CN110669772A - 一种烟草新烟碱合成调控基因NtERF91的克隆和应用 - Google Patents
一种烟草新烟碱合成调控基因NtERF91的克隆和应用 Download PDFInfo
- Publication number
- CN110669772A CN110669772A CN201911005045.7A CN201911005045A CN110669772A CN 110669772 A CN110669772 A CN 110669772A CN 201911005045 A CN201911005045 A CN 201911005045A CN 110669772 A CN110669772 A CN 110669772A
- Authority
- CN
- China
- Prior art keywords
- nterf91
- tobacco
- gene
- neonicotinoid
- culture medium
- 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.)
- Pending
Links
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 89
- 241000208125 Nicotiana Species 0.000 title claims abstract description 86
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 31
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 31
- 108700005075 Regulator Genes Proteins 0.000 title claims abstract description 19
- 238000010367 cloning Methods 0.000 title claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 239000013598 vector Substances 0.000 claims abstract description 37
- 241000196324 Embryophyta Species 0.000 claims abstract description 36
- 230000009261 transgenic effect Effects 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000002299 complementary DNA Substances 0.000 claims abstract description 16
- 230000002018 overexpression Effects 0.000 claims abstract description 16
- 241000589158 Agrobacterium Species 0.000 claims abstract description 15
- 230000009466 transformation Effects 0.000 claims abstract description 14
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 claims abstract description 13
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960002715 nicotine Drugs 0.000 claims abstract description 13
- 230000001105 regulatory effect Effects 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000010839 reverse transcription Methods 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 230000002194 synthesizing effect Effects 0.000 claims abstract 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000001963 growth medium Substances 0.000 claims description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 239000012634 fragment Substances 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 19
- 239000013604 expression vector Substances 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 230000003321 amplification Effects 0.000 claims description 14
- 238000001976 enzyme digestion Methods 0.000 claims description 14
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 12
- 230000004069 differentiation Effects 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 claims description 10
- 238000009630 liquid culture Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 9
- 238000010276 construction Methods 0.000 claims description 9
- 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 claims description 9
- 229960001225 rifampicin Drugs 0.000 claims description 9
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 claims description 9
- 229960000268 spectinomycin Drugs 0.000 claims description 9
- 239000012264 purified product Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- 241001002356 Valeriana edulis Species 0.000 claims description 7
- 229930027917 kanamycin Natural products 0.000 claims description 7
- 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 claims description 7
- 229960000318 kanamycin Drugs 0.000 claims description 7
- 229930182823 kanamycin A Natural products 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000005520 cutting process Methods 0.000 claims description 6
- 238000012215 gene cloning Methods 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 230000002068 genetic effect Effects 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 230000001965 increasing effect Effects 0.000 claims description 4
- 230000028744 lysogeny Effects 0.000 claims description 4
- 239000012266 salt solution Substances 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 3
