叶片分别于放入培养皿前和取出培养皿后用扫描仪扫描叶片大小(分辨率设定为200),用ImageJ软件计算扫描到的叶片面积,并计算幼虫取食叶片的面积。
试验组和对照组棉铃虫取食叶片的面积(单位为cm2)(进行三次重复试验,每次重复试验中,试验组和对照组各设置5个培养皿进行试验,结果取所有重复试验和重复样本的平均值)。结果表明,野生型植株叶片被进食的面积显著大于转基因植株叶片被进食的面积,即导入棉铃虫RNAi干扰载体后,棉花植株对棉铃虫的抗性显著增加。
将pGreen-HY104载体代替棉铃虫RNAi干扰载体进行步骤一,然后作为步骤一得到的棉花的平行对照进行步骤三的试验,结果均与野生型棉花植株的结果一致,即导入pGreen-HY104载体不能增加植物对棉铃虫的抗性(结果见图10)。
按照制备步骤一的(8)中转基因植株的方式,制备含有dsEGFF的棉花叶片,然后按照上述步骤试验,结果如图11所示。表明导入ds EGFF的棉花叶片没有抗虫性质,而本实施例制备的含有ds sNPF的棉花植株可以显著增加抗虫特性。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
SEQUENCE LISTING
<110> 江苏仁明生物科技有限公司
<120> 一种双链RNA的应用
<130> CPCN20110612
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 178
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ser Pro Ser Phe Val Cys Ala Leu Ala Val Cys Gly Phe Ala Ala
1 5 10 15
Ile Cys Ser Leu Pro Val Ser Thr Ala Gln Ala Leu Val Ser Pro Tyr
20 25 30
Glu Ser Ser Ala Ala Ser Asp Ser Gln Asn Gly Trp Gly Ala Leu Gly
35 40 45
Gly Leu Tyr Ala Leu Leu Ala Gln His Asp Ala Leu Gly Gly His Ala
50 55 60
Leu Ala Arg Lys Ser Val Arg Ser Pro Ser Arg Arg Leu Arg Phe Gly
65 70 75 80
Arg Arg Ser Asp Pro Asp Met Pro Pro Gln Ala Pro Leu Asp Glu Met
85 90 95
Asn Glu Leu Leu Ser Leu Arg Glu Val Arg Thr Pro Val Arg Leu Arg
100 105 110
Phe Gly Arg Arg Ser Glu Glu Arg Ala Val Pro His Ile Phe Pro Gln
115 120 125
Glu Glu Gln Asp Arg Ala Val Arg Ala Pro Ser Ile Arg Leu Arg Phe
130 135 140
Gly Arg Arg Ser Asp Asn Asn Met Phe Leu Leu Pro Tyr Glu Ser Ala
145 150 155 160
Leu Pro Gln Glu Val Lys Ala Asn Gly Ser Val Glu Asp Asp Arg Gln
165 170 175
Gln Glu
<210> 2
<211> 537
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtctccga gcttcgtttg cgcgctggct gtctgcggtt tcgcggcgat ctgctcattg 60
cccgtgtcca ccgcgcaggc gcttgtttct ccttatgaat catcagcagc gtcggacagt 120
caaaacggtt ggggcgcgtt aggcgggctg tacgctctac tagcgcagca cgacgcactg 180
ggcggccacg cgctcgcccg caagtcggtc cgctcgccct cccgccgtct gcgcttcgga 240
cgccgatccg accccgacat gcctcctcaa gctcccctgg acgagatgaa tgagctgctc 300
tctctgcgcg aggtccgcac gcccgtgcgt ctgcgattcg gccgccgctc ggaggaacgt 360
gccgtgcccc acatcttccc ccaggaggag caggaccgtg ccgtgcgtgc cccgtccatc 420
cgtctccggt tcggacgccg gtccgacaac aacatgttct tactgcccta cgaatcggct 480
ctgccccagg aagtgaaagc caacggctcc gtggaagacg acagacaaca ggaataa 537
<210> 3
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaatcatc agcagcgtcg gac 23
<210> 4
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
acaacaacat gttcttactg ccc 23
<210> 5
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
aagcagtggt atcaacgcag agt 23
<210> 6
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (15)..(15)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (21)..(21)
<223> n is a, c, g, t or u
<400> 6
aancknarnc kdatnswngg ngc 23
<210> 7
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gggcagtaag aacatgttgt tgt 23
<210> 8
<211> 26
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgtctccga gcttcgtttg cgcgct 26
<210> 9
<211> 27
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
ttattcctgt tgtctgtcgt cttccac 27
<210> 10
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgaatcatc agcagcgtcg gac 23
<210> 11
