CN113881685B - 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 - Google Patents
促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 Download PDFInfo
- Publication number
- CN113881685B CN113881685B CN202111177712.7A CN202111177712A CN113881685B CN 113881685 B CN113881685 B CN 113881685B CN 202111177712 A CN202111177712 A CN 202111177712A CN 113881685 B CN113881685 B CN 113881685B
- Authority
- CN
- China
- Prior art keywords
- like1
- pphsp20
- gene
- red
- peach
- 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.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 80
- 210000000056 organ Anatomy 0.000 title claims abstract description 17
- 230000001737 promoting effect Effects 0.000 title claims abstract description 14
- 235000006040 Prunus persica var persica Nutrition 0.000 claims abstract description 68
- 244000144730 Amygdalus persica Species 0.000 claims abstract description 56
- 241000196324 Embryophyta Species 0.000 claims abstract description 51
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 36
- 241001164374 Calyx Species 0.000 claims abstract description 16
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 230000009261 transgenic effect Effects 0.000 claims description 29
- 239000013598 vector Substances 0.000 claims description 27
- 241000219194 Arabidopsis Species 0.000 claims description 24
- 239000013612 plasmid Substances 0.000 claims description 21
- 239000013604 expression vector Substances 0.000 claims description 16
- 241000286209 Phasianidae Species 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 239000002299 complementary DNA Substances 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 7
- 241000589158 Agrobacterium Species 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 108700026244 Open Reading Frames Proteins 0.000 claims description 2
- 229930002877 anthocyanin Natural products 0.000 abstract description 22
- 235000010208 anthocyanin Nutrition 0.000 abstract description 22
- 239000004410 anthocyanin Substances 0.000 abstract description 22
- 150000004636 anthocyanins Chemical class 0.000 abstract description 22
- 241000219195 Arabidopsis thaliana Species 0.000 abstract description 18
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 238000009395 breeding Methods 0.000 abstract description 12
- 230000001488 breeding effect Effects 0.000 abstract description 12
- 230000009466 transformation Effects 0.000 abstract description 11
- 230000015572 biosynthetic process Effects 0.000 abstract description 10
- 108010004889 Heat-Shock Proteins Proteins 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 16
- 240000005809 Prunus persica Species 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 235000020989 red meat Nutrition 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 238000011144 upstream manufacturing Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 101710100489 Heat shock protein beta-6 Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000003259 recombinant expression Methods 0.000 description 6
- 102100039170 Heat shock protein beta-6 Human genes 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- YTMNONATNXDQJF-UBNZBFALSA-N chrysanthemin Chemical compound [Cl-].O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 YTMNONATNXDQJF-UBNZBFALSA-N 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 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 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000001131 transforming effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000207746 Nicotiana benthamiana Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000006870 ms-medium Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 3
- 244000020518 Carthamus tinctorius Species 0.000 description 3
- USNPULRDBDVJAO-YRBSALHSSA-O Cyanidin 3-rutinoside Natural products O(C[C@@H]1[C@@H](O)[C@@H](O)[C@@H](O)[C@H](Oc2c(-c3cc(O)c(O)cc3)[o+]c3c(c(O)cc(O)c3)c2)O1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](C)O1 USNPULRDBDVJAO-YRBSALHSSA-O 0.000 description 3
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- USNPULRDBDVJAO-FXCAAIILSA-O cyanidin 3-O-rutinoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC=2C(=[O+]C3=CC(O)=CC(O)=C3C=2)C=2C=C(O)C(O)=CC=2)O1 USNPULRDBDVJAO-FXCAAIILSA-O 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 229960001331 keracyanin Drugs 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 230000004960 subcellular localization Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 2