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 244000061176 Nicotiana tabacum Species 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000006911 enzymatic reaction Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 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 claims description 2
- 238000003287 bathing Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- 238000013518 transcription Methods 0.000 claims description 2
- 230000035897 transcription Effects 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- ZNJFBWYDHIGLCU-UHFFFAOYSA-N jasmonic acid Natural products CCC=CCC1C(CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-UHFFFAOYSA-N 0.000 claims 1
- 238000011144 upstream manufacturing Methods 0.000 claims 1
- MTXSIJUGVMTTMU-JTQLQIEISA-N (S)-anabasine Chemical compound N1CCCC[C@H]1C1=CC=CN=C1 MTXSIJUGVMTTMU-JTQLQIEISA-N 0.000 abstract description 13
- MTXSIJUGVMTTMU-UHFFFAOYSA-N Neonicotine Natural products N1CCCCC1C1=CC=CN=C1 MTXSIJUGVMTTMU-UHFFFAOYSA-N 0.000 abstract description 11
- 230000001404 mediated effect Effects 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 18
- 229930013930 alkaloid Natural products 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 7
- 108091061403 ERF family Proteins 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 229930014345 anabasine Natural products 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- MYKUKUCHPMASKF-VIFPVBQESA-N (S)-nornicotine Chemical compound C1CCN[C@@H]1C1=CC=CN=C1 MYKUKUCHPMASKF-VIFPVBQESA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 101100388299 Arabidopsis thaliana DTX54 gene Proteins 0.000 description 1
- 101100294133 Arabidopsis thaliana NIC2 gene Proteins 0.000 description 1
- KJGNDQCYBNBXDA-GUBZILKMSA-N Arg-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N KJGNDQCYBNBXDA-GUBZILKMSA-N 0.000 description 1
- GHNDBBVSWOWYII-LPEHRKFASA-N Arg-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GHNDBBVSWOWYII-LPEHRKFASA-N 0.000 description 1
- OGSQONVYSTZIJB-WDSOQIARSA-N Arg-Leu-Trp Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCN=C(N)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O OGSQONVYSTZIJB-WDSOQIARSA-N 0.000 description 1
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 1
- UYXXMIZGHYKYAT-NHCYSSNCSA-N Asn-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)N)N UYXXMIZGHYKYAT-NHCYSSNCSA-N 0.000 description 1
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 1
- ZJIFRAPZHAGLGR-MELADBBJSA-N Asn-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(=O)N)N)C(=O)O ZJIFRAPZHAGLGR-MELADBBJSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- 108010009685 Cholinergic Receptors Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- TUTIHHSZKFBMHM-WHFBIAKZSA-N Glu-Asn Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O TUTIHHSZKFBMHM-WHFBIAKZSA-N 0.000 description 1
- ZHNHJYYFCGUZNQ-KBIXCLLPSA-N Glu-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O ZHNHJYYFCGUZNQ-KBIXCLLPSA-N 0.000 description 1
- IALQAMYQJBZNSK-WHFBIAKZSA-N Gly-Ser-Asn Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O IALQAMYQJBZNSK-WHFBIAKZSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- BXOLYFJYQQRQDJ-MXAVVETBSA-N His-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CN=CN1)N BXOLYFJYQQRQDJ-MXAVVETBSA-N 0.000 description 1
- ZNOBVZFCHNHKHA-KBIXCLLPSA-N Ile-Ser-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZNOBVZFCHNHKHA-KBIXCLLPSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- LLBQJYDYOLIQAI-JYJNAYRXSA-N Leu-Glu-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LLBQJYDYOLIQAI-JYJNAYRXSA-N 0.000 description 1
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 1
- KPYAOIVPJKPIOU-KKUMJFAQSA-N Leu-Lys-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O KPYAOIVPJKPIOU-KKUMJFAQSA-N 0.000 description 1