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
gggcagtaag aacatgttgt tgt 23
<210> 12
<211> 52
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
gatcactaat acgactcact atagggagaa tgaatcatca gcagcgtcgg ac 52
<210> 13
<211> 52
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
gatcactaat acgactcact atagggagag ggcagtaaga acatgttgtt gt 52
<210> 14
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgaatcatc agcagcgtcg gac 23
<210> 15
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
gggcagtaag aacatgttgt tgt 23
<210> 16
<211> 50
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 16
gatcactaat acgactcact atagggagat ggtcccaatt ctcgtggaac 50
<210> 17
<211> 50
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 17
gatcactaat acgactcact atagggagac ttgaagttga ccttgatgcc 50
<210> 18
<211> 35
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 18
gcccgggaat tcatgaatca tcagcagcgt cggac 35
<210> 19
<211> 36
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 19
cggatccaag cttgggcagt aagaacatgt tgttgt 36
<210> 20
<211> 6442
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccgccgtcga gcgggctttt ttatccccgg aagcctgtgg atagagggta gttatccacg 60
tgaaaccgct aatgccccgc aaagccttga ttcacggggc tttccggccc gctccaaaaa 120
ctatccacgt gaaatcgcta atcagggtac gtgaaatcgc taatcggagt acgtgaaatc 180
gctaataagg tcacgtgaaa tcgctaatca aaaaggcacg tgagaacgct aatagccctt 240
tcagatcaac agcttgcaaa cacccctcgc tccggcaagt agttacagca agtagtatgt 300
tcaattagct tttcaattat gaatatatat atcaattatt ggtcgccctt ggcttgtgga 360
caatgcgcta cgcgcaccgg ctccgcccgt ggacaaccgc aagcggttgc ccaccgtcga 420
gcgcctttgc ccacaacccg gcggccggcc gcaacagatc gttttataaa tttttttttt 480
tgaaaaagaa aaagcccgaa aggcggcaac ctctggggct tctggatttc cgatccccgg 540
aattagatct tggcaggata tattgtggtg taacgttatc agcttgcatg ccggtggatg 600
tagtaacata gatgacaccg cgcgcgataa tttatcctag tttgcgcggt atattttgtt 660
ttctatcggg tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa 720
taacgtcatg cattacatgt taattattac atgcttaacg taattcaaca gaaattatat 780
gataatcatc gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt 840
tgaacgatct gcttgactct aggggtcatc agatttcggt gacgggcagg accggacggg 900
gcggcaccgg caggctgaag tccagctgcc agaaacccac gtcatgccag ttcccgtgct 960
tgaagccggc cgcccgcagc atgccgcggg gggcatatcc gagcgcctcg tgcatgcgca 1020
cgctcgggtc gttgggcagc ccgatgacag cgaccacgct cttgaagccc tgtgcctcca 1080
gggacttcag caggtgggtg tagagcgtgg agcccagtcc cgtccgctgg tggcgggggg 1140
agacgtacac ggtcgactcg gccgtccagt cgtaggcgtt gcgtgccttc cagggacccg 1200
cgtaggcgat gccggcgacc tcgccgtcca cctcggcgac gagccaggga tagcgctccc 1260
gcagacggac gaggtcgtcc gtccactcct gcggttcctg cggctcggta cggaagttga 1320
ccgtgcttgt ctcgatgtag tggttgacga tggtgcagac cgccggcatg tccgcctcgg 1380
tggcacggcg gatgtcggcc gggcgtcgtt ctgggctcat ggtagatccc ctcgatcgag 1440
ttgagagtga atatgagact ctaattggat accgagggga atttatggaa cgtcagtgga 1500
gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt ttgaacgcgc 1560
aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg cggctcagtg 1620
gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc 1680
ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcgataac attaacgctt 1740
acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 1800
cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 1860
cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg 1920
actcactata gggcgaattg ggtaccccta ctccaaaaat gtcaaagata cagtctcaga 1980
agaccaaagg gctattgaga cttttcaaca aagggtaatt tcgggaaacc tcctcggatt 2040
ccattgccca gctatctgtc acttcatcga aaggacagta gaaaaggaag gtggctccta 2100
caaatgccat cattgcgata aaggaaaggc tatcattcaa gatgcctctg ccgacagtgg 2160
tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 2220
gtcttcaaag caagtggatt gatgtgacat ctccactgac gtaagggatg acgcacaatc 2280
ccacccctac tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac 2340
ttttcaacaa agggtaattt cgggaaacct cctcggattc cattgcccag ctatctgtca 2400
cttcatcgaa aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa 2460
aggaaaggct atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 2520
cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 2580
atgtgacatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc 2640
ttcctctata taaggaagtt catttcattt ggagaggaca gcccaagcta cgcgtctcga 2700
ggtcgacggt atcgataagc ttgatatcga attcgatatc tacccgcttc gcgtcggcat 2760
ccggtcagtg gcagtgaagg gcgaacagtt cctgattaac cacaaaccgt tctactttac 2820
tggctttggt cgtcatgaag atgcggactt gcgtggcaaa ggattcgata acgtgctgat 2880
ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta 2940
cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga 3000
aactgctgct gtcggcttta acctctcttt aggcattggt ttcgaagcgg gcaacaagcc 3060
gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc 3120
gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc 3180
caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc 3240
aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc 3300
tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg 3360
gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc 3420
ctggcaagag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc 3480
cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga 3540
tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt 3600
cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt 3660
cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat 3720
gaacttcggt gaaaaaccgc agcagggagg caacaatgac tgcagcccgg gggatccact 3780
agttctagat aattcgctga aatcaccagt ctctctctac aaatctatct ctctctattt 3840
tctccataaa taatgtgtga gtagtttccc gataagggaa attagggttc ttatagggtt 3900
tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 3960
ctatcaataa aatttctaat tcctaaaacc aaaatccagt aactaaaatc cagatcacta 4020
gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg 4080
cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 4140
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 4200
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 4260
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 4320
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 4380
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 4440
atgaaggcct tgacaggata tattggcggg taaactaagt cgctgtatgt gtttgtttga 4500
gatctcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4560