- 102000005431 Molecular Chaperones Human genes 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000003208 gene overexpression Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 102200111112 rs397514590 Human genes 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- BRPMXFSTKXXNHF-IUCAKERBSA-N (2s)-1-[2-[[(2s)-pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H]1NCCC1 BRPMXFSTKXXNHF-IUCAKERBSA-N 0.000 description 1
- 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
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- KYDYGANDJHFBCW-DRZSPHRISA-N Ala-Phe-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N KYDYGANDJHFBCW-DRZSPHRISA-N 0.000 description 1
- VQAVBBCZFQAAED-FXQIFTODSA-N Ala-Pro-Asn Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)N)C(=O)O)N VQAVBBCZFQAAED-FXQIFTODSA-N 0.000 description 1
- UCDOXFBTMLKASE-HERUPUMHSA-N Ala-Ser-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N UCDOXFBTMLKASE-HERUPUMHSA-N 0.000 description 1
- 102100028292 Aladin Human genes 0.000 description 1
- 101710065039 Aladin Proteins 0.000 description 1
- 235000011446 Amygdalus persica Nutrition 0.000 description 1
- 101100080548 Arabidopsis thaliana NRPB1 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
- ITVINTQUZMQWJR-QXEWZRGKSA-N Arg-Asn-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ITVINTQUZMQWJR-QXEWZRGKSA-N 0.000 description 1
- UBCPNBUIQNMDNH-NAKRPEOUSA-N Arg-Ile-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O UBCPNBUIQNMDNH-NAKRPEOUSA-N 0.000 description 1
- IIAXFBUTKIDDIP-ULQDDVLXSA-N Arg-Leu-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IIAXFBUTKIDDIP-ULQDDVLXSA-N 0.000 description 1
- FSNVAJOPUDVQAR-AVGNSLFASA-N Arg-Lys-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FSNVAJOPUDVQAR-AVGNSLFASA-N 0.000 description 1
- CTAPSNCVKPOOSM-KKUMJFAQSA-N Arg-Tyr-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O CTAPSNCVKPOOSM-KKUMJFAQSA-N 0.000 description 1
- LUVODTFFSXVOAG-ACZMJKKPSA-N Asn-Cys-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N LUVODTFFSXVOAG-ACZMJKKPSA-N 0.000 description 1
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 1
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 1
- NJSNXIOKBHPFMB-GMOBBJLQSA-N Asn-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N)N NJSNXIOKBHPFMB-GMOBBJLQSA-N 0.000 description 1
- GWTLRDMPMJCNMH-WHFBIAKZSA-N Asp-Asn-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GWTLRDMPMJCNMH-WHFBIAKZSA-N 0.000 description 1
- UGKZHCBLMLSANF-CIUDSAMLSA-N Asp-Asn-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UGKZHCBLMLSANF-CIUDSAMLSA-N 0.000 description 1
- PSLSTUMPZILTAH-BYULHYEWSA-N Asp-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PSLSTUMPZILTAH-BYULHYEWSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- OEDJQRXNDRUGEU-SRVKXCTJSA-N Asp-Leu-His Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O OEDJQRXNDRUGEU-SRVKXCTJSA-N 0.000 description 1
- BKOIIURTQAJHAT-GUBZILKMSA-N Asp-Pro-Pro Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 BKOIIURTQAJHAT-GUBZILKMSA-N 0.000 description 1
- DINOVZWPTMGSRF-QXEWZRGKSA-N Asp-Pro-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O DINOVZWPTMGSRF-QXEWZRGKSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- SFUUYRSAJPWTGO-SRVKXCTJSA-N Cys-Asn-Phe Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SFUUYRSAJPWTGO-SRVKXCTJSA-N 0.000 description 1
- SDWZYDDNSMPBRM-AVGNSLFASA-N Cys-Gln-Phe Chemical compound SC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SDWZYDDNSMPBRM-AVGNSLFASA-N 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- ORYMMTRPKVTGSJ-XVKPBYJWSA-N Gln-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O ORYMMTRPKVTGSJ-XVKPBYJWSA-N 0.000 description 1
- SHAUZYVSXAMYAZ-JYJNAYRXSA-N Gln-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N SHAUZYVSXAMYAZ-JYJNAYRXSA-N 0.000 description 1
- RLZBLVSJDFHDBL-KBIXCLLPSA-N Glu-Ala-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RLZBLVSJDFHDBL-KBIXCLLPSA-N 0.000 description 1