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- ILDSIMPXNFWKLH-KATARQTJSA-N Leu-Thr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- PBIPLDMFHAICIP-DCAQKATOSA-N Lys-Glu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PBIPLDMFHAICIP-DCAQKATOSA-N 0.000 description 1
- ALEVUGKHINJNIF-QEJZJMRPSA-N Lys-Phe-Ala Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ALEVUGKHINJNIF-QEJZJMRPSA-N 0.000 description 1
- JPCHYAUKOUGOIB-HJGDQZAQSA-N Met-Glu-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPCHYAUKOUGOIB-HJGDQZAQSA-N 0.000 description 1
- 101001055239 Nicotiana tabacum Isoflavone reductase homolog A622 Proteins 0.000 description 1
- 206010057852 Nicotine dependence Diseases 0.000 description 1
- MYKUKUCHPMASKF-UHFFFAOYSA-N Nornicotine Natural products C1CCNC1C1=CC=CN=C1 MYKUKUCHPMASKF-UHFFFAOYSA-N 0.000 description 1
- YUPRIZTWANWWHK-DZKIICNBSA-N Phe-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N YUPRIZTWANWWHK-DZKIICNBSA-N 0.000 description 1
- PZSCUPVOJGKHEP-CIUDSAMLSA-N Pro-Gln-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PZSCUPVOJGKHEP-CIUDSAMLSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- DMNANGOFEUVBRV-GJZGRUSLSA-N Pro-Trp-Gly Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)O)C(=O)[C@@H]1CCCN1 DMNANGOFEUVBRV-GJZGRUSLSA-N 0.000 description 1
- WWXNZNWZNZPDIF-SRVKXCTJSA-N Pro-Val-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 WWXNZNWZNZPDIF-SRVKXCTJSA-N 0.000 description 1
- JCLAFVNDBJMLBC-JBDRJPRFSA-N Ser-Ser-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JCLAFVNDBJMLBC-JBDRJPRFSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 description 1
- SOUPNXUJAJENFU-SWRJLBSHSA-N Thr-Trp-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O SOUPNXUJAJENFU-SWRJLBSHSA-N 0.000 description 1
- 208000025569 Tobacco Use disease Diseases 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- IBBBOLAPFHRDHW-BPUTZDHNSA-N Trp-Asn-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N IBBBOLAPFHRDHW-BPUTZDHNSA-N 0.000 description 1
- AKXBNSZMYAOGLS-STQMWFEESA-N Tyr-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AKXBNSZMYAOGLS-STQMWFEESA-N 0.000 description 1
- KOVXHANYYYMBRF-IRIUXVKKSA-N Tyr-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KOVXHANYYYMBRF-IRIUXVKKSA-N 0.000 description 1
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 1
- PVPAOIGJYHVWBT-KKHAAJSZSA-N Val-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N)O PVPAOIGJYHVWBT-KKHAAJSZSA-N 0.000 description 1
- CGGVNFJRZJUVAE-BYULHYEWSA-N Val-Asp-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CGGVNFJRZJUVAE-BYULHYEWSA-N 0.000 description 1
- FEFZWCSXEMVSPO-LSJOCFKGSA-N Val-His-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](C)C(O)=O FEFZWCSXEMVSPO-LSJOCFKGSA-N 0.000 description 1
- 102000034337 acetylcholine receptors Human genes 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- -1 alkaloid compounds Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 244000038280 herbivores Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- GWWNCLHJCFNTJA-UHFFFAOYSA-N nicandrenone-2 Natural products C12OC2C2(O)CC=CC(=O)C2(C)C(CCC23C)C1C3CCC2(O)C(C)C1OC(O)C2(C)OC2(C)C1 GWWNCLHJCFNTJA-UHFFFAOYSA-N 0.000 description 1
- 239000004069 plant analysis Substances 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 206010043778 thyroiditis Diseases 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了烟草新烟碱合成调控基因NtERF91及其克隆方法与应用,烟碱合成调控基因NtERF91核苷酸序列如SEQ ID:No.1所示,编码的氨基酸序列如SEQ ID:No.2所示。本发明还公开了新烟碱合成调控基因NtERF91的克隆方法,具体步骤包括:A、确定NtERF91基因序列;B、提取烟草RNA,反转录得到第一链cDNA;C、根据NtERF91基因序列设计合成特异性引物,以cDNA作为模板,进行PCR扩增;D、回收和纯化PCR产物;E、构建了含NtERF91基因的过表达载体。将过表达载体通过农杆菌介导的转化在烟草中过表达,制备转基因植株。获得的转基因植株新烟碱含量是对照的4倍以上。说明烟草NtERF91基因在培育高新烟碱含量的烟草方面具有较大的应用前景。
Description
技术领域
本发明属于遗传工程技术领域,具体涉及一种烟草新烟碱合成调控基因NtERF91的克隆方法与应用。
背景技术
新烟碱是茄科植物(如烟草、番茄)中的一种生物碱。在烟草中,新烟碱在烟草总生物碱中的比例较低。烟草生物碱占烟草总干重的2%-4%。在烟草四种生物碱当中,烟碱占到了总生物碱的95%左右,新烟碱、降烟碱以及假木贼碱仅占剩余的5%。在自然界当中,这些烟草生物碱类化合物具有生物活性,在烟草防御系统当中作为天然毒素来防御昆虫或食草动物的捕食。