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4620
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4680
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4740
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4800
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4860
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4920
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4980
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 5040
gttaccttcg gaagaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5100
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5160
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5220
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5280
tttaaatcaa tctaaagtat atatgtgtaa cattggtcta gtgattagaa aaactcatcg 5340
agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 5400
agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 5460
tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 5520
tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 5580
ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 5640
tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 5700
aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 5760
aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 5820
aatgctgttt tccctgggat cgcagtggtg agtaaccatg catcatcagg agtacggata 5880
aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 5940
tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 6000
ggcttcccat acaatcggta gattgtcgca cctgattgcc cgacattatc gcgagcccat 6060
ttatacccat ataaatcagc atccatgttg gaatttaatc gcggccttga gcaagacgtt 6120
tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 6180
attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 6240
acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt 6300
cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc 6360
tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga tggggcgatt 6420
caggcgatcc ccatccaaca gc 6442
<210> 21
<211> 178
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Ser Pro Ser Phe Val Cys Ala Leu Ala Val Cys Gly Phe Ala Ala
1 5 10 15
Ile Cys Ser Leu Pro Val Ser Thr Ala Gln Ala Leu Val Ser Pro Tyr
20 25 30
Glu Ser Ser Ala Ala Ser Asp Ser Gln Asn Gly Trp Gly Ala Leu Gly
35 40 45
Gly Leu Tyr Ala Leu Leu Ala Gln His Asp Ala Leu Gly Gly His Ala
50 55 60
Leu Ala Arg Lys Ser Val Arg Ser Pro Ser Arg Arg Leu Arg Phe Gly
65 70 75 80
Arg Arg Ser Asp Pro Asp Met Pro Pro Gln Ala Pro Leu Asp Glu Met
85 90 95
Asn Glu Leu Leu Ser Leu Arg Glu Val Arg Thr Pro Val Arg Leu Arg
100 105 110
Phe Gly Arg Arg Ser Glu Glu Arg Ala Val Pro His Ile Phe Pro Gln
115 120 125
Glu Glu Gln Asp Arg Ala Val Arg Ala Pro Ser Ile Arg Leu Arg Phe
130 135 140
Gly Arg Arg Ser Asp Asn Asn Met Phe Leu Leu Pro Tyr Glu Ser Ala
145 150 155 160
Leu Pro Gln Glu Val Lys Ala Asn Gly Ser Val Glu Asp Asp Arg Gln
165 170 175
Gln Glu
<210> 22
<211> 537
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgtctccga gcttcgtttg cgcgctggct gtctgcggtt tcgcggcgat ctgctcattg 60
cccgtgtcca ccgcgcaggc gcttgtttct ccttatgaat catcagcagc gtcggacagt 120
