CN112501142A - 一种柳杉抗寒调控基因CfAPX及其应用 - Google Patents
一种柳杉抗寒调控基因CfAPX及其应用 Download PDFInfo
- Publication number
- CN112501142A CN112501142A CN202011266568.XA CN202011266568A CN112501142A CN 112501142 A CN112501142 A CN 112501142A CN 202011266568 A CN202011266568 A CN 202011266568A CN 112501142 A CN112501142 A CN 112501142A
- Authority
- CN
- China
- Prior art keywords
- cfapx
- gene
- cryptomeria fortunei
- cold
- plants
- 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
- 240000005286 Cryptomeria japonica var. sinensis Species 0.000 title claims abstract description 33
- 108700005075 Regulator Genes Proteins 0.000 title claims abstract description 12
- 241000196324 Embryophyta Species 0.000 claims abstract description 60
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 17
- 241000218645 Cedrus Species 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 5
- 241000589158 Agrobacterium Species 0.000 claims description 4
- 230000002000 scavenging effect Effects 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 abstract description 34
- 230000014509 gene expression Effects 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 6
- 239000001301 oxygen Substances 0.000 abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 3
- 238000012795 verification Methods 0.000 abstract description 3
- 108060006004 Ascorbate peroxidase Proteins 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000035882 stress Effects 0.000 description 22
- 241000219194 Arabidopsis Species 0.000 description 14
- 102000003992 Peroxidases Human genes 0.000 description 14
- 108040007629 peroxidase activity proteins Proteins 0.000 description 13
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 8
- 229940118019 malondialdehyde Drugs 0.000 description 8
- 235000010323 ascorbic acid Nutrition 0.000 description 6
- 239000011668 ascorbic acid Substances 0.000 description 6
- 229960005070 ascorbic acid Drugs 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229930002875 chlorophyll Natural products 0.000 description 3
- 235000019804 chlorophyll Nutrition 0.000 description 3
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108700003863 Arabidopsis ENT1 Proteins 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 108010092854 aspartyllysine Proteins 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 108010009298 lysylglutamic acid Proteins 0.000 description 2
- 108010015796 prolylisoleucine Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- HMRWQTHUDVXMGH-GUBZILKMSA-N Ala-Glu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HMRWQTHUDVXMGH-GUBZILKMSA-N 0.000 description 1
- KMGOBAQSCKTBGD-DLOVCJGASA-N Ala-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CN=CN1 KMGOBAQSCKTBGD-DLOVCJGASA-N 0.000 description 1
- NJWJSLCQEDMGNC-MBLNEYKQSA-N Ala-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C)N)O NJWJSLCQEDMGNC-MBLNEYKQSA-N 0.000 description 1