新烟碱经常在动物模型和细胞系中被用于研究其是否对可以治疗烟碱致瘾性、阿尔茨海默病、甲状腺炎、多发性硬化症、以及抑制乙酰胆碱受体活性。在烟草生物碱次生代谢方面,ERF(Ethylene Response Factor)类型的转录因子基因被认为是烟草中调控烟碱合成重要调控因子(Rushton et al.,2008,Plant Physiology,147:280-295)。调控烟碱合成的NIC2遗传位点已被发现是由至少7个ERF基因组成,如NtERF189,NtERF179等(Shoji etal.,2010,Plant Cell,22:3390-3409)。这些ERF转录因子属于ERF家族中I的X亚族,它们可以通过直接结合烟碱合成途径中关键代谢限速酶基因(如NtPMT、NtQPT)的启动子中的GCC-box来转录激活该基因的表达,进而增强烟碱的合成与积累(Shoji et al.,2010,PlantCell,22:3390-3409)。然而,新烟碱在烟草悬浮细胞系中主要积累,主要原因是由于烟碱合成途径中的MPO代谢酶表达量在悬浮细胞系中表达偏低造成的(Shoji和Hashimoto,2008,Plant Cell Physiology,49:1209-1216)。另一方面,新烟碱作为烟草生物碱当中积累量较少的生物碱,针对调控其代谢和积累的转录调控因子基因尚未见报道。
由于新烟碱在医学方面的功效,科学家正开展利用生物技术手段提高烟草烟碱含量进行研究,从而增加烟草的商业用途,也为降低新烟碱原料的生产成本。目前,我国也亟需深入研究研究新烟碱的合成调控机理,为培育新烟碱含量高烟草品种提供理论基础。
发明内容
本发明的第一目的在于提供一种烟草新烟碱合成调控基因NtERF91;第二目的在于提供所述的烟草新烟碱合成调控基因NtERF91的克隆方法;第三目的在于提供所述的烟草新烟碱合成调控基因NtERF91的应用。
本发明的第一目的是这样实现的,所述的烟草新烟碱合成调控基因NtERF91的核苷酸序列如序列表SEQ ID NO:1所示。
本发明的第二目的是这样实现的,包括以下步骤:
A、确定NtERF91基因序列;
通过分析茉莉素处理烟草根部组织的转录组数据,获得受茉莉素正向调控ERF家族基因1个,序列比对发现该基因为NtERF91。根据该基因序列设计基因克隆引物:
正向引物NtERF91-BamH I:
GGATCCATGTTAACTAGTGTCAATACCACGT;
反向引物NtERF91-Xho I:CTCGAGTCAATTTTCAGCCAATTTTCTCTTC;
B、提取烟草根组织RNA,反转录得到第一链cDNA;
C、以反转录得到的第一链cDNA作为模板,利用NtERF91基因克隆引物进行PCR扩增,回收和纯化PCR产物;
D、纯化产物与载体连接,连接体系与过程如下:4μL纯化产物、1μL saltsolution、1μL-BluntⅡ-TOPO(Invitrogen)混匀,25℃,水浴30min;将连接好的载体通过热激转化大肠杆菌DH5α,加液体培养基振荡培养后涂布至含100mg/L卡那霉素的LB平板上过夜培养,挑取菌落进行菌液培养,质粒提取和PCR检测。筛选阳性克隆,对阳性克隆进行测序。
本发明的第三目的是这样实现的,所述的烟草新烟碱合成调控基因NtERF91在获得新烟碱含量显著提高的转基因植株中的应用;即所述的烟草烟碱合成调控基因NtERF91用于提高烟草烟叶的新烟碱含量。
本发明从烟草中得到一个烟草新烟碱合成调控基因NtERF91,具体步骤为:通过分析茉莉素处理烟草根部组织的转录组数据,获得受茉莉素正向调控ERF家族基因1个,序列比对发现该基因为NtERF91。根据该基因序列信息设计基因特异引物进行PCR反应,得到目的产物;对目的产物测序,得到NtERF91基因序列;利用农杆菌介导的遗传转化方法获得NtERF91基因的过表达(OE)株系,对NtERF91进行功能鉴定,结果表明NtERF91基因具有显著提高烟草叶片中新烟碱的积累量,获得的转基因植株新烟碱含量是对照的4倍以上。说明烟草NtERF91基因在培育提高新烟碱含量的烟草方面具有较大的应用前景。
附图说明
图1为NtERF91基因PCR扩增产物琼脂糖凝胶电泳图,NtERF91大小为435bp,M为DL2000DNA Marker;
图2为菌落PCR检测重组过表达载体pK2GW7-NtERF91的农杆菌转化效果,M为DL2000DNA Marker,1和2为PCR产物;
图3为T0代NtERF91基因转基因植株基因表达及烟碱合成途径基因表达水平分析;NtERF91-OE-2,NtERF91-OE-4,NtERF91-OE-5为转基因株系,EV为转空载体对照,A为NtPMT,B为NtQPT,C为NtAO,D为NtMPO,E为NtA622,F为NtQS,G为NtODC,H为NtADC;
图4为T0代NtERF91基因转基因植株烟叶新烟碱含量分析;NtERF91-OE-2,NtERF91-OE-4,NtERF91-OE-5为转基因株系,A为上部叶,B为中部叶,C为下部叶,Ⅰ为EV为转空载体对照,Ⅱ为NtERF91-OE-2转基因株系,Ⅲ为NtERF91-OE-4转基因株系,Ⅳ为NtERF91-OE-5转基因株系,“*”表示该植株假木贼碱含量与空载对照EV在0.05水平上差异显著。。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
本发明所述的烟草新烟碱合成调控基因NtERF91的核苷酸序列如序列表SEQ IDNO:1所示。
所述的烟草新烟碱合成调控基因NtERF91编码的氨基酸序列如SEQ ID NO:2所示。
本发明所述的烟草新烟碱合成调控基因NtERF91的克隆方法,其特征在于包括以下步骤:
A、确定NtERF91基因序列;
通过分析茉莉素处理后烟草根部组织转录组数据,获得受茉莉素正向调控ERF家族基因1个,序列比对发现该基因为NtERF91。根据该基因序列设计基因克隆引物:
正向引物NtERF91-BamH I:
GGATCCATGTTAACTAGTGTCAATACCACGT;
反向引物NtERF91-Xho I:5’-CTCGAGTCAATTTTCAGCCAATTTTCTCTTC;
B、提取烟草根组织RNA,反转录得到第一链cDNA;
C、以反转录得到的第一链cDNA作为模板,利用NtERF91基因克隆引物进行PCR扩增,回收和纯化PCR产物;
D、纯化产物与载体连接,通过试剂盒反应与TOPO载体连接,连接体系与过程如下:4μL纯化产物、1μL salt solution、1μL-BluntⅡ-TOPO混匀,25℃,水浴30min;将连接好的载体通过热激转化大肠杆菌,加液体培养基振荡培养后涂布至含100mg/L卡那霉素的LB平板上过夜培养,挑取菌落进行培养,取2mL菌液提取质粒,进行质粒PCR检测。筛选阳性克隆,对阳性克隆进行测序。
C步骤中PCR扩增的反应体系是选用Phusion高保真扩增酶反应体系,体系总体积50μL,包括:200ng cDNA,5×Phusion HF反应缓冲液10μL,10mM dNTP 1μL,2U的High-Fidelity DNA Polymerase,10μM的正反向引物各1μL,补水至50μL。
本发明所述的烟草新烟碱合成调控基因NtERF91的应用为所述的烟草新烟碱合成调控基因NtERF91在用于获得烟叶烟碱含量显著提高的转基因植株中的应用。
所述获得烟叶新烟碱含量显著提高转基因植株的方法包括以下步骤:
A、构建过表达载体
(1)以烟草品种Coker176根部组织的cDNA为模板,利用基因特异引物进行扩增,得到大小约为0.5kb的基因片段,纯化回收;
(2)将回收的NtERF91基因片段进行TOPO克隆,连接到-BluntⅡ-TOPO(3.5kb)载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为0.5kb的质粒提取DNA,构建的载体命名为pTOPO-NtERF91;
(3)由于基因正反游引物分别带有BamH I、Xho I的识别位点,因此选择这两个酶对质粒DNA样品进行双酶切检测,酶切结果产生两条片段,大小分别为3.5kb和0.5kb左右,说明目的片段已插入TOPO载体;
2、植物过表达载体的构建
a、入门克隆pENTRTM2B-NtERF91的构建
(1)BamH I/Xho I酶切pTOPO-NtNtERF91和pENTRTM2B得到目的基因片段NtERF91和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;
(2)挑取转化DH5α后的克隆,提取质粒DNA,BamH I/Xho I酶切,酶切结果为3.8kb的载体片段和0.5kb左右的片段为正确的克隆,正确的克隆命名为pENTRTM2B-NtERF91;
b、通过LR反应得到植物表达载体
(1)入门克隆pENTRTMNtERF91和表达载体pK2GW7 LR反应后转化大肠杆菌DH5α感受态细胞,得到植物表达载体pK2GW7-NtERF91。利用BamH I/Xho I酶切鉴定pK2GW7-NtNtERF91,正确的克隆可以切出两条片段大约0.5kb和11kb。
上述LR反应体系:构建成功的入门载体pENTRTMNtERF91(50-150ng)1-7μL,0.5μLDestination Vector,TE Buffer至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LRCloneaseTMⅡenzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min;