caaaacggtt ggggcgcgtt aggcgggctg tacgctctac tagcgcagca cgacgcactg 180
ggcggccacg cgctcgcccg caagtcggtc cgctcgccct cccgccgtct gcgcttcgga 240
cgccgatccg accccgacat gcctcctcaa gctcccctgg acgagatgaa tgagctgctc 300
tctctgcgcg aggtccgcac gcccgtgcgt ctgcgattcg gccgccgctc ggaggaacgt 360
gccgtgcccc acatcttccc ccaggaggag caggaccgtg ccgtgcgtgc cccgtccatc 420
cgtctccggt tcggacgccg gtccgacaac aacatgttct tactgcccta cgaatcggct 480
ctgccccagg aagtgaaagc caacggctcc gtggaagacg acagacaaca ggaataa 537
<210> 23
<211> 38
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 23
aagcagtggt atcaacgcag agtggccatt acggccgg 38
<210> 24
<211> 27
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 24
attctagagg ccgaggcggc cgacatg 27
<210> 25
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 25
aagcagtggt atcaacgcag agt 23
<210> 26
<211> 27
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 26
attctagagg ccgaggcggc cgacatg 27
<210> 27
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 27
atgaatcatc agcagcgtcg gac 23
<210> 28
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 28
tggtcccaat tctcgtggaa c 21
<210> 29
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 29
cttgaagttg accttgatgc c 21
<210> 30
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 30
atgaatcatc agcagcgtcg gac 23
Claims (10)
1.一种双链RNA在制备抗虫剂或培育抗虫的转基因植物中的应用,其特征在于,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
2.一种包含双链RNA的载体或重组菌株,其特征在于,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
3.根据权利要求2所述的载体或重组菌株,其特征在于,所述的载体包含SEQ ID NO:20所示的核苷酸序列。
4.一种双链RNA的制备方法,其特征在于,所述的制备方法包括采用靶向靶序列SEQ IDNO:3和/或SEQ ID NO:4的引物,扩增棉铃虫sNPF基因的cDNA序列,合成双链RNA。
5.根据权利要求4所述的制备方法,其特征在于,所述的制备方法包括:
获取棉铃虫sNPF基因的总RNA,反转录获得cDNA;优选为棉铃虫肠总RNA或脑总RNA,进一步优选为肠总RNA;
扩增所述的cDNA;优选扩增引物为SEQ ID NO:10、11;
将扩增获得的cDNA连接至载体后,转入细胞,提取质粒;
采用靶向靶序列SEQ ID NO:3和/或SEQ ID NO:4的引物,扩增提取的质粒,合成双链RNA;优选先采用引物SEQ ID NO:14和SEQ ID NO:15,然后采用引物SEQ ID NO:12和SEQ IDNO:13扩增提取的质粒,合成双链RNA。
6.一种包含双链RNA核苷酸序列的RNAi干扰载体的制备方法,其特征在于,所述的制备方法包括采用上游引物:包含酶切位点I、酶切位点II和SEQ ID NO:10,下游引物:包含酶切位点III、酶切位点IV和SEQ ID NO:11,扩增棉铃虫sNPF基因的cDNA,然后经酶切与载体连接获得RNAi干扰载体。
7.根据权利要求6所述的制备方法,其特征在于,所述的制备方法包括:
获取棉铃虫sNPF基因的总RNA,反转录获得cDNA;优选为棉铃虫肠总RNA或脑总RNA,进一步优选为肠总RNA;
采用上游引物:包含酶切位点I、酶切位点II和SEQ ID NO:10,下游引物:包含酶切位点III、酶切位点IV和SEQ ID NO:11,扩增cDNA,获得扩增产物;优选酶切位点I为SmaI识别位点、酶切位点II为EcoRI识别位点、酶切位点III为BamHI识别位点、酶切位点IV为HindIII识别位点;进一步优选采用引物SEQ ID NO:18、19扩增cDNA;
用酶切位点I对应的酶与酶切位点III对应的酶双酶切获得的扩增产物,获得目的片段1;
用酶切位点I对应的酶与酶切位点III对应的酶双酶切载体,获得载体骨架1;优选载体为pGreen-HY104;
将获得的目的片段1连接至载体骨架1上,获得重组质粒1;
用酶切位点II对应的酶与酶切位点IV对应的酶双酶切获得的扩增产物,获得目的片段2;
用酶切位点II对应的酶与酶切位点IV对应的酶双酶切获得的重组质粒1,获得载体骨架2;
将获得的目的片段2连接至载体骨架2上,获得RNAi干扰载体。
8.一种抗虫剂,其特征在于,所述的抗虫剂包含双链RNA或权利要求2-3任一所述的载体或重组菌株,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
9.一种抗虫的转基因植物的培育方法,其特征在于,所述的培育方法包括:
将双链RNA、权利要求2-3任一所述的载体或重组菌株,导入出发植物,得到抗虫的转基因植物,所述的双链RNA包含SEQ ID NO:22所示的核苷酸序列。
10.根据权利要求9所述的培育方法,其特征在于,所述的抗虫为抗鳞翅目昆虫,所述的出发植物为单子叶植物或双子叶植物,优选为棉花出发植物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010754468.5A CN112029766B (zh) | 2020-07-30 | 2020-07-30 | 一种双链rna的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010754468.5A CN112029766B (zh) | 2020-07-30 | 2020-07-30 | 一种双链rna的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112029766A true CN112029766A (zh) | 2020-12-04 |