- RZZMZYZXNJRPOJ-BJDJZHNGSA-N Ala-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](C)N RZZMZYZXNJRPOJ-BJDJZHNGSA-N 0.000 description 1
- WUHJHHGYVVJMQE-BJDJZHNGSA-N Ala-Leu-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WUHJHHGYVVJMQE-BJDJZHNGSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- WQLDNOCHHRISMS-NAKRPEOUSA-N Ala-Pro-Ile Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WQLDNOCHHRISMS-NAKRPEOUSA-N 0.000 description 1
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 1
- HJVGMOYJDDXLMI-AVGNSLFASA-N Arg-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N HJVGMOYJDDXLMI-AVGNSLFASA-N 0.000 description 1
- XLWSGICNBZGYTA-CIUDSAMLSA-N Arg-Glu-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XLWSGICNBZGYTA-CIUDSAMLSA-N 0.000 description 1
- UPKMBGAAEZGHOC-RWMBFGLXSA-N Arg-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O UPKMBGAAEZGHOC-RWMBFGLXSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- NECWUSYTYSIFNC-DLOVCJGASA-N Asp-Ala-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 NECWUSYTYSIFNC-DLOVCJGASA-N 0.000 description 1
- KNMRXHIAVXHCLW-ZLUOBGJFSA-N Asp-Asn-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N)C(=O)O KNMRXHIAVXHCLW-ZLUOBGJFSA-N 0.000 description 1
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 1
- YVHGKXAOSVBGJV-CIUDSAMLSA-N Asp-Lys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N YVHGKXAOSVBGJV-CIUDSAMLSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000218691 Cupressaceae Species 0.000 description 1
- HAYVLBZZBDCKRA-SRVKXCTJSA-N Cys-His-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N HAYVLBZZBDCKRA-SRVKXCTJSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- IKDOHQHEFPPGJG-FXQIFTODSA-N Gln-Asp-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O IKDOHQHEFPPGJG-FXQIFTODSA-N 0.000 description 1
- OZEQPCDLCDRCGY-SOUVJXGZSA-N Gln-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCC(=O)N)N)C(=O)O OZEQPCDLCDRCGY-SOUVJXGZSA-N 0.000 description 1
- FITIQFSXXBKFFM-NRPADANISA-N Gln-Val-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FITIQFSXXBKFFM-NRPADANISA-N 0.000 description 1
- WOSRKEJQESVHGA-CIUDSAMLSA-N Glu-Arg-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O WOSRKEJQESVHGA-CIUDSAMLSA-N 0.000 description 1
- BUAKRRKDHSSIKK-IHRRRGAJSA-N Glu-Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BUAKRRKDHSSIKK-IHRRRGAJSA-N 0.000 description 1
- LRPXYSGPOBVBEH-IUCAKERBSA-N Glu-Gly-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O LRPXYSGPOBVBEH-IUCAKERBSA-N 0.000 description 1
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- WZSHYFGOLPXPLL-RYUDHWBXSA-N Gly-Phe-Glu Chemical compound NCC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(O)=O)C(O)=O WZSHYFGOLPXPLL-RYUDHWBXSA-N 0.000 description 1