B、烟草的遗传转化:
(1)表达载体转化农杆菌
从-80℃冰箱中取出农杆菌感受态细胞,放置冰上溶解后加入重组表达载体pK2GW7-NtERF91 4μL;液氮速冻1分钟,转入37℃水浴5分钟,再冰浴2分钟,向混合物中加入1mL LB液体培养基,28℃、220rpm培养3~4小时;培养物涂布于含有壮观霉素100mg/L和利福平25mg/L的LB固体培养基上,28℃倒置培养2~3天,可见含有目的载体的农杆菌克隆;
(2)烟草转化
a、挑取含有目标载体的农杆菌克隆,在含有壮观霉素和利福平的LB平板上划线,28℃培养2~3天;刮取划线菌斑接菌于含有壮观霉素和利福平的MS培养基中,28℃,220rpm振荡培养,菌液浓度达到OD=0.5~0.8时6,000rpm离心5分钟富集菌体,弃上清,再用20mL液体MS培养基重悬菌体,得到含目标载体的农杆菌悬浮菌液;
b、将烟草叶片置于500mL广口瓶中,加入适量75%乙醇,漂洗1min;弃乙醇,加入0.1%的HgCl2溶液,置摇床上室温振荡15~30分钟;弃HgCl2溶液,用无菌水冲洗6遍;
c、将烟草叶片取出,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,将切成小片的烟草叶片放入含目标载体的无菌MS液体培养基悬浮菌液中,静置15~20min;取出烟草叶片,使用无菌滤纸吸去多余菌液,于含有6-BA(0.02mg/L)、NAA(2mg/L)的MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,切口接触培养基,于温室条件下分化培养;分化培养基为含有6-BA(0.5mg/L)、NAA(0.1mg/L)、卡那霉素(100mg/L)、头孢霉素(500mg/L)的MS培养基,每2~3周继代培养1次,切口处逐渐长出愈伤组织,最终分化出芽;
d、将长至3~5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净培养基,移植于灭菌的营养土中;
e、转基因植株经NPTII基因特异引物(NPTII-F:TCGGCTATGACTGGGCACAACAGA,NPTII-R:AAGAAGGCGATAGAAGGCGATGCG)PCR验证扩增,鉴定转基因阳性植株。
所述的0.1%的HgCl2溶液配制方法为称取升汞0.1克,用少许酒精溶解,再加水定容至100mL。
下面以具体实施例对本发明做进一步说明:
实施例1——NtERF91基因的分离克隆,包括以下几个步骤:
1、确定NtERF91基因序列:
通过分析茉莉素处理后烟草根部组织的转录组数据,获得受茉莉素正向调控ERF家族基因1个,序列比对发现该基因为NtERF91。根据该基因序列设计基因克隆引物进行PCR反应,得到目的产物;正向引物NtERF91-BamH I:
GGATCCATGTTAACTAGTGTCAATACCACGT;
反向引物NtERF91-Xho I:CTCGAGTCAATTTTCAGCCAATTTTCTCTTC;为了构建过表达载体,在上述引物中引物酶切位点(下划线所示)BamH I和Xho I;
2、使用Trizol试剂盒(Invitrogen)提取烟草烟草品种Coker176根部RNA,按试剂盒提供的说明书进行操作。
3、以反转录得到的第一链cDNA作为模板,用引物NtERF91
-BamH I F/NtERF91-Xho I R对进行PCR扩增,选用Phusion高保真扩增酶反应体系,体系总体积50μL,包括:200ng cDNA,5×Phusion HF反应缓冲液10μL,10mM dNTP 1μL,2U的High-Fidelity DNA Polymerase,10μM的正反向引物各1μL,补水至50μL。PCR反应在pro扩增仪上进行,反应程序为:98℃,30秒;98℃,7秒,58℃,30秒,72℃,30秒,35个循环;72℃延伸7分钟;回收和纯化PCR产物,产物经1%(1g/100mL电泳缓冲液)琼脂糖凝胶电泳分离,扩增结果产生一条单一PCR条带,见图1。
4、纯化产物与载体连接:连接体系与过程如下:4μL纯化产物、1μL saltsolution、1μL-BluntⅡ-TOPO(Invitrogen)混匀,25℃,水浴30min;将连接好的载体通过热激转化大肠杆菌DH5α,加液体培养基振荡培养后涂布至含100mg/L卡那霉素的LB平板上过夜培养,挑取菌落进行菌液培养,质粒提取和PCR检测,见图2。筛选阳性克隆,对阳性克隆进行测序。
步骤4中所述的培养基配方及制备方法是称取胰蛋白胨8~12g,酵母提取物5g,NaCl 10g溶解于1L蒸馏水中,121℃灭菌25min得到。
实施例2
过表达载体构建
1、克隆的NtERF91连接TOPO载体
(1)以烟草品种Coker176根部的cDNA为模板,利用NtERF91基因特异引物进行扩增,得到大小约为0.5kb的基因片段,纯化回收;
(2)将回收的NtERF91基因片段进行TOPO克隆,连接到-BluntⅡ-TOPO(3.5kb)载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为0.5kb的质粒提取DNA,构建的载体命名为pTOPO-NtERF91;
(3)由于基因正反游引物分别带有BamH I、Xho I的识别位点,因此选择这两个酶对质粒DNA样品进行双酶切检测,酶切结果产生两条片段,大小分别为3.5kb和0.5kb左右,说明目的片段已插入TOPO载体;
2、植物过表达载体的构建
a、入门克隆pENTRTM2B-NtERF91的构建
(1)BamH I/Xho I酶切pTOPO-NtERF91和pENTRTM2B得到目的基因片段NtERF91和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;
(2)挑取转化DH5α后的克隆,提取质粒DNA,BamH I/Xho I酶切,酶切结果为3.8kb的载体片段和0.5kb左右的片段为正确的克隆,正确的克隆命名为pENTRTM2B-NtERF91;
b、通过LR反应得到植物表达载体
(1)入门克隆pENTRTMNtERF91和表达载体pK2GW7 LR反应后转化大肠杆菌DH5α感受态细胞,得到植物表达载体pK2GW7-NtERF91。利用BamH I/Xho I酶切鉴定pK2GW7-NtERF91,正确的克隆可以切出两条片段大约0.5kb和11kb。
上述LR反应体系:构建成功的入门载体pENTRTMNtERF91(50-150ng)1-7μL,0.5μLDestination Vector,TE Buffer至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LRCloneaseTMⅡenzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min。
实施例3
烟草的遗传转化
(1)表达载体转化农杆菌
从-80℃冰箱中取出农杆菌感受态细胞,放置冰上溶解后加入重组表达载体pK2GW7-NtERF91 4μL;液氮速冻1分钟,转入37℃水浴5分钟,再冰浴2分钟,向混合物中加入1mL LB液体培养基,28℃、220rpm培养3~4小时;培养物涂布于含有壮观霉素100mg/L和利福平25mg/L的LB固体培养基上,28℃倒置培养2~3天,可见含有目的载体的农杆菌克隆;
(2)烟草转化
a、挑取含有目标载体的农杆菌克隆,在含有壮观霉素和利福平的LB平板上划线,28℃培养2~3天;刮取划线菌斑接菌于含有壮观霉素和利福平的MS培养基中,28℃,220rpm振荡培养,菌液浓度达到OD=0.5~0.8时6,000rpm离心5分钟富集菌体,弃上清,再用20mL液体MS培养基重悬菌体,得到含目标载体的农杆菌悬浮菌液;
b、将烟草叶片置于500mL广口瓶中,加入适量75%乙醇,漂洗1min;弃乙醇,加入0.1%的HgCl2溶液,置摇床上室温振荡15~30分钟;弃HgCl2溶液,用无菌水冲洗6遍;
c、将烟草叶片取出,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,将切成小片的烟草叶片放入含目标载体的无菌MS液体培养基悬浮菌液中,静置15~20min;取出烟草叶片,使用无菌滤纸吸去多余菌液,于含有6-BA(0.02mg/L)、NAA(2mg/L)的MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,切口接触培养基,于温室条件下分化培养;分化培养基为含有6-BA(0.5mg/L)、NAA(0.1mg/L)、卡那霉素(100mg/L)、头孢霉素(500mg/L)的MS培养基,每2~3周继代培养1次,切口处逐渐长出愈伤组织,最终分化出芽;
d、将长至3~5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净培养基,移植于灭菌的营养土中;