- KVBPDJIFRQUQFY-ACZMJKKPSA-N Glu-Cys-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O KVBPDJIFRQUQFY-ACZMJKKPSA-N 0.000 description 1
- UMIRPYLZFKOEOH-YVNDNENWSA-N Glu-Gln-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UMIRPYLZFKOEOH-YVNDNENWSA-N 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- HILMIYALTUQTRC-XVKPBYJWSA-N Glu-Gly-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HILMIYALTUQTRC-XVKPBYJWSA-N 0.000 description 1
- CBEUFCJRFNZMCU-SRVKXCTJSA-N Glu-Met-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O CBEUFCJRFNZMCU-SRVKXCTJSA-N 0.000 description 1
- YTRBQAQSUDSIQE-FHWLQOOXSA-N Glu-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 YTRBQAQSUDSIQE-FHWLQOOXSA-N 0.000 description 1
- QOXDAWODGSIDDI-GUBZILKMSA-N Glu-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N QOXDAWODGSIDDI-GUBZILKMSA-N 0.000 description 1
- LURCIJSJAKFCRO-QWRGUYRKSA-N Gly-Asn-Tyr Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LURCIJSJAKFCRO-QWRGUYRKSA-N 0.000 description 1
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 1
- GJHWILMUOANXTG-WPRPVWTQSA-N Gly-Val-Arg Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GJHWILMUOANXTG-WPRPVWTQSA-N 0.000 description 1
- SBVMXEZQJVUARN-XPUUQOCRSA-N Gly-Val-Ser Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O SBVMXEZQJVUARN-XPUUQOCRSA-N 0.000 description 1
- 101150051208 HSPH1 gene Proteins 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 102100031624 Heat shock protein 105 kDa Human genes 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 1
- BCZFOHDMCDXPDA-BZSNNMDCSA-N His-Lys-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CN=CN2)N)O BCZFOHDMCDXPDA-BZSNNMDCSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- QYZYJFXHXYUZMZ-UGYAYLCHSA-N Ile-Asn-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N QYZYJFXHXYUZMZ-UGYAYLCHSA-N 0.000 description 1
- SPQWWEZBHXHUJN-KBIXCLLPSA-N Ile-Glu-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O SPQWWEZBHXHUJN-KBIXCLLPSA-N 0.000 description 1
- CMNMPCTVCWWYHY-MXAVVETBSA-N Ile-His-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(C)C)C(=O)O)N CMNMPCTVCWWYHY-MXAVVETBSA-N 0.000 description 1
- CSQNHSGHAPRGPQ-YTFOTSKYSA-N Ile-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)O)N CSQNHSGHAPRGPQ-YTFOTSKYSA-N 0.000 description 1
- HXIDVIFHRYRXLZ-NAKRPEOUSA-N Ile-Ser-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)O)N HXIDVIFHRYRXLZ-NAKRPEOUSA-N 0.000 description 1
- NJGXXYLPDMMFJB-XUXIUFHCSA-N Ile-Val-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N NJGXXYLPDMMFJB-XUXIUFHCSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 1
- VCSBGUACOYUIGD-CIUDSAMLSA-N Leu-Asn-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O VCSBGUACOYUIGD-CIUDSAMLSA-N 0.000 description 1
- HUEBCHPSXSQUGN-GARJFASQSA-N Leu-Cys-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N HUEBCHPSXSQUGN-GARJFASQSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- NEEOBPIXKWSBRF-IUCAKERBSA-N Leu-Glu-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O NEEOBPIXKWSBRF-IUCAKERBSA-N 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- LVTJJOJKDCVZGP-QWRGUYRKSA-N Leu-Lys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LVTJJOJKDCVZGP-QWRGUYRKSA-N 0.000 description 1
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 1
- YIRIDPUGZKHMHT-ACRUOGEOSA-N Leu-Tyr-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YIRIDPUGZKHMHT-ACRUOGEOSA-N 0.000 description 1
- FZIJIFCXUCZHOL-CIUDSAMLSA-N Lys-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN FZIJIFCXUCZHOL-CIUDSAMLSA-N 0.000 description 1
- QIJVAFLRMVBHMU-KKUMJFAQSA-N Lys-Asp-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QIJVAFLRMVBHMU-KKUMJFAQSA-N 0.000 description 1
- LLSUNJYOSCOOEB-GUBZILKMSA-N Lys-Glu-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O LLSUNJYOSCOOEB-GUBZILKMSA-N 0.000 description 1
- HAUUXTXKJNVIFY-ONGXEEELSA-N Lys-Gly-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAUUXTXKJNVIFY-ONGXEEELSA-N 0.000 description 1
- OWRUUFUVXFREBD-KKUMJFAQSA-N Lys-His-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O OWRUUFUVXFREBD-KKUMJFAQSA-N 0.000 description 1
- ONPDTSFZAIWMDI-AVGNSLFASA-N Lys-Leu-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ONPDTSFZAIWMDI-AVGNSLFASA-N 0.000 description 1