- WDXLKVQATNEAJQ-BQBZGAKWSA-N Gly-Pro-Asp Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WDXLKVQATNEAJQ-BQBZGAKWSA-N 0.000 description 1
- IXHQLZIWBCQBLQ-STQMWFEESA-N Gly-Pro-Phe Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IXHQLZIWBCQBLQ-STQMWFEESA-N 0.000 description 1
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 1
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- BIAKMWKJMQLZOJ-ZKWXMUAHSA-N His-Ala-Ala Chemical compound C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)Cc1cnc[nH]1)C(O)=O BIAKMWKJMQLZOJ-ZKWXMUAHSA-N 0.000 description 1
- YADRBUZBKHHDAO-XPUUQOCRSA-N His-Gly-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](C)C(O)=O YADRBUZBKHHDAO-XPUUQOCRSA-N 0.000 description 1
- QCBYAHHNOHBXIH-UWVGGRQHSA-N His-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CN=CN1 QCBYAHHNOHBXIH-UWVGGRQHSA-N 0.000 description 1
- UIEZQYNXCYHMQS-BJDJZHNGSA-N Ile-Lys-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)O)N UIEZQYNXCYHMQS-BJDJZHNGSA-N 0.000 description 1
- CIJLNXXMDUOFPH-HJWJTTGWSA-N Ile-Pro-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 CIJLNXXMDUOFPH-HJWJTTGWSA-N 0.000 description 1
- APQYGMBHIVXFML-OSUNSFLBSA-N Ile-Val-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N APQYGMBHIVXFML-OSUNSFLBSA-N 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 1
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 1
- KTFHTMHHKXUYPW-ZPFDUUQYSA-N Leu-Asp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KTFHTMHHKXUYPW-ZPFDUUQYSA-N 0.000 description 1
- LOLUPZNNADDTAA-AVGNSLFASA-N Leu-Gln-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LOLUPZNNADDTAA-AVGNSLFASA-N 0.000 description 1
- OGUUKPXUTHOIAV-SDDRHHMPSA-N Leu-Glu-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGUUKPXUTHOIAV-SDDRHHMPSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- QLDHBYRUNQZIJQ-DKIMLUQUSA-N Leu-Ile-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QLDHBYRUNQZIJQ-DKIMLUQUSA-N 0.000 description 1
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- XOQMURBBIXRRCR-SRVKXCTJSA-N Lys-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN XOQMURBBIXRRCR-SRVKXCTJSA-N 0.000 description 1
- CNGOEHJCLVCJHN-SRVKXCTJSA-N Lys-Pro-Glu Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O CNGOEHJCLVCJHN-SRVKXCTJSA-N 0.000 description 1
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 1
- MYAPQOBHGWJZOM-UWVGGRQHSA-N Met-Gly-Leu Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C MYAPQOBHGWJZOM-UWVGGRQHSA-N 0.000 description 1
- ALTHVGNGGZZSAC-SRVKXCTJSA-N Met-Val-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCNC(N)=N ALTHVGNGGZZSAC-SRVKXCTJSA-N 0.000 description 1
- LPNWWHBFXPNHJG-AVGNSLFASA-N Met-Val-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN LPNWWHBFXPNHJG-AVGNSLFASA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- LSXGADJXBDFXQU-DLOVCJGASA-N Phe-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 LSXGADJXBDFXQU-DLOVCJGASA-N 0.000 description 1