e、转基因植株经NPTII基因特异引物(NPTII-F:TCGGCTATGACTGGGCACAACAGA,NPTII-R:AAGAAGGCGATAGAAGGCGATGCG)PCR验证扩增,鉴定转基因阳性植株。
实施例4
转基因植株分析
T0代转基因株系提取总RNA,利用TaKaRa公司PrimeScriptTM RT reagent Kit反转录试剂盒合成cDNA作为模板进行实时荧光定量PCR分析,获得过表达(EV)株系进行表型分析(图3)。T0代转基因株系实验结果表明,NtERF91基因过表达植株与对照相比烟叶新烟碱含量提高了4倍(图4),表明NtERF91基因正向调控烟草新烟碱的合成。
SEQUENCE LISTING
<110> 云南省烟草农业科学研究院
<120> 一种烟草新烟碱合成调控基因NtERF91的克隆和应用
<130> 20191019
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 435
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgttaacta gtgtcaatac cacgtggcaa cgagtcgata atttggagta caaggaggaa 60
acaaagaaga accatgtggt ggcgcgtggt gtgcacgcac ctcaggattg gaaccggtac 120
aggggtgtta ggcggaggcc gtggggtaaa tttgcggcgg agataagaaa cccggatagg 180
aaaggcgccc ggctttggct gggaacttac gagacacccg aagatgcagc attggcttat 240
gaccaagccg catataagat ccgtggctct aaggctcggc tcaacttccc tcacttaatc 300
ggctcgaaca tatccgagcc ggttagagtg gctccgagac ggcgttgcct atcgccggag 360
atttcatcat ctattttttc gtcgtcattt gttgaaaata tacctctaaa gaagagaaaa 420
ttggctgaaa attga 435
<210> 2
<211> 146
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Glu Thr Leu Thr Ser Val Asn Thr Thr Trp Gln Arg Val Asp Asn
1 5 10 15
Leu Glu Tyr Lys Glu Glu Thr Lys Lys Asn His Val Val Ala Arg Gly
20 25 30
Val His Ala Pro Gln Asp Trp Asn Arg Tyr Arg Gly Val Arg Arg Arg
35 40 45
Pro Trp Gly Lys Phe Ala Ala Glu Ile Arg Asn Pro Asp Arg Lys Gly
50 55 60
Ala Arg Leu Trp Leu Gly Thr Tyr Glu Thr Pro Glu Asp Ala Ala Leu
65 70 75 80
Ala Tyr Asp Gln Ala Ala Tyr Lys Ile Arg Gly Ser Lys Ala Arg Leu
85 90 95
Asn Phe Pro His Leu Ile Gly Ser Asn Ile Ser Glu Pro Val Arg Val
100 105 110
Ala Pro Arg Arg Arg Cys Leu Ser Pro Glu Ile Ser Ser Ser Ile Phe
115 120 125
Ser Ser Ser Phe Val Glu Asn Ile Pro Leu Lys Lys Arg Lys Leu Ala
130 135 140
Glu Asn
145
Claims (9)
1.一种烟草新烟碱合成调控基因NtERF91,其特征在于所述的烟草新烟碱合成调控基因NtERF91的核苷酸序列如序列表SEQ ID NO:1所示。
2.根据权利要求1所述的烟草新烟碱合成调控基因NtERF91,其特征在于所述的烟草新烟碱合成调控基因NtERF91编码的氨基酸序列如SEQ ID NO:2所示。
3.一种权利要求1所述的烟草新烟碱合成调控基因NtERF91的克隆方法,其特征在于包括以下步骤:
A、确定NtERF91基因序列;
根据茉莉酸处理烟草根部组织转录组分析获得NtERF91基因序列,利用此序列设计基因克隆引物:
正向引物NtERF91-BamH I:
GGATCCATGTTAACTAGTGTCAATACCACGT;
反向引物NtERF91-XhoI:CTCGAGTCAATTTTCAGCCAATTTTCTCTTC;
B、提取烟草根组织RNA,反转录得到第一链cDNA;
C、根据NtERF91基因序列设计合成特异性引物,以反转录得到的第一链cDNA作为模板,进行PCR扩增,回收和纯化PCR产物;
4.根据权利要求3所述的烟草新烟碱合成调控基因NtERF91的克隆方法,其特征在于C步骤中PCR扩增的反应体系是选用Phusion高保真扩增酶反应体系,体系总体积50μL,包括:200ng cDNA,5×Phusion HF反应缓冲液10μL,10mM dNTP 1μL,2U的High-Fidelity DNA Polymerase,10μM的正反向引物各1μL,补水至50μL。
6.一种权利要求1或2所述的调控烟草叶片新烟碱含量的基因NtERF91的过表达载体,其特征在于所述的调控烟草叶片烟碱含量的基因NtERF91的过表达载体为pK2GW7-NtERF91。
7.一种权利要求1所述的烟草新烟碱合成调控基因NtERF91的应用,其特征在于所述的烟草新烟碱合成调控基因NtERF91在用于获得新烟碱含量显著提高的转基因植株中的应用。
8.根据权利要求7所述的烟草新烟碱合成调控基因NtERF91的应用,其特征在于所述获得NtERF91转基因烟草的方法包括以下步骤:
A、过表达隆载体的构建:
1、克隆的NtERF91连接TOPO载体
(1)以烟草品种Coker176根部组织的cDNA为模板,利用NtERF91基因特异引物进行扩增,得到大小约为0.5kb的基因片段;纯化回收;
(2)将回收的NtERF91基因片段进行TOPO克隆,连接到-BluntⅡ-TOPO(3.5kb)载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为0.5kb的质粒提取DNA,构建的载体命名为pTOPO-NtERF91;
(3)由于基因上下游引物分别带有BamH I、Xho I的识别位点,因此选择这两个酶对检测正确的质粒样品进行双酶切检测,将目的基因片段胶回收;对pTOPO进行BamH I、Xho I双酶切检测,酶切结果产生两条片段,大小分别为3.5kb和0.5kb左右,说明目的片段已插入TOPO载体;
2、植物过表达载体的构建
a、入门克隆pENTRTM2B-NtERF91的构建
(1)BamH I/Xho I酶切pTOPO-NtERF91和pENTRTM2B得到目的基因片段NtERF91和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;
(2)挑取转化DH5α后的克隆,提取质粒DNA,BamH I/Xho I酶切,酶切结果为3.8kb的载体片段和0.5kb左右的片段为正确的克隆,正确的克隆命名为pENTRTM2B-NtNtERF91;
b、通过LR反应得到植物表达载体
(1)入门克隆pENTRTMNtERF91和表达载体pK2GW7 LR反应后转化大肠杆菌DH5α感受态细胞,得到植物表达载体pK2GW7-NtERF91;利用BamH I/Xho I酶切鉴定pK2GW7-NtERF91,正确的克隆可以切出两条片段大约0.5kb和11kb;
上述LR反应体系:构建成功的入门载体pENTRTMNtERF91(50-150ng)1-7μL,0.5μLDestination Vector,TE Buffer至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LRCloneaseTMⅡ enzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min;
B、烟草的遗传转化:
(1)表达载体转化农杆菌
从-80℃冰箱中取出农杆菌感受态细胞,放置冰上溶解后加入重组表达载体pK2GW7-NtERF91 4μL;液氮速冻1分钟,转入37℃水浴5分钟,再冰浴2分钟,向混合物中加入1mL LB液体培养基,28℃、220rpm培养3~4小时;培养物涂布于含有壮观霉素100mg/L和利福平25mg/L的LB固体培养基上,28℃倒置培养2~3天,可见含有目的载体的农杆菌克隆;
(2)烟草转化