- JYVCOTWSRGFABJ-DCAQKATOSA-N Lys-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCCN)N JYVCOTWSRGFABJ-DCAQKATOSA-N 0.000 description 1
- AZOFEHCPMBRNFD-BZSNNMDCSA-N Lys-Phe-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 AZOFEHCPMBRNFD-BZSNNMDCSA-N 0.000 description 1
- WGILOYIKJVQUPT-DCAQKATOSA-N Lys-Pro-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WGILOYIKJVQUPT-DCAQKATOSA-N 0.000 description 1
- TVOOGUNBIWAURO-KATARQTJSA-N Lys-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N)O TVOOGUNBIWAURO-KATARQTJSA-N 0.000 description 1
- RQILLQOQXLZTCK-KBPBESRZSA-N Lys-Tyr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O RQILLQOQXLZTCK-KBPBESRZSA-N 0.000 description 1
- MIMXMVDLMDMOJD-BZSNNMDCSA-N Lys-Tyr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O MIMXMVDLMDMOJD-BZSNNMDCSA-N 0.000 description 1
- WINFHLHJTRGLCV-BZSNNMDCSA-N Lys-Tyr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=C(O)C=C1 WINFHLHJTRGLCV-BZSNNMDCSA-N 0.000 description 1
- 101100071630 Mesocentrotus franciscanus HSP110 gene Proteins 0.000 description 1
- MTBVQFFQMXHCPC-CIUDSAMLSA-N Met-Glu-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MTBVQFFQMXHCPC-CIUDSAMLSA-N 0.000 description 1
- HOZNVKDCKZPRER-XUXIUFHCSA-N Met-Lys-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HOZNVKDCKZPRER-XUXIUFHCSA-N 0.000 description 1
- YLDSJJOGQNEQJK-AVGNSLFASA-N Met-Pro-Leu Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O YLDSJJOGQNEQJK-AVGNSLFASA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100451677 Mus musculus Hspa4 gene Proteins 0.000 description 1
- 102100031787 Myosin regulatory light polypeptide 9 Human genes 0.000 description 1
- 101710107065 Myosin regulatory light polypeptide 9 Proteins 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- IILUKIJNFMUBNF-IHRRRGAJSA-N Phe-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O IILUKIJNFMUBNF-IHRRRGAJSA-N 0.000 description 1
- YEEFZOKPYOUXMX-KKUMJFAQSA-N Phe-Gln-Met Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O YEEFZOKPYOUXMX-KKUMJFAQSA-N 0.000 description 1
- NPLGQVKZFGJWAI-QWHCGFSZSA-N Phe-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O NPLGQVKZFGJWAI-QWHCGFSZSA-N 0.000 description 1
- GYEPCBNTTRORKW-PCBIJLKTSA-N Phe-Ile-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O GYEPCBNTTRORKW-PCBIJLKTSA-N 0.000 description 1
- WWPAHTZOWURIMR-ULQDDVLXSA-N Phe-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=CC=C1 WWPAHTZOWURIMR-ULQDDVLXSA-N 0.000 description 1
- ILGCZYGFYQLSDZ-KKUMJFAQSA-N Phe-Ser-His Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O ILGCZYGFYQLSDZ-KKUMJFAQSA-N 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- GNZCMRRSXOBHLC-JYJNAYRXSA-N Phe-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N GNZCMRRSXOBHLC-JYJNAYRXSA-N 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 101100306008 Plasmodium falciparum (isolate CDC / Honduras) RPII gene Proteins 0.000 description 1
- CQZNGNCAIXMAIQ-UBHSHLNASA-N Pro-Ala-Phe Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O CQZNGNCAIXMAIQ-UBHSHLNASA-N 0.000 description 1
- AMBLXEMWFARNNQ-DCAQKATOSA-N Pro-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 AMBLXEMWFARNNQ-DCAQKATOSA-N 0.000 description 1
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-N 0.000 description 1
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 1
- SXJOPONICMGFCR-DCAQKATOSA-N Pro-Ser-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O SXJOPONICMGFCR-DCAQKATOSA-N 0.000 description 1
- OABLKWMLPUGEQK-JYJNAYRXSA-N Pro-Tyr-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(O)=O OABLKWMLPUGEQK-JYJNAYRXSA-N 0.000 description 1
- 108010003201 RGH 0205 Proteins 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- UCOYFSCEIWQYNL-FXQIFTODSA-N Ser-Cys-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(O)=O UCOYFSCEIWQYNL-FXQIFTODSA-N 0.000 description 1
- PVDTYLHUWAEYGY-CIUDSAMLSA-N Ser-Glu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PVDTYLHUWAEYGY-CIUDSAMLSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-N 0.000 description 1
- FZNNGIHSIPKFRE-QEJZJMRPSA-N Ser-Trp-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZNNGIHSIPKFRE-QEJZJMRPSA-N 0.000 description 1
- HAYADTTXNZFUDM-IHRRRGAJSA-N Ser-Tyr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HAYADTTXNZFUDM-IHRRRGAJSA-N 0.000 description 1