CN112029766B CN112029766B (zh) | 2024-06-18 |
Family
ID=73583611
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010754468.5A Active CN112029766B (zh) | 2020-07-30 | 2020-07-30 | 一种双链rna的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112029766B (zh) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101370940A (zh) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | 作为昆虫防治剂的dsRNA |
CN103665132A (zh) * | 2012-09-12 | 2014-03-26 | 中国农业大学 | 棉铃虫和烟夜蛾的npy神经肽及其编码基因和应用 |
CN103665131A (zh) * | 2012-09-12 | 2014-03-26 | 中国农业大学 | 棉铃虫和烟夜蛾的npf神经肽及其编码基因和应用 |
CN105440124A (zh) * | 2016-01-07 | 2016-03-30 | 西南大学 | 神经肽sNPF和其受体基因及在桔小实蝇特异性控制剂中的应用 |
CN108129559A (zh) * | 2017-11-30 | 2018-06-08 | 浙江大学 | 小菜蛾神经肽Px-sNPF和其受体以及在调控小菜蛾体内海藻糖含量中的应用 |
US20190343122A1 (en) * | 2016-05-27 | 2019-11-14 | Provivi, Inc. | Improved insect control strategies utilizing pheromones and rnai |
-
2020
- 2020-07-30 CN CN202010754468.5A patent/CN112029766B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101370940A (zh) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | 作为昆虫防治剂的dsRNA |
CN103665132A (zh) * | 2012-09-12 | 2014-03-26 | 中国农业大学 | 棉铃虫和烟夜蛾的npy神经肽及其编码基因和应用 |
CN103665131A (zh) * | 2012-09-12 | 2014-03-26 | 中国农业大学 | 棉铃虫和烟夜蛾的npf神经肽及其编码基因和应用 |
CN105440124A (zh) * | 2016-01-07 | 2016-03-30 | 西南大学 | 神经肽sNPF和其受体基因及在桔小实蝇特异性控制剂中的应用 |
US20190343122A1 (en) * | 2016-05-27 | 2019-11-14 | Provivi, Inc. | Improved insect control strategies utilizing pheromones and rnai |
CN108129559A (zh) * | 2017-11-30 | 2018-06-08 | 浙江大学 | 小菜蛾神经肽Px-sNPF和其受体以及在调控小菜蛾体内海藻糖含量中的应用 |
Non-Patent Citations (1)
Title |
---|
LIU,X.G., 等: "GenBank: KC340936.1", pages 1 - 2 * |
Also Published As
Publication number | Publication date |
---|---|
CN112029766B (zh) | 2024-06-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7683238B2 (en) | Production of pharmaceutically active proteins in sprouted seedlings | |
CN110452908A (zh) | 在昆虫害虫中下调基因表达 | |
CN104824010B (zh) | 杀虫蛋白的用途 | |
WO2018090715A1 (zh) | 杀虫蛋白组合及其管理昆虫抗性的方法 | |
JP2004535804A (ja) | タバコバイオマスの利用 | |
CN104488945B (zh) | 杀虫蛋白的用途 | |
CN110295185A (zh) | 一种基于CsWAKL08超量表达提高柑橘对溃疡病抗性的方法 | |
CN101128591A (zh) | 用于植物中种子偏好性表达的表达盒 | |
CN112239491A (zh) | 与抗锈病相关的蛋白及其编码基因与应用 | |
CN102782141A (zh) | 用于在植物中胚-特异性表达的表达盒 | |
CN110041416A (zh) | GmABCA9基因在提高大豆蛋白含量和粒重中的应用 | |
CN106497966A (zh) | 杀虫蛋白的用途 | |
BR102013031734B1 (pt) | método para controlar athetis lepigone | |
KR20180137558A (ko) | 유전자내 식물 형질전환을 위한 구조체 및 벡터 | |
CN106496313A (zh) | 抗病相关蛋白IbSWEET10及其编码基因与应用 | |
CN103734169B (zh) | 控制害虫的方法 | |
CN104886111A (zh) | 杀虫蛋白的用途 | |
CN103703131B (zh) | 诱导根的伸长或增加生物质的量的新基因及其应用 | |
CN112029766B (zh) | 一种双链rna的应用 | |
CN104920425B (zh) | 杀虫蛋白的用途 | |
CN104621171B (zh) | 杀虫蛋白的用途 | |
CN107531766A (zh) | 葡聚糖酶的生产和使用其的方法 | |
WO2018090714A1 (zh) | 杀虫蛋白组合及其管理昆虫抗性的方法 | |
CN104798802B (zh) | 杀虫蛋白的用途 | |
Tsygankova et al. | Increasing the resistance of rape plants to the parasitic nematode Heterodera schachtii using RNAi technology |
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 |