a、挑取含有目标载体的农杆菌克隆,在含有壮观霉素和利福平的LB平板上划线,28℃培养2~3天;刮取划线菌斑接菌于含有壮观霉素和利福平的MS培养基中,28℃,220rpm振荡培养,菌液浓度达到OD=0.5~0.8时6,000rpm离心5分钟富集菌体,弃上清,再用20mL液体MS培养基重悬菌体,得到含目标载体的农杆菌悬浮菌液;
b、将烟草叶片置于500mL广口瓶中,加入适量75%乙醇,漂洗1min;弃乙醇,加入0.1%的HgCl2溶液,置摇床上室温振荡15~30分钟;弃HgCl2溶液,用无菌水冲洗6遍;
c、将烟草叶片取出,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,将切成小片的烟草叶片放入含目标载体的无菌MS液体培养基悬浮菌液中,静置15~20min;取出烟草叶片,使用无菌滤纸吸去多余菌液,于含有6-BA(0.02mg/L)、NAA(2mg/L)的MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,切口接触培养基,于温室条件下分化培养;分化培养基为含有6-BA(0.5mg/L)、NAA(0.1mg/L)、卡那霉素(100mg/L)、头孢霉素(500mg/L)的MS培养基,每2~3周继代培养1次,切口处逐渐长出愈伤组织,最终分化出芽;
d、将长至3~5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净培养基,移植于灭菌的营养土中;
e、转基因植株经NPTII基因特异引物(NPTII-F:TCGGCTATGACTGGGCACAACAGA,NPTII-R:AAGAAGGCGATAGAAGGCGATGCG)PCR验证扩增,鉴定转基因阳性植株。
9.根据权利要求8所述的提高烟草叶片新烟碱含量的基因NtERF91的应用,其特征在于所述的0.1%的HgCl2溶液配制方法为称取升汞0.1克,用少许酒精溶解,再加水定容至100mL。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911005045.7A CN110669772A (zh) | 2019-10-22 | 2019-10-22 | 一种烟草新烟碱合成调控基因NtERF91的克隆和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911005045.7A CN110669772A (zh) | 2019-10-22 | 2019-10-22 | 一种烟草新烟碱合成调控基因NtERF91的克隆和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110669772A true CN110669772A (zh) | 2020-01-10 |
Family
ID=69083539
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911005045.7A Pending CN110669772A (zh) | 2019-10-22 | 2019-10-22 | 一种烟草新烟碱合成调控基因NtERF91的克隆和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110669772A (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110592103A (zh) * | 2019-10-22 | 2019-12-20 | 云南省烟草农业科学研究院 | 一种烟草假木贼碱合成调控基因NtERF91的克隆和应用 |
CN111118031A (zh) * | 2020-02-17 | 2020-05-08 | 云南省烟草农业科学研究院 | 一种烟草生物碱合成负向调控基因NtARF6的克隆和应用 |
CN112877355A (zh) * | 2021-01-22 | 2021-06-01 | 杜云龙 | 一种利用烟草表达三七皂苷的方法 |
CN113999857A (zh) * | 2021-11-22 | 2022-02-01 | 云南中烟工业有限责任公司 | 一种烟草烟碱合成调控相关的基因及其应用 |
Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140246036A1 (en) * | 2013-03-01 | 2014-09-04 | North Carolina State University | Transcription factors that regulate nicotine biosynthesis in tobacco |
CN107828795A (zh) * | 2017-11-09 | 2018-03-23 | 云南省烟草农业科学研究院 | 一种提高烟草叶片钾含量的基因NtNHX1‑2及其克隆方法与应用 |
CN108374014A (zh) * | 2018-02-08 | 2018-08-07 | 云南省烟草农业科学研究院 | 一种提高烟草叶片钾含量的基因NtTPKa及其克隆方法与应用 |
CN109295072A (zh) * | 2018-10-17 | 2019-02-01 | 云南省烟草农业科学研究院 | 一种烟草烟碱合成调控基因NtERF115及其克隆方法与应用 |
US20190203215A1 (en) * | 2016-09-02 | 2019-07-04 | 22Nd Century Limited, Llc | TRANSCRIPTION FACTOR NtERF241 AND METHODS OF USING THE SAME |
CN110592103A (zh) * | 2019-10-22 | 2019-12-20 | 云南省烟草农业科学研究院 | 一种烟草假木贼碱合成调控基因NtERF91的克隆和应用 |
CN110643616A (zh) * | 2019-10-22 | 2020-01-03 | 云南省烟草农业科学研究院 | 一种烟草降烟碱合成调控基因NtERF91的克隆和应用 |
CN110684779A (zh) * | 2019-10-22 | 2020-01-14 | 云南省烟草农业科学研究院 | 一种烟草烟碱合成调控基因NtERF91的克隆和应用 |
EP3642346A1 (en) * | 2017-06-23 | 2020-04-29 | University of Kentucky Research Foundation | Method |
CN111118031A (zh) * | 2020-02-17 | 2020-05-08 | 云南省烟草农业科学研究院 | 一种烟草生物碱合成负向调控基因NtARF6的克隆和应用 |
WO2021026119A1 (en) * | 2019-08-05 | 2021-02-11 | University Of Virginia Patent Foundation | TRANSCRIPTION FACTOR NtERF221 AND METHODS OF USING THE SAME |
WO2021113337A1 (en) * | 2019-12-03 | 2021-06-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
WO2021126978A1 (en) * | 2019-12-17 | 2021-06-24 | North Carolina State University | Materials and methods related to transgenic plants having reduced levels of alkaloids and carcinogens |
US20220010324A1 (en) * | 2020-06-03 | 2022-01-13 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
-
2019
- 2019-10-22 CN CN201911005045.7A patent/CN110669772A/zh active Pending
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140246036A1 (en) * | 2013-03-01 | 2014-09-04 | North Carolina State University | Transcription factors that regulate nicotine biosynthesis in tobacco |
US20190203215A1 (en) * | 2016-09-02 | 2019-07-04 | 22Nd Century Limited, Llc | TRANSCRIPTION FACTOR NtERF241 AND METHODS OF USING THE SAME |
EP3642346A1 (en) * | 2017-06-23 | 2020-04-29 | University of Kentucky Research Foundation | Method |