- XEXSSIBQYNKFBX-KBPBESRZSA-N Phe-Gly-His Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CC=CC=C1 XEXSSIBQYNKFBX-KBPBESRZSA-N 0.000 description 1
- 235000017339 Pinus palustris Nutrition 0.000 description 1
- 241000204936 Pinus palustris Species 0.000 description 1
- 235000005097 Pinus roxburghii Nutrition 0.000 description 1
- RFWXYTJSVDUBBZ-DCAQKATOSA-N Pro-Pro-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RFWXYTJSVDUBBZ-DCAQKATOSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- VBZXFFYOBDLLFE-HSHDSVGOSA-N Pro-Trp-Thr Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H]([C@H](O)C)C(O)=O)C(=O)[C@@H]1CCCN1 VBZXFFYOBDLLFE-HSHDSVGOSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- DKKGAAJTDKHWOD-BIIVOSGPSA-N Ser-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)C(=O)O DKKGAAJTDKHWOD-BIIVOSGPSA-N 0.000 description 1
- VAIZFHMTBFYJIA-ACZMJKKPSA-N Ser-Asp-Gln Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O VAIZFHMTBFYJIA-ACZMJKKPSA-N 0.000 description 1
- 241001116495 Taxaceae Species 0.000 description 1
- 241001116500 Taxus Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- BURPTJBFWIOHEY-UWJYBYFXSA-N Tyr-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BURPTJBFWIOHEY-UWJYBYFXSA-N 0.000 description 1
- IELISNUVHBKYBX-XDTLVQLUSA-N Tyr-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 IELISNUVHBKYBX-XDTLVQLUSA-N 0.000 description 1
- NZFCWALTLNFHHC-JYJNAYRXSA-N Tyr-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NZFCWALTLNFHHC-JYJNAYRXSA-N 0.000 description 1
- VBFVQTPETKJCQW-RPTUDFQQSA-N Tyr-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VBFVQTPETKJCQW-RPTUDFQQSA-N 0.000 description 1
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 1
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 1
- PMKQKNBISAOSRI-XHSDSOJGSA-N Val-Tyr-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N PMKQKNBISAOSRI-XHSDSOJGSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 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 1
- 230000006851 antioxidant defense Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010009932 leucyl-alanyl-glycyl-valine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010087810 leucyl-seryl-glutamyl-leucine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010029384 tryptophyl-histidine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01011—L-ascorbate peroxidase (1.11.1.11)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请公开了一种柳杉抗寒调控基因CfAPX及其应用，属于植物分子生物学和基因工程技术领域。本申请的柳杉抗寒调控基因CfAPX具有如SEQIDNO.1所示的核苷酸序列，其表达蛋白的氨基酸序列如SEQIDNO.2所示。本申请通过实验发现CfAPX基因在低温胁迫下柳杉叶片中的基因表达量在‑16℃达到峰值。通过构建表达载体PBI121‑CfAPX对CfAPX进行功能验证，发现，转基因植株CfAPX基因的表达量上调，与野生型相比均达到了显著或极显著水平。因此，CfAPX基因可以调节植物抗坏血酸过氧化物酶活性，从而提高细胞清除活性氧，维持稳态的能力，进一步提高植株的抗逆性，对植物的生长发育具有重要意义。