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 102000008063 Small Heat-Shock Proteins Human genes 0.000 description 1
- 108010088928 Small Heat-Shock Proteins Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- MQCPGOZXFSYJPS-KZVJFYERSA-N Thr-Ala-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MQCPGOZXFSYJPS-KZVJFYERSA-N 0.000 description 1
- VASYSJHSMSBTDU-LKXGYXEUSA-N Thr-Asn-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)O VASYSJHSMSBTDU-LKXGYXEUSA-N 0.000 description 1
- JBHMLZSKIXMVFS-XVSYOHENSA-N Thr-Asn-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JBHMLZSKIXMVFS-XVSYOHENSA-N 0.000 description 1
- ZSPQUTWLWGWTPS-HJGDQZAQSA-N Thr-Lys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZSPQUTWLWGWTPS-HJGDQZAQSA-N 0.000 description 1
- JWQNAFHCXKVZKZ-UVOCVTCTSA-N Thr-Lys-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JWQNAFHCXKVZKZ-UVOCVTCTSA-N 0.000 description 1
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- ICPRIGUXAFULPH-ILWGZMRPSA-N Trp-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)N)C(=O)O ICPRIGUXAFULPH-ILWGZMRPSA-N 0.000 description 1
- KHCSOLAHNLOXJR-BZSNNMDCSA-N Tyr-Leu-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHCSOLAHNLOXJR-BZSNNMDCSA-N 0.000 description 1
- TYGHOWWWMTWVKM-HJOGWXRNSA-N Tyr-Tyr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 TYGHOWWWMTWVKM-HJOGWXRNSA-N 0.000 description 1
- IDKGBVZGNTYYCC-QXEWZRGKSA-N Val-Asn-Pro Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(O)=O IDKGBVZGNTYYCC-QXEWZRGKSA-N 0.000 description 1
- XLDYBRXERHITNH-QSFUFRPTSA-N Val-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)C(C)C XLDYBRXERHITNH-QSFUFRPTSA-N 0.000 description 1
- DDNIHOWRDOXXPF-NGZCFLSTSA-N Val-Asp-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DDNIHOWRDOXXPF-NGZCFLSTSA-N 0.000 description 1
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 1
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- PDDJTOSAVNRJRH-UNQGMJICSA-N Val-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](C(C)C)N)O PDDJTOSAVNRJRH-UNQGMJICSA-N 0.000 description 1
- JAIZPWVHPQRYOU-ZJDVBMNYSA-N Val-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O JAIZPWVHPQRYOU-ZJDVBMNYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000004640 cellular pathway Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000004292 cytoskeleton Anatomy 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
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010025153 lysyl-alanyl-alanine Proteins 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000010839 reverse transcription Methods 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
- 229960004555 rutoside Drugs 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 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 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 108010077037 tyrosyl-tyrosyl-phenylalanine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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
- C12N15/825—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 involving pigment biosynthesis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Nutrition Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种促进植物器官产生红色色泽的PpHSP20‑like1基因及其应用,该基因核苷酸序列为SEQ ID NO.1,编码的蛋白氨基酸序列为SEQ ID NO.2。本发明的PpHSP20‑like1基因是一个热激蛋白基因,完整编码框长度为1179个碱基,编码蛋白含393个氨基酸。PpHSP20‑like1异源转化拟南芥使叶片和花萼变红,花色苷含量从无到有;同源瞬时转化白肉桃果实,果肉变红,说明本发明首次克隆的PpHSP20‑like1基因与其编码的蛋白促进如果实红肉色泽形成,结果对于丰富红肉色泽成因基础理论、开发分子标记、改良现有红肉桃缺陷、提高优质红肉桃育种效率等实际应用具有重要意义。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及促进植物器官产生红色色泽的PpHSP20-like1基因及其应用。
背景技术
果肉颜色是果树果实的一个重要农艺性状,是评价果实品质的关键指标之一。桃(Prunus persica)原产中国,分布广泛,种质资源丰富。桃果肉颜色有白、黄、绿、红、紫五种。红肉桃是桃亚属植物中的特异资源,与非红肉桃相比,红肉桃不仅具有特殊的视觉冲击力和感官吸引力,而且其富含的花色苷和酚类物质具有抗氧化、抗衰老、预防心脑血管疾病等对人体健康非常有益的功能。随着人们对果品多样化、功能化需求的不断提高,红肉桃的营养价值日益受到重视,对优质红肉桃的需求不断上升。但是现有红肉桃资源及品种普遍存在风味偏酸、果形偏小等缺陷,亟待选育优质红肉桃品种。当前,桃红肉色泽形成的分子机制虽然在个别红肉桃种质资源中有了一定进展,但是红肉桃种质资源多样、遗传规律复杂、一些种质间存在遗传背景差异,其余红肉桃种质资源的红肉色泽形成分子机制仍不清楚,在一定程度上制约了利用现代分子标记技术提高优质红肉桃育种效率。
热激蛋白(heat shock protein,HSP)普遍存在于植物、动物以及人、果蝇等生物体中。根据分子量大小以及氨基酸序列的同源性,热激蛋白被分成HSP110、HSP90、HSP70、小分子质量HSP(small heat shock protein,缩写为sHSP)等多个家族。因sHSP家族蛋白分子量大多在15~22kDa,故也被称之为HSP20家族蛋白。HSP20家族蛋白的典型特征是有一个保守的α-crystalline结构域(ACD)或具有一个与ACD结构域非常相似的被称为ACD-like或HSP20-like的结构域。也有一些文献把含有HSP20-like结构域的HSP20蛋白分为单独的一类,称为HSP20-like家族。