CN111315891A (zh) * | 2017-06-23 | 2020-06-19 | 肯塔基大学研究基金会 | 方法 |
US20200291413A1 (en) * | 2017-06-23 | 2020-09-17 | University Of Kentucky Research Foundation | Method |
CN107828795A (zh) * | 2017-11-09 | 2018-03-23 | 云南省烟草农业科学研究院 | 一种提高烟草叶片钾含量的基因NtNHX1‑2及其克隆方法与应用 |
CN108374014A (zh) * | 2018-02-08 | 2018-08-07 | 云南省烟草农业科学研究院 | 一种提高烟草叶片钾含量的基因NtTPKa及其克隆方法与应用 |
CN109295072A (zh) * | 2018-10-17 | 2019-02-01 | 云南省烟草农业科学研究院 | 一种烟草烟碱合成调控基因NtERF115及其克隆方法与应用 |
WO2021026119A1 (en) * | 2019-08-05 | 2021-02-11 | University Of Virginia Patent Foundation | TRANSCRIPTION FACTOR NtERF221 AND METHODS OF USING THE SAME |
CN110643616A (zh) * | 2019-10-22 | 2020-01-03 | 云南省烟草农业科学研究院 | 一种烟草降烟碱合成调控基因NtERF91的克隆和应用 |
CN110684779A (zh) * | 2019-10-22 | 2020-01-14 | 云南省烟草农业科学研究院 | 一种烟草烟碱合成调控基因NtERF91的克隆和应用 |
CN110592103A (zh) * | 2019-10-22 | 2019-12-20 | 云南省烟草农业科学研究院 | 一种烟草假木贼碱合成调控基因NtERF91的克隆和应用 |
WO2021113337A1 (en) * | 2019-12-03 | 2021-06-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
WO2021126978A1 (en) * | 2019-12-17 | 2021-06-24 | North Carolina State University | Materials and methods related to transgenic plants having reduced levels of alkaloids and carcinogens |
CN111118031A (zh) * | 2020-02-17 | 2020-05-08 | 云南省烟草农业科学研究院 | 一种烟草生物碱合成负向调控基因NtARF6的克隆和应用 |
US20220010324A1 (en) * | 2020-06-03 | 2022-01-13 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
Non-Patent Citations (7)
Title |
---|
SHOJI,T.等: "Nicotiana tabacum ERF91 mRNA for ethylene response factor 91, complete cds", 《GENBANK DATABASE》 * |
TSUBASA SHOJI等: "Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco", 《THE PLANT CELL》 * |
XUEYI SUI等: "Ethylene response factor NtERF91 positively regulates alkaloid accumulations in tobacco (Nicotiana tabacum L.)", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
李文正等: "烟碱合成分子调控研究进展", 《安徽农业科学》 * |
熊安平等: "调控植物生物碱生物合成的转录因子研究进展", 《分子植物育种》 * |
白戈等: "烟草(Nicotiana tabacum)生长素响应基因NtIAA27的克隆及功能分析", 《分子植物育种》 * |
金云峰等: "烟草烟碱代谢的生化和分子机制及其调控", 《基因组学与应用生物学》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110592103A (zh) * | 2019-10-22 | 2019-12-20 | 云南省烟草农业科学研究院 | 一种烟草假木贼碱合成调控基因NtERF91的克隆和应用 |
CN111118031A (zh) * | 2020-02-17 | 2020-05-08 | 云南省烟草农业科学研究院 | 一种烟草生物碱合成负向调控基因NtARF6的克隆和应用 |
CN112877355A (zh) * | 2021-01-22 | 2021-06-01 | 杜云龙 | 一种利用烟草表达三七皂苷的方法 |
CN113999857A (zh) * | 2021-11-22 | 2022-02-01 | 云南中烟工业有限责任公司 | 一种烟草烟碱合成调控相关的基因及其应用 |
CN113999857B (zh) * | 2021-11-22 | 2024-02-02 | 云南中烟工业有限责任公司 | 一种烟草烟碱合成调控相关的基因及其应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111118031A (zh) | 一种烟草生物碱合成负向调控基因NtARF6的克隆和应用 | |
CN110684779A (zh) | 一种烟草烟碱合成调控基因NtERF91的克隆和应用 | |
CN110669772A (zh) | 一种烟草新烟碱合成调控基因NtERF91的克隆和应用 | |
CN110643616A (zh) | 一种烟草降烟碱合成调控基因NtERF91的克隆和应用 | |
CN107541520B (zh) | 与水稻根发育和抗逆性相关OsSAUR11基因及编码蛋白与应用 | |
CN113831397B (zh) | 一种原花青素物质调控因子NtMYB330及其表达载体、转化体、试剂盒与方法 | |
CN112831478B (zh) | 一种调控水稻垩白的蛋白质OsCAT8及其编码基因和应用 | |
CN109295072A (zh) | 一种烟草烟碱合成调控基因NtERF115及其克隆方法与应用 | |
CN110592103A (zh) | 一种烟草假木贼碱合成调控基因NtERF91的克隆和应用 | |
CN109207484A (zh) | 一种烟草尼古丁含量调控基因iaa27及其克隆方法与应用 | |
CN107267528B (zh) | 一种烟草腋芽生长调控基因NtMOC1及其克隆方法与应用 | |
CN109468329B (zh) | 一种烟草外向整流钾离子通道基因NtSKOR1及其克隆方法与应用 | |
CN112143738B (zh) | 一种烟草受体蛋白基因及其克隆方法与应用 | |
CN107365777B (zh) | 一种烟草尼古丁含量调控基因NtCLC-b及其克隆方法与应用 | |
CN112080507B (zh) | 一种调控银杏类黄酮合成的关键基因GbMYB4及其表达的蛋白、载体和应用 | |
CN108707614B (zh) | 一种花生抗逆性基因及其应用 | |
CN113943740B (zh) | 一种可调控烟叶钾含量的NtCHA1基因及其应用 | |
CN113461794B (zh) | 一种调控种子萌发的试剂盒、方法及其应用 | |
CN113481210B (zh) | 棉花GhDof1.7基因在促进植物耐盐中的应用 | |
CN110078805B (zh) | 枇杷EjAG基因及其编码的蛋白与应用 | |
CN111560055B (zh) | 水稻基因OsLAT3在调节敌草快的吸收累积中的应用 | |
CN110885844B (zh) | 紫花苜蓿基因MsCYP20-3B及应用 | |
CN108103075B (zh) | 一种延缓植物衰老的柳枝稷基因PvC3H29及其应用 | |
CN113151315A (zh) | 烟草多酚代谢途径蛋白基因NtPPOE及其应用 | |
CN107365775B (zh) | 一种烟草腋芽生长调控基因NtMAX3-2及其克隆方法与应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200110 |
|
RJ01 | Rejection of invention patent application after publication |