Description
技术领域
本发明属于植物分子生物学和基因工程技术领域，具体涉及一种柳杉抗寒调控基因CfAPX及其应用。
背景技术
柳杉(Cryptomeriafortunei)为杉科(Taxodiaceae)柳杉属(Cryptomeria)常绿高大乔木，又称为长叶孔雀松，喜山地气候及条件良好的酸性土壤，具有侧根发达、生长迅速、适应性强、树形优美且能净化空气、改善环境等特性，是中国南方优良的用材和园林树种。柳杉的生长和分布常受到干旱、寒冷等极端环境的制约，随着全球气候变化以及极端天气频繁发生，柳杉的生长发育受到了极大的影响。
抗坏血酸过氧化物酶(APX)是含有血红素的过氧化物酶家族，它以抗坏血酸作为特异性电子供体，催化H2O2生成水，通过清除活性氧维持细胞氧化还原稳态。生物或非生物胁迫(干旱、盐、高温、低温等)会使植物产生过多的活性氧物质，对细胞膜和细胞器造成不同程度的损伤。抗坏血酸过氧化物酶作为细胞抗氧化防御最重要的组成部分之一，当植物受到各种环境胁迫时，在调节活性氧(ROS)水平方面发挥着至关重要的作用。
发明内容
针对现有技术中存在的不足，本发明所要解决的技术问题是提供一种柳杉抗寒调控基因CfAPX，其基因蛋白为抗坏血酸过氧化物酶，能高效的清除植物体内的过氧化氢，维持细胞稳态，当柳杉受到各种环境胁迫时，能够调节活性氧水平从而提高抗逆性。本发明所要解决的另一技术间题在于提供所述柳杉CfAPX基因的应用。
为了解决上述技术问题，本发明所采用的技术方案如下：
一种柳杉抗寒调控基因CfAPX，其核苷酸序列如SEQIDNO.1所示。
所述的柳杉CfAPX基因的编码蛋白，其氨基酸序列如SEQIDNO.2所示。
含有所述的柳杉CfAPX基因的载体和宿主细胞。
所述的载体为植物重组表达载体。
所述的植物重组表达载体为PBI121+CfAPX。
所述的宿主细胞为农杆菌GV3101。
所述的柳杉抗寒调控基因CfAPX在提高植物抗逆性中的应用。
所述的应用，基因CfAPX在清除植物体内的过氧化氢，调节活性氧水平从而提高植物抗逆性。
所述的植株为柳杉或拟南芥。
有益效果：本申请通过实验发现CfAPX基因在低温胁迫下柳杉叶片中的基因表达量在-16℃达到峰值。通过构建表达载体PBI121-CfAPX对CfAPX进行功能验证，发现，转基因植株CfAPX基因的表达量上调，与野生型相比均达到了显著或极显著水平。因此，CfAPX基因可以调节植物抗坏血酸过氧化物酶活性，从而提高细胞清除活性氧，维持稳态的能力，进一步提高植株的抗逆性。本申请提供的柳杉CfAPX基因的表达蛋白是抗坏血酸过氧化物酶，其表达受到盐胁迫、干旱胁迫、低温胁迫等多种环境刺激的调节，在逆境条件下，APX的活性明显增强，参与将H2O2催化形成H2O的过程，通过清除活性氧维持细胞氧化还原稳态，提高植物的抗逆性，对植物的生长发育具有重要意义。
附图说明
图1是CfAPX基因在低温胁迫下柳杉叶片中的基因表达量柱状图；
图2是本发明的拟南芥转基因植株和野生型植株4℃处理后表型图；
图3是本发明的CfAPX基因在拟南芥中不同时间的相对表达量柱状图；
图4是低温胁迫下转基因植株和野生型植株Fv/Fm的变化柱状图；
图5是低温胁迫下转基因植株和野生型植株Y(II)的变化柱状图；
图6是低温胁迫下转基因植株和野生型植株MDA含量的变化柱状图；
图7是低温胁迫下转基因植株和野生型植株POD含量的变化柱状图。
具体实施方法
下面结合附图对本发明作进一步详细描述。下述实施案例中所使用的方法如无特别说明均为常规方法，本实施例所有引物合成及测序工作均由南京金斯瑞生物技术公司完成。
实施例1：柳杉CfAPX基因的克隆和表达分析
1、柳杉CfAPX基因的克隆
选取生长良好的柳杉扦插苗，放于培养箱中15℃预冷1d，4℃培养3d后，采取幼嫩叶片，提取总RNA并反转录成cDNA。在NCBI网站中根据柳杉高通量测序结果，建立柳杉本地BLAST数据库，最后以本地BLAST结果得分最高的序列作为APX同源序列设计引物CfAPX-F和CfAPX-R，以cDNA为模板进行PCR扩增，PCR产物回收后连接载体PMD19-T。该特异性扩增引物CfAPX-F和CfAPX-R的序列为：
CfAPX-F：5′-ATCCTGCGGAATTCTATCGAG-3′，
CfAPX-R：5′-CTTAGAAATTTAGGTAGCGACT-3′。
以反转录合成的cDNA为模板，利用引物CfAPX-F和CfAPX-R进行扩增。反应体系如下(50μl)：2μlcDNAtemplate，32.5μldH2O，8μldNTPMixture，5μl10×LAPCRBuffer(Mg2++)，1μlForwardPrimer(10μM)，1μlReversePrimer(10μM)，0.5μlLATaq。扩增反应程序为：94℃4min，94℃30s，56℃1min，72℃2min，35个循环，72℃10min，4℃保持。
PCR产物经1.5％琼脂糖凝胶电泳检测后，对目的片段进行回收，并连接到载体PMD19-T上，然后转移到大肠杆菌感受态细胞，经阳性克隆检测后送至南京金斯瑞生物公司测序。
将测序得到的柳杉APX基因中间片段，3′、5′Race片段用BioXM2.6软件拼接，得到CfAPX基因全长，用NCBI网站BLAST在线工具进行比对，以确保序列的完整正确。结果表明：CfAPX基因全长有1226bp，其核苷酸序列如下：
序列中：下划线为引物序列，加粗、斜体与下划线部分为起始、终止密码子，具体序列见SEQIDNO.1所示，其中有750个碱基编码249个氨基酸(SEQIDNO.2)。
2、柳杉CfAPX基因实时荧光定量表达分析
柳杉选择GAPDH为内参基因，实时定量引物如下：
RT-APX-F：5′-ACTACAGATAGCAAAAGCATCG-3′
RT-APX-R：5′-TTCTTGTATTCCTCGCTCACTT-3′
实时荧光定量PCR反应体系(20μL)：10.0μl2×ChamQSYBRqPCRMasterMix(LowROXPremixed)，1μlPrimer-F(10μM)，1μlPrimer-R(10μM)，2μlTemplateDNA/cDNA，6μlddH2O。扩增程序为：95℃20s，95℃10s，95℃10s，60℃34s，40个循环；95℃15s，60℃60s，95℃15s，4℃保持。
荧光定量结果(图1)显示：柳杉CfAPX基因的表达量在低温胁迫下，随着温度的降低呈现先升高后下降的趋势。在-16℃达到峰值，-20℃相较于-16℃表现略有下降，即-16℃时柳杉CfAPX基因表达量最高。