HSP20蛋白能够阻止蛋白变性,保持细胞正常的生理活性,使细胞免受热胁迫伤害。HSP20蛋白作为分子伴侣,其功能研究多基于草本植物等实验材料,在正常生长条件下和应激条件(高温、寒冷、干旱、病毒侵袭及化学刺激等)下的多种多样的细胞路径中起着复杂且重要的作用,例如细胞增殖、分化、迁移、信号转导、细胞骨架、纤毛动力学、免疫学、病原抵抗等。而在木本植物,尤其桃中的研究罕见。
发明内容
发明目的:针对现有技术中存在的不足,本发明提供一个正调控花色苷合成的PpHSP20-like1基因,其可以促进植物器官红色色泽的形成,如促进植物拟南芥叶和花萼、桃果肉红色色泽的形成。
本发明的另一个目的是提供PpHSP20-like1基因及其编码蛋白序列、过表达载体、宿主菌以及该PpHSP20-like1基因在促进植物器官红色中的应用。本发明构建PpHSP20-like1植物过表达载体,通过蘸花法异源转化拟南芥和同源瞬时转化白肉桃果实进行其生物学功能的验证。表明本发明克隆的PpHSP20-like1基因具有促进如果实红肉色泽形成的功能。
为了实现上述目的,本发明采用以下技术方案:
本发明利用克隆技术从桃地方品种‘野鸡红’克隆得到一个与拟南芥HSP20-like同源的基因PpHSP20-like1。PpHSP20-like1核苷酸序列如SEQ ID NO.1所示,包含1179个碱基,编码393个氨基酸,其氨基酸序列如SEQ ID NO.2所示,分子量为98.24997kDa,等电点为5.07,亚细胞定位在细胞核。
本发明所述的基因PpHSP20-like1在促进植物器官产生红色色泽中的应用。
本发明所述的基因PpHSP20-like1在促进植物拟南芥叶和花萼、桃果肉红色中的应用。
本发明所述含基因PpHSP20-like1的表达载体在促进植物器官产生红色色泽中的应用。
本发明所述的表达载体的构建包括如下步骤:
以红肉桃品种‘野鸡红’的cDNA为模板,设计引物,扩增得PpHSP20-like1基因片段,PCR产物纯化后连接到pENTR/D-TOPO载体,构建的质粒命名为pENTR/D-TOPO:PpHSP20-like1;通过GATEWAY方法用LR酶将pENTR/D-TOPO:PpHSP20-like1连接到R05载体上,构建的质粒命名为R05-PpHSP20-like1。
本发明设计克隆上述基因Pplike1PpHSP20-like1核苷酸开放阅读框序列引物对,其碱基序列如下所示:
P1:5’-CACCATGGAAGATGACCCTCCTAAAATGTCCGTC-3’
P2:5’-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’。
利用含有重组载体R05-PpHSP20-like1的农杆菌异源转化拟南芥和同源转化白肉桃果实,获得拟南芥红叶、红色花萼;桃果实红肉表型,验证其具有促进红色色泽形成的功能。
含有上述基因的重组表达载体、转基因重组菌。
上述的基因或上述的蛋白在提高优质红肉桃育种效率方面的应用。
上述的基因或上述的蛋白在鉴定红肉桃资源、基因分型、分子标记方面的应用。
本发明从红肉桃品种‘野鸡红’果实中克隆得到的功能基因PpHSP20-like1,并实现了其在促进植物器官产生红色色泽中的应用,尤其是促进果肉红色色泽形成方面的应用。申请人利用植物基因克隆技术从‘野鸡红’果实中克隆得到一个新基因PpHSP20-like1。PpHSP20-like1属于HSP20基因家族成员,具有一个与ACD结构域非常相似的被称为ACD-like或HSP20-like的结构域,在果实中表达。将PpHSP20-like1连接到含有GFP标签的R05载体上,并在烟草叶中瞬时转化,结果显示PpHSP20-like1位于细胞核,是一个核蛋白。同时把R05-PpHSP20-like1异源转化拟南芥和同源瞬时转化白肉桃果实,结果显示:阳性株叶片花色苷含量和PpHSP20-like1基因相对表达水平显著高于野生型,阳性株叶片和花萼呈红色,高分辨率质谱(LC-QTOF/MS)分析表明阳性株叶片含有花色苷矢车菊素-3-葡萄糖苷和矢车菊素-3-芸香糖苷;与对照相比,白肉桃注射部位乳白色果肉变成红色。PpHSP20-like1促进红色色泽产生,在植物基因工程以及红肉桃育种的基因工程改良中具有广阔的应用前景。
本发明涉及一个赋予植物器官红色色泽的PpHSP20-like1基因。PpHSP20-like1的功能研究揭示其表达调控机理以及作用机制,可应用于植物基因工程以及红肉桃育种的基因工程改良中,也可以利用本发明的基因构建成各种植物表达载体,应用于农业生物技术育种以提高如桃果果实红肉色泽改良效率。
本发明通过红肉桃地方品种‘野鸡红’品种与多个非红肉桃分别进行杂交、QTL定位、基因组重测序、转录组测定等分析,发现一个果实红肉色泽性状相关基因PpHSP20-like1,该基因与拟南芥HSP20-like chaperones superfamily protein高度同源(登录号AT1G20870.1),E-value是2e-31;与桃‘Lovell’参考基因组序列中的Prupe.5G005900.1(XM_007209161.2)相似度最高,达99.92%,以Prupe.5G005900.1序列为参考,设计引物从红肉桃品种‘野鸡红’果肉中将PpHSP20-like1基因克隆,并将该基因转化拟南芥和同源瞬时转化白肉桃果实,获得的转基因拟南芥植株叶片和花萼均呈现红色、转基因桃果肉红色。桃果肉色泽性状相关基因PpHSP20-like1的分离可以进一步丰富果实品质性状基因资源,对其功能研究为该类基因的进一步利用奠定基础。
有益效果:与现有技术相比,本发明具有的优点和效果:
(1)本发明首次克隆到促进植物器官产生红色色泽的PpHSP20-like1基因该基因可以使得桃果肉红色色泽形成,为提高红肉桃育种效率提供了新的思路,为实现分子育种和节省大量人力、物力、地力等成本提供了基础。
(2)通过异源转化拟南芥和同源瞬时转化白肉桃,获得红肉色泽表型,功能验证结果表明本发明克隆的基因具有促进红色色泽产生的功能。
(3)PpHSP20-like1基因与红肉性状形成相关,将PpHSP20-like1与构建植物表达载体转化拟南芥,结果该基因在转基因植株中过表达后,与野生型和空载体对照株相比,叶片和花萼显现红色;转化白肉桃果实,与对照相比,白肉桃注射部位乳白色果肉变成红色。PpHSP20-like1表达量显著提高。PpHSP20-like1的分离可以进一步丰富果肉红色色泽基因资源,其功能研究为该类基因的进一步利用奠定基础。
(4)本发明有助于更好地了解果实红肉色泽形成的分子作用机制。PpHSP20-like1的克隆为进一步了解红肉色泽相关基因编码蛋白之间互作、花色苷代谢信号传导通路奠定基础。可以利用该基因的转基因植株进一步分析PpHSP20-like1是如何传递信号以及传导过程,从而获得PpHSP20-like1参与果肉花色苷信号传导通路的机制。PpHSP20-like1的分离以及功能鉴定为研究果肉红色色泽基因的作用机制奠定基础。
(5)本发明应用于果肉红色色泽育种,针对PpHSP20-like1开发分子标记,为在苗期淘汰掉非目标性状小苗奠定基础,大幅提高育种效率,在果实红肉转基因育种中有较大的应用价值。
附图说明
图1为‘野鸡红’品种果实发育四个阶段的红肉表型图。
图2为提取的‘野鸡红’成熟果实总RNA的琼脂糖凝胶电泳图。
图3为扩增PpHSP20-like1基因全长PCR产物的琼脂糖凝胶电泳图,其中M是Marker分子量标准,条带从上至下依次为5000bp、3000bp、2000bp、1500bp、1000bp、750bp、500bp、250bp、100bp;泳道1和泳道2均是PpHSP20-like1全长。
图4为pENTR/D-TOPO:PpHSP20-like1菌液PCR鉴定阳性克隆琼脂糖凝胶电泳图,其中M是Marker分子量标准,条带从上至下依次为5000bp、3000bp、2000bp、1500bp、1000bp、750bp、500bp、250bp、100bp;泳道1至泳道8是pENTR/D-TOPO:PpHSP20-like1菌液PCR产物1至8号管。
图5为PpHSP20-like1的亚细胞定位分析;采用转盘式激光共聚焦显微镜(UltraView VOX,USA),20X物镜情况下拍照。图上方英文标出了不同光质设置,从左至右分别是明场(Bright field)、绿色荧光(PpHSP20-like1-GFP,488nm)、红色荧光(Right field,561nm)、以上三种情况重合在一起(Merge)的结果图。
图6为PCR检测pENTR/D-TOPO:PpHSP20-like1转基因拟南芥阳性株琼脂糖凝胶电泳图,其中图A,M是Marker分子量标准,条带从上至下依次为4500bp、30000bp、2000bp、1200bp、800bp、500bp、200bp;泳道1至泳道4是pENTR/D-TOPO:PpHSP20-like1转基因株编号1-4号,WT是野生型对照株;图B中M是Marker分子量标准,条带从上至下依次为2000bp、1500bp、1000bp、750bp、500bp、250bp、100bp;泳道5和泳道6是pENTR/D-TOPO:PpHSP20-like1转基因株5和6号苗,泳道空载体是侵染R05空载体株。
图7为拟南芥转基因阳性株系和对照株叶片表型;WT代表野生型对照,空载体代表空载体对照,OE1至OE5分别代表过表达株系1至5号。A:整体颜色表型;B:茎上花萼颜色表型;C:放大的茎上花萼颜色表型;D:顶花颜色表型。
图8为拟南芥转基因阳性株系和对照株叶片中PpHSP20-like1的相对表达水平;WT代表野生型对照叶片中PpHSP20-like1的相对表达水平,空载体代表空载体对照株叶片中PpHSP20-like1的相对表达水平,OE1至OE5分别代表PpHSP20-like1过表达株系1至5号叶片中PpHSP20-like1的相对表达水平。RPII为看家基因;以OE1株系叶片中PpHSP20-like1表达水平为1的情况下,其他株系及对照株中的相对表达水平。