3、PBI121表达载体和宿主细胞的构建
根据已扩增得到的CfAPX基因编码区利用Oligo7设计引物，以柳杉cDNA为模板，利用引物进行编码区扩增，同时将PBI121表达载体也用BamHI酶、XbaI酶进行双酶切，再用T4连接酶将两个酶切产物连接、转化、挑取单克隆测序，将测序结果正确的菌液提取重组表达载体质粒。
将构建好的表达载体PBI121+CfAPX转化到农杆菌GV3101感受态细胞中，挑取单克隆进行培养，使用PCR检测阳性菌落，获得宿主细胞。
实施例2：拟南芥的遗传转化、筛选和鉴定
将生长良好的哥伦比亚野生型拟南芥的顶生花序放入农杆菌GV3101浸染液中进行浸染，重复浸染直至花期结束，继续培养收获转基因T1代拟南芥种子。
将T1代拟南芥种子点播到含有卡那霉素的1/2MS培养基中，叶片绿色、生长健壮的幼苗为转基因拟南芥，未转基因的拟南芥会逐渐黄化死亡，继续培养收获拟南芥T2代种子，培养的植株为T2代转基因植株。
待拟南芥植株长到10片叶子左右时，收集转基因植株叶片，提取DNA并进行PCR检测。
实施例3：转基因拟南芥的功能验证
1、拟南芥表型观察
将CfAPX转基因拟南芥和野生型拟南芥幼苗分别放入4℃培养箱中培养两周(图2)，观察发现转基因植株与对照植株相比，其叶片绿色变深，而野生型拟南芥叶片发黄变红，其叶绿素合成受到了破坏。
2、转基因拟南芥基因表达量分析
将CfAPX转基因拟南芥放入4℃处理，分别于0、4、8、12h取叶片提取RNA，保证反转录前每个处理RNA的总量相同，进行实时荧光定量，拟南芥内参采用Actin基因。引物设计如下：
RT-APX-F：5′-ACTACAGATAGCAAAAGCATCG-3′
RT-APX-R：5′-TTCTTGTATTCCTCGCTCACTT-3′
Actin-F：5′-AATTACCCGATGGGCA-3′
Acfin-R：5′-TCATACTCGGCCTTGGA-3′
采用SYBRGreenI荧光染料法进行qRT-PCR定量分析，所加试剂如下(20μl)：10μl2×SYBRPremixExTaq，5.2μldH2O，0.4μlForwardPrimer，0.4μlReversePrimer，4μlcDNA。
CfAPX转基因拟南芥基因表达量分析如图3所示，结果显示：CfAPX基因的表达量在处理的0～8h内随着时间的增加而呈现出上调的状态，但当处理时间达到12h，相对基因表达量上升缓慢，但转基因植株CfAPX基因的表达量上调，与野生型相比均达到了显著或极显著水平。
3、低温胁迫对转基因拟南芥和野生型拟南芥叶绿素荧光的影响
如图4、图5所示，低温处理后，野生型植株和转基因拟南芥植株叶绿素荧光参数Fv/Fm和Y(II)均显著降低。在4℃时随着处理时间的延长，Fv/Fm和Y(II)逐渐降低且转基因植株均低于野生型，在-6℃时转基因拟南芥植株Fv/Fm高于野生型，转基因植株和野生型植株在Y(II)参数上4℃时间延时(4h，12h，24h)及-6℃(8h)均表现出显著差异，说明CfAPX基因在低温下发挥作用，抵御逆境胁迫，使拟南芥抗寒性增强。
4、低温胁迫对转基因拟南芥和野生型拟南芥丙二醛(MDA)含量的影响
如图6所示，随着温度的降低，转基因植株和野生型植株丙二醛含量显著增加。4℃时两者MDA含量逐渐增加，野生型植株变化均高于转基因植株，从4℃到-6℃，MDA含量迅速积累，-6℃时转基因植株含量低于野生型，说明在低温胁迫下，野生型拟南芥体内MDA含量的积累大于转基因拟南芥，野生型植株受伤害的程度更大，抗寒性较弱。
5、低温胁迫对转基因拟南芥和野生型拟南芥过氧化物酶(POD)含量的影响
图7显示在低温胁迫下，POD含量呈现逐渐增加的趋势。在-6℃处理时POD含量变化显著，转基因植株POD含量大于野生型，随着4℃处理时间的增加，转基因植株和野生型植株体内POD含量逐渐积累，总体来说在低温胁迫下转基因拟南芥POD含量大于野生型，酶活性较高，能有效的清除细胞内积累的活性氧，维持细胞稳态，提高抗逆性。
序列表
<110> 南京林业大学
<120> 一种柳杉抗寒调控基因CfAPX及其应用
<130> 100
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1309
<212> DNA
<213> Cryptomeria fortunei
<400> 1
ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagtacatg gggactacag 60
atagcaaaag catcgttgat cctgcggaat tctatcgagg cgaagtaaag agagcaatca 120
tggtgaaggt atacccacaa gtgagcgagg aatacaagaa ggctattgac aaatgcagga 180
ggaagctgcg agctctcatt gctgagaaaa actgtgctcc tattatggtt cgtctcgcat 240
ggcatgctgc tgggacttat gatgtaaaga gcaagactgg agggcctttt ggaactatta 300
gacatccttc tgagttggct catggggcta acagtggctt ggatatcgcc attaagttgc 360
ttgagcctat taaggcacag ttccccattg tcacttatgc tgatctctat gagttggctg 420
gagttgtggc tgttgaggtc actggtggac ctgatattcc attccaccct ggcagagaag 480
acaagcctga gccaccagaa gagggacggt tgcccgatgc caccaaagga gctgatcatt 540
tgagagatgt cttcggtcac atgggattga gtgaccagga cattgtggct ttgtctggtg 600
ctcacacttt gggaagttgc cacaaggaga gatctggatt tgaaggtcca tggacgtcta 660
acccgctcat ctttgacaac tcctatttca cagaacttgt cactggagag aaggaaggtc 720
tgcttcaatt gccttctgac aaggctttac taactgatcc taagtttgca ccccttgtac 780
acaagtatgc tcaggatgaa gatgcttttt ttgctgacta tgctgaggct catttgaaac 840