图9为HPLC检测过表达PpHSP20-like1拟南芥阳性株系和野生型对照株叶片中花色苷成分;A图代表野生型对照株的结果,B图代表转基因阳性株的结果;WT代表野生型对照株;OE代表PpHSP20-like1过表达株。
图10为LC-QTOF/MS检测和分析拟南芥转基因阳性株系叶片中花色苷成分;A代表矢车菊素-3-葡萄糖苷质谱TOFMS二级图,B代表矢车菊素-3-芸香糖苷质谱TOFMS二级图。
图11为PpHSP20-like1过表达白肉桃果肉色泽和对照桃果肉色泽表型;OE1和OE2分别代表2个白肉桃瞬时过表达PpHSP20-like1的果肉色泽表型,CK1代表不含PpHSP20-like1基因的空载体对照的果肉色泽表型,CK2代表不注射任何液体对照的果肉色泽表型。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
下述实施例便于更好的理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。实施例中所用到的实验耗材,如无特殊说明,均为自常规生化试剂公司购买得到。
所用的引物合成和测序均由通用生物***有限公司合成。
本发明中品种‘野鸡红’由江苏省农业科学院提供(许建兰,马瑞娟,张斌斌,丁辉,严娟,俞明亮.不同肉色桃杂交后代主要性状遗传规律研究.果树学报,2019,36(1):21-30)。
瞬时表达品种‘小红花’由江苏省农业科学院提供(沈志军,桃初级核心种质、关联分析和红肉性状的图谱定位,2013)。
野生型拟南芥(Arabidopsis thaliana)Col-0种子和野生型本氏烟草(Nicotianabenthamiana)种子均由江苏省农业科学院提供。
实施例1
PpHSP20-like1克隆以及序列分析
桃果实红肉色泽相关蛋白PpHSP20-like1编码基因PpHSP20-like1的克隆。
以参考基因组Prupe.5G005900.1序列为参考,设计引物对(上游引物P1和下游引物P2)用于从红肉桃‘野鸡红’(图1)果肉中扩增果实红肉色泽相关like1PpHSP20-like1基因。
按照Minibest RNA提取试剂盒(Minibest RNA Kit,宝生物工程(大连)有限公司)说明书方法提取桃‘野鸡红’成熟果实总RNA(图2)。采用cDNA第一链合成试剂盒(PrimeScriptTMII 1st Strand cDNA Synthesis Kit,宝生物工程(大连)有限公司)按说明书方法反转录成cDNA,并稀释10倍,-20℃保存。以此cDNA为模板,采用上游引物P1(5’-CACCATGGAAGATGACCCTCCTAAAATGTCCGTC-3’)和下游引物P2(5’-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’)进行PCR扩增。每管PCR反应体系50μL,包含2xHigh-Fidelity Master Mix 25μL,上下游引物各1μL(10μM),cDNA 2μL,ddH2O 21μL。PCR反应程序为:98℃预变性1min,98℃变性15s,55℃退火15s和72℃延伸20s,72℃2min,35个循环,4℃保存,合计3管。
取2管PCR产物进行1.5%琼脂糖凝胶电泳检测,利用紫外凝胶成像仪看到约为1179bp的条带(图3),与预期大小一致。用PCR产物纯化试剂盒(TaKaRa MiniBEST DNAFragment Purification Kit Ver4.0,宝生物工程(大连)有限公司)回收相同的另一管PCR产物。将该回收纯化DNA片段与克隆载体质粒pENTR/D-TOPO(赛默飞世尔科技(中国)有限公司)连接,得到重组质粒pENTR/D-TOPO:PpHSP20-like1。将重组质粒pENTR/D-TOPO:PpHSP20-like1转化大肠杆菌DH5a感受态细胞(宝生物工程(大连)有限公司),用卡那霉素(50.0mg·L-1)标记筛选阳性克隆,并进行菌液PCR鉴定(上游引物5’-TGTAAAACGACGGCCAGT-3’和下游引物P2:5’-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’),得到含有预期片段大小的条带(图4),并提取阳性重组质粒送通用生物***有限公司测序。测序比对结果表明PpHSP20-like1 ORF长度是1179bp,将该基因命名为PpHSP20-like1,序列如SEQ ID NO:1所示,其与参考基因组‘Lovell’有1个SNP变异。PpHSP20-like1基因编码的蛋白序列如SEQ ID NO:2所示。以上实验步骤均按试剂盒说明书进行操作。
SEQ ID NO:1
ATGGAAGATGACCCTCCTAAAATGTCCGTCAATCCATCGTTTGCCGACCAGCTTTTTCTACTGAACTTCGTAATGGGAAATTATTTGGGCCCTGATGTCACGTTTGATAACCTCGAATGTTCAGCATTTCAAAGGATAGCTGAAGGTTCACCTCCGTATATGTCAAGTTATTTGGGGCCTTCTTATGTTAGTGTGTCTCTTTTGGAGGGTTTATATTACTATCTCTTGAGAAATGTTCACCCTAGTCTCATTTTGAGACCAGAAATGTTGCATAAGTATTTAAAGGGCAGCCTACCTTTGCCAAATTCAGGGCTGACGAAAGACAGTTGGCAGTTCACAAATTTTTTCCCTTTGGATCTTCATGAACAGATATGGTACCCAGAAAGCTTCAGAATTGTTAAGGGGATTCTTTTTATTGATGATCCTGTTACATCATGTATGAAAGAGGACGATCTGGAAAAGTTCAAATCTTTATCAGGTATAAATAACATGAAAATTGATATAGATGAAGCCATACGATATCAACACAAGTACCTGGATAATGGGGAAAGTAAGACGAATTGCTTGAATGATGATTGCAATTTCACAAAGACAGAATTTTTTTCCAATGGAAATGGAAATTGGTCGGAAAGATTTCAGCAAAAATATAAGAGAAGGTGCTTCTCTCATTCTCCATCAAAGCCAGCATTTCCCCAAGTTGTTACTACAAAACATTTAAGCAAATATGGAGCTTCATGGAAGACCTGCAAACCAGATGGACCTGTGTTCATGCCCCTTATCTCTGTTCCTAATTTGGAAGAATGTACTTCAGATTCTTCTGTTGTTTTGTCTGGGACTGCCAGGAAAGGGGTAGTTGGGCCACCTGTTGGTGTTGTGGACATTGGTGTTAGCAAGGCTGCATATTACTTCCGGGTTGCTCTACCAGGGGTCAGGAAGGATTTTTGTCAATTCAATTGTGAGATTGAATCTAATGGCAAGATTCATCTCCAAGGCGTCACAAGTGGTGGAAATCCCATAAGGAAACGTTCGCGTGTATTCCAAATGAAATTGCAGCAATTATGCCCACCCGGACCATTCACGCTCTCGTTCAGCCTTCCAGGACCTGTTGATCCAAGGCTCTTTGCACCAAATTTTGGACCTGATGGCATTTTTGAAGGAGTTATCATAAAAGACGAGTGA
SEQ ID NO:2MEDDPPKMSVNPSFADQLFLLNFVMGNYLGPDVTFDNLECSAFQRIAEGSPPYMSSYLGPSYVSVSLLEGLYYYLLRNVHPSLILRPEMLHKYLKGSLPLPNSGLTKDSWQFTNFFPLDLHEQIWYPESFRIVKGILFIDDPVTSCMKEDDLEKFKSLSGINNMKIDIDEAIRYQHKYLDNGESKTNCLNDDCNFTKTEFFSNGNGNWSERFQQKYKRRCFSHSPSKPAFPQVVTTKHLSKYGASWKTCKPDGPVFMPLISVPNLEECTSDSSVVLSGTARKGVVGPPVGVVDIGVSKAAYYFRVALPGVRKDFCQFNCEIESNGKIHLQGVTSGGNPIRKRSRVFQMKLQQLCPPGPFTLSFSLPGPVDPRLFAPNFGPDGIFEGVIIKDE*
实施例2
构建PpHSP20-like1基因过表达载体
采用GATEWAY方法将pENTR/D-TOPO:PpHSP20-like1重组质粒中的PpHSP20-like1按照说明书步骤连接到R05载体(赛默飞世尔科技(中国)有限公司),冻融法转化大肠杆菌DH5ɑ感受态细胞(宝生物工程(大连)有限公司),次日挑单克隆进行菌液PCR鉴定,并取4管(技术重复)具有预期条带大小的菌液送通用生物***有限公司测序鉴定。测序比对结果表明:成功获得了正确的含有PpHSP20-like1基因的重组表达载体R05-PpHSP20-like1。根据AxyPrep质粒回收试剂盒(康宁生命科学有限公司)说明书回收R05-PpHSP20-like1质粒,-20℃保存。
实施例3
PpHSP20-like1基因过表达载体转化农杆菌
采用冻融法,将重组质粒R05-PpHSP20-like1和R05空载体质粒按照GV3101说明书步骤分别转化农杆菌GV3101感受态细胞(宝生物工程(大连)有限公司),28℃条件下倒置培养24h后挑单克隆置于1.5mL离心管中,内含有1.0mL的YEB液体培养基(含庆大霉素50μg/mL、利福平50μg/mL、壮观霉素100μg/mL),培养条件为28℃,200rpm,振荡培养过夜后,以1μL菌液为模板、用rTaq酶(宝生物工程(大连)有限公司)和载体及PpHSP20-like1基因特异性引物(P35S(上游引物):5’-GACGTTCCAACCACGTCTTCAAAG-3’和P2(下游引物):5’-CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’)进行PCR检测,产物片段与预期目的片段大小一致,表明成功获得了含重组表达载体R05-PpHSP20-like1的农杆菌菌株。
实施例4
PpHSP20-like1蛋白亚细胞定位将实施例3中制备的含有重组质粒R05-PpHSP20-like1的阳性克隆菌液扩大至50mL YEB液体培养基体系,28℃,200rpm,继续震荡培养至OD600为0.8~1.0,离心弃上清。用注射缓冲液(含10mM MES,10mM MgCl2,100μM乙酰丁香酮)洗一次,然后用上述缓冲液将菌体GV3101-R05-PpHSP20-like1重悬至OD600为0.7,并在室温条件下静置4h。随后,选取长势优良本氏烟草,用一次性灭菌注射器吸取1mL重悬液注射叶片两侧,注射3株合计9片叶,烟草于16h:8h光暗周期、22℃和70%空气湿度的条件下暗培养1天,正常培养2天。用转盘式激光共聚焦显微镜(型号:Ultra View VOX,美国珀金埃尔默股份有限公司)观察PpHSP20-like1的亚细胞定位。如图5所示,PpHSP20-like1蛋白位于细胞核,是一个核蛋白。
实施例5
R05-PpHSP20-like1重组表达载体转化拟南芥
1)转PpHSP20-like1基因拟南芥株系的获得和鉴定:
将实施例3中制备含有重组质粒R05-PpHSP20-like1的阳性克隆菌液和含有R05空载体质粒的菌液分别扩大至100mL YEB液体培养基体系,28℃,200rpm,继续震荡培养至OD600为0.8~1.0,离心弃上清。用缓冲液(含MS液体培养基,100μM乙酰丁香酮)洗一次,然后用上述缓冲液将沉淀重悬至OD600为1.0,并在室温条件下静置4h。通过蘸花法转化拟南芥,具体为将携带重组质粒R05-PpHSP20-like1的农杆菌悬浮液放在灭菌烧杯中,将拟南芥的含苞待放花浸在烧杯液体10s,合计转染6株,放在暗处一天后在16h/8h昼夜光周期、22℃和70%空气湿度的植物培养室中继续生长。R05空载体质粒的菌体也采用同样的方法转化拟南芥6株。成熟后分别收获种子,干燥后4℃密封保存,命名为T0种子。
把野生型对照种在不含抗生素的MS培养基上,R05空载体对照和T0种子种到MS培养基(含有终浓度为25.0mg·L-1卡那霉素)的培养皿中,待长出4个叶片时移栽到方形土钵中,在16h/8h昼夜光周期、22℃和70%空气湿度的植物培养室中生长。一个月后分别提取PpHSP20-like1过表达、空载体、野生型对照拟南芥叶片的基因组DNA,利用引物P35S:GACGTTCCAACCACGTCTTCAAAG和P2CTCGTCTTTTATGATAACTCCTTCAAAAAT,通过PCR进行阳性苗鉴定(图6),将转PpHSP20-like1基因阳性拟南芥株系做好标记并命名为T0代转基因株系。待种子成熟,单株收获种子T1代。各单株种子分别播于添加有终浓度为25.0mg·L-1卡那霉素的MS培养基内,待T1代植株长出真叶移至方形营养钵中生长,在同样条件下的植物培养室培养。将T1代单株收获种子并干燥后4℃密封保存用添加有终浓度为25.0mg·L-1卡那霉素的MS培养基继续筛选,以观察T2代植株分离情况。T2代种子在含有卡那霉素的MS培养基上发芽率为100%。结果:该T2代种子是纯合转PpHSP20-like1基因株系种子,共获得稳定的6包纯合转PpHSP20-like1基因株系种子;T2种子种下去,转基因阳性株的叶片和花萼均呈现红色(图7)。其中:T0代表示转基因当代植株,T1代表示T0代自交产生的种子及由它所长成的植株,T2代表示T1代自交产生的种子及由它所长成的植株。
2)PpHSP20-like1在转PpHSP20-like1基因阳性株系和对照中的表达水平检测
qRT-PCR检测转基因株和对照株叶片中PpHSP20-like1相对表达量。选取上述转基因阳性株的5个T2代纯合转基因株系种子(OE1、OE2、OE3、OE4、OE5)、1个R05空载体株种子、2个野生型对照株种子在统一条件下播种后培养,分别提取叶片总RNA,分别反转录成cDNA,稀释10倍备用(具体步骤同实施例1),PpHSP20-like1以稀释的各株系cDNA为模板,为PpHSP20-like1设计实时荧光定量PCR引物(上游引物:5’-CCTACCTTTGCCAAATTCAG-3’和下游引物5’-CTTTTCCAGATCGTCCTCTT-3’),采用Premix Ex TaqTM(Tli RNaseH Plus)试剂盒(宝生物工程(大连)有限公司)进行荧光定量PCR反应,反应体系是20μL:包含10.0μLSYBR Premix Ex Taq(2×),0.4μL ROX dye,上下游引物各0.4μL,cDNA模板2.0μL,ddH2O6.8μL,密封在96孔板中,仪器采用Applied Biosystems 7500real-time PCR***。反应程序按照/>Premix Ex TaqTM(Tli RNaseH Plus)说明书设置,看家基因采用RNApolymerase II(RP II)。设置3个生物学重复和3个技术重复。每个基因的相对表达水平分析采用2-ΔΔCT方法,使用SPSS20.0对数据进行差异显著性分析,所有数据以邓肯氏新复极差法进行测验。
qRT-PCR结果表明(图8),PpHSP20-like1基因在OE5中表达量最高,OE3中次之,OE2和OE1再次之,OE4中最弱(图8),但是拟南芥阳性苗株OE1~OE5的PpHSP20-like1基因表达量均显著高于空载体对照和野生型WT对照。
实施例6
HPLC检测拟南芥转基因阳性株系和野生型对照株叶片中花色苷成分
采用高效液相色谱(HPLC)方法,分别称取拟南芥转基因阳性株系和野生型对照株新鲜叶片0.2g,液氮研磨至粉末状,加入2.0mL溶液(丙酮:水:乙酸=70:29.5:0.5),4℃冰箱中避光浸提12h。取上清液放到新的离心管中,10000rpm 4℃离心10min,采用微孔滤膜(0.22μm)过滤3次,放在棕色进样瓶中备测。
采用Agilent高效液相色谱***(1100系列,VWD紫外检测器)和Agilent ZORBAXSB-C18色谱分析柱(4.6mm×250mm,5μm)进行花色苷成分检测。标准品为矢车菊素-3-葡萄糖苷(Aladdin,27661-36-5),甲醇甲酸等试剂均为色谱纯级。甲醇是分析纯级磷酸、甲酸是HPLC级;色谱条件:流动相A:甲醇:磷酸:三氟乙酸=97:2:1(V/V);;流动相B:水:磷酸:三氟乙酸=97:2:1(V/V);进样量为20μL,流速为1.0mL/min;检测波长:515nm;柱温:25℃。
结果发现,野生型对照中无花色苷(图9A),阳性拟南芥叶片在14.589min、15.411min、19.195min时分别出峰(图9B),其中15.411min峰最高,其次是14.589min,再次是19.195min;通过与标准品相比,发现14.589min峰是矢车菊素-3-葡萄糖苷,但是15.411min峰代表的花色苷物质未知。
这些结果说明拟南芥野生型对照中无花色苷,过表达PpHSP20-like1拟南芥阳性株中产生了花色苷中的一种矢车菊素-3-葡萄糖苷,这与红肉桃‘野鸡红’果肉红色是由花色苷矢车菊素-3-葡萄糖苷决定的结果相互印证,15.411min峰代表的花色苷物质,需要进一步进行定性分析。
PpHSP20-like1基因过表达使叶片和花萼呈现红色,野生型对照和空载体对照株叶片和花萼不呈现红色,即PpHSP20-like1基因过表达使叶片和花萼中产生了花色苷中的矢车菊素-3-葡萄糖苷及另外一种未知花色苷物质。
实施例7
质谱检测拟南芥转基因阳性株系叶片中15.411min峰代表的花色苷成分
称取拟南芥转基因阳性株系新鲜叶片0.2g,液氮研磨至粉末状,加入2.0mL提取液(丙酮:水:乙酸=70:29.5:0.5),4℃冰箱中避光浸提12h。取上清液与放到新的离心管中,10000rpm 4℃离心10min,采用微孔滤膜(0.22μm)过滤,放在棕色进样瓶中备用备测。
色谱条件:Shimadzu LC20+SCIEX TripleTOF 5600+;液相条件:柱子采用watersBEH Xbridge 3.5um,2.1*150mm;流动相A:乙腈,流动相B:0.1%甲酸水,进样量5uL,流速0.3ml/min;LC-QTOF/MS质谱条件:IDA方法,mass range:m/z 100-1000,正模式。采用串联飞行时间质谱仪(规格型号:triple tof 5600+,美国国AB SCIEX公司)进行高分辨率质谱检测。甲酸、乙腈是HPLC级;实验用水来自Milli-Q纯水***。
根据质谱二级TOFMS图(图10A和图10B)、文献中各种花色苷成分的分子量综合判定转基因阳性株系15.411min峰代表花色苷矢车菊素-3-芸香苷。因此,过表达PpHSP20-like1拟南芥阳性株中产生了花色苷中的矢车菊素-3-葡萄糖和矢车菊素-3-芸香糖苷。
实施例8
重组表达质粒瞬时转化白肉桃果实
将实施例3中制备含有重组质粒R05-PpHSP20-like1的阳性克隆菌液、空载体质粒菌液分别扩大至50mL YEB液体培养基体系,28℃,200rpm继续震荡培养至OD600为0.8~1.0,离心弃上清。用注射缓冲液(含10mM MES,10mM MgCl2,100μM乙酰丁香酮)洗2次,然后用上述缓冲液将沉淀重悬至OD600为0.7,并在室温条件下静置4h。随后,选取江苏省农业科学院提供的白肉桃品种‘小红花’160个果实,设计处理:注射空载体质粒(作为对照1)60个果,注射重组质粒R05-PpHSP20-like1的阳性克