tttctgaact tgggtttgct gaagcataga tacttaattt gggaaggact ctgtctctgt 900
tcaccattcc aagaaactac tatcatatag tgaacagaga atgtcatttt ggttcaccgt 960
ttcgatcagc agaatcgtat catttcgatt tttcgtaatt gtctgttttg tggattgata 1020
gttaatgtaa aacgtgtaca tttttggagc cttttatcta tataagtgac ttagtcgcta 1080
cctaaatttc taagattgaa ataaaatgtg ggtcttgact ttgtgaaagc acttagaagt 1140
taaaacattt ccattttttt taagaaatac ctacccaagg gttaagtttt ccacactttt 1200
taatggaaat gcaaatcaaa actttgtaat ggaatgttga aaaaaaaaaa aaaaaaaaaa 1260
aaaaaaaaag tactctgcgt tgataccact gcttgcccta tagtgagtc 1309
<210> 2
<211> 249
<212> PRT
<213> Cryptomeria fortunei
<400> 2
Met Val Lys Val Tyr Pro Gln Val Ser Glu Glu Tyr Lys Lys Ala Ile
1 5 10 15
Asp Lys Cys Arg Arg Lys Leu Arg Ala Leu Ile Ala Glu Lys Asn Cys
20 25 30
Ala Pro Ile Met Val Arg Leu Ala Trp His Ala Ala Gly Thr Tyr Asp
35 40 45
Val Lys Ser Lys Thr Gly Gly Pro Phe Gly Thr Ile Arg His Pro Ser
50 55 60
Glu Leu Ala His Gly Ala Asn Ser Gly Leu Asp Ile Ala Ile Lys Leu
65 70 75 80
Leu Glu Pro Ile Lys Ala Gln Phe Pro Ile Val Thr Tyr Ala Asp Leu
85 90 95
Tyr Glu Leu Ala Gly Val Val Ala Val Glu Val Thr Gly Gly Pro Asp
100 105 110
Ile Pro Phe His Pro Gly Arg Glu Asp Lys Pro Glu Pro Pro Glu Glu
115 120 125
Gly Arg Leu Pro Asp Ala Thr Lys Gly Ala Asp His Leu Arg Asp Val
130 135 140
Phe Gly His Met Gly Leu Ser Asp Gln Asp Ile Val Ala Leu Ser Gly
145 150 155 160
Ala His Thr Leu Gly Ser Cys His Lys Glu Arg Ser Gly Phe Glu Gly
165 170 175
Pro Trp Thr Ser Asn Pro Leu Ile Phe Asp Asn Ser Tyr Phe Thr Glu
180 185 190
Leu Val Thr Gly Glu Lys Glu Gly Leu Leu Gln Leu Pro Ser Asp Lys
195 200 205
Ala Leu Leu Thr Asp Pro Lys Phe Ala Pro Leu Val His Lys Tyr Ala
210 215 220
Gln Asp Glu Asp Ala Phe Phe Ala Asp Tyr Ala Glu Ala His Leu Lys
225 230 235 240
Leu Ser Glu Leu Gly Phe Ala Glu Ala
245
<210> 3
<211> 21
<212> DNA
<213> CfAPX-F引物序列(Artificial Sequence)
<400> 3
atcctgcgga attctatcga g 21
<210> 4
<211> 22
<212> DNA
<213> CfAPX-R引物序列(Artificial Sequence)
<400> 4
cttagaaatt taggtagcga ct 22
<210> 5
<211> 22
<212> DNA
<213> RT-APX-F引物序列(Artificial Sequence)
<400> 5
actacagata gcaaaagcat cg 22
<210> 6
<211> 22
<212> DNA
<213> RT-APX-R引物序列(Artificial Sequence)
<400> 6
ttcttgtatt cctcgctcac tt 22
<210> 7
<211> 22
<212> DNA
<213> RT-APX-F引物序列(Artificial Sequence)
<400> 7
actacagata gcaaaagcat cg 22
<210> 8
<211> 22
<212> DNA
<213> RT-APX-R引物序列(Artificial Sequence)
<400> 8
ttcttgtatt cctcgctcac tt 22
<210> 9
<211> 16
<212> DNA
<213> Actin-F引物序列(Artificial Sequence)
<400> 9
aattacccga tgggca 16
<210> 10
<211> 17
<212> DNA
<213> Actin-R引物序列(Artificial Sequence)
<400> 10
tcatactcgg ccttgga 17
Claims (9)
1.一种柳杉抗寒调控基因CfAPX，其核苷酸序列如SEQIDNO.1所示。
2.权利要求1所述的柳杉CfAPX基因的编码蛋白，其氨基酸序列如SEQIDNO.2所示。
3.含有权利要求1所述的柳杉CfAPX基因的载体和宿主细胞。
4.根据权利要求3所述的载体，其特征在于，所述的载体为植物重组表达载体。
5.根据权利要求4所述的载体，其特征在于，所述的植物重组表达载体为PBI121+CfAPX。
6.根据权利要求3所述的宿主细胞，其特征在于，所述的宿主细胞为农杆菌GV3101。
7.权利要求1所述的柳杉抗寒调控基因CfAPX在提高植物抗逆性中的应用。
8.权利要求7所述的应用，其特征在于，基因CfAPX在清除植物体内的过氧化氢，调节活性氧水平从而提高植物抗逆性。
9.权利要求7或8所述的应用，其特征在于，所述的植株为柳杉或拟南芥。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011266568.XA CN112501142A (zh) | 2020-11-12 | 2020-11-12 | 一种柳杉抗寒调控基因CfAPX及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011266568.XA CN112501142A (zh) | 2020-11-12 | 2020-11-12 | 一种柳杉抗寒调控基因CfAPX及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112501142A true CN112501142A (zh) | 2021-03-16 |
Family
ID=74957397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011266568.XA Pending CN112501142A (zh) | 2020-11-12 | 2020-11-12 | 一种柳杉抗寒调控基因CfAPX及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112501142A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763683A (zh) * | 2020-06-30 | 2020-10-13 | 南京林业大学 | 一种柳杉CfICE1基因及其应用 |
-
2020
- 2020-11-12 CN CN202011266568.XA patent/CN112501142A/zh active Pending
Non-Patent Citations (1)
| Title |
|---|
| GENBANK: AB254809.1: "Cryptomeria japonica mRNA for putative ascorbate peroxidase, complete cds", 《NCBI》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763683A (zh) * | 2020-06-30 | 2020-10-13 | 南京林业大学 | 一种柳杉CfICE1基因及其应用 |
| CN111763683B (zh) * | 2020-06-30 | 2022-06-03 | 南京林业大学 | 一种柳杉CfICE1基因及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111560058B (zh) | 枳抗寒基因PtrMYC2及其在植物抗寒遗传改良中的应用 | |
| CN112608928B (zh) | 一种龙眼单果重性状调控基因DlCNR8及其蛋白与应用 | |
| CN113717983A (zh) | 龙眼基因DlGRAS34、蛋白及其在调控植株开花中的应用 | |
| CN108341859B (zh) | 植物抗旱、耐盐和提高小麦产量相关蛋白及其基因和应用 | |
| LU504522B1 (en) | Gene related to low potassium stress of tobacco, promoter and application thereof | |
| CN111979253B (zh) | TrFQR1基因及其克隆、表达载体构建方法和应用 | |
| CN113249388A (zh) | 一种假俭草EoPHR2基因及其表达蛋白和应用 | |
| CN112501142A (zh) | 一种柳杉抗寒调控基因CfAPX及其应用 | |
| CN109867716B (zh) | 蜡梅CpVIN3基因及其应用 | |
| CN101050462B (zh) | 来源于拟南芥的缺磷诱导基因及其编码蛋白与应用 | |
| CN114292856B (zh) | 一种调控胡杨耐盐性的基因PeCLH2及其应用 | |
| CN111454340B (zh) | 长穗偃麦草外整流钾通道蛋白及其编码基因与应用 | |
| CN111499708B (zh) | 葡萄VabHLH036基因在提高植物抗寒能力中的应用 | |
| CN115838734A (zh) | C2h2型锌指蛋白基因hstl在调控水稻耐盐性中的应用 | |
| CN111676227B (zh) | 大豆核糖体蛋白编码基因GmRPL12的基因工程应用 | |
| CN108948162B (zh) | 一种花生逆境胁迫基因AhDOG1L及其应用 | |
| CN114181946B (zh) | 与植物耐低氮胁迫相关的基因、启动子及其应用 | |
| CN113234731A (zh) | 编码大豆ARF转录因子的GmARF16基因及应用 | |
| CN108410829B (zh) | 蒙古沙冬青叶绿体ω-3脂肪酸脱饱和酶AmFAD7编码基因与应用 | |
| CN111423500A (zh) | SiMYB56蛋白及其编码基因在调控植物耐干旱能力中的应用 | |
| CN114605514B (zh) | 蛋白质VvANN1在提高植物抗旱性中的应用 | |
| CN116732048B (zh) | 水稻转录因子基因OsbZIP48在获得高锌水稻籽粒和/或调节氮素吸收中的应用 | |
| CN114524865B (zh) | 一种提高玉米开花期抗旱性的dna分子及其相关生物材料与应用 | |
| CN112029745B (zh) | 植物tmk家族基因及其抗非生物胁迫功能的应用 | |
| CN114196679B (zh) | 铜离子转运蛋白基因OsCOPT7在水稻选育中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210316 |
|
| RJ01 | Rejection of invention patent application after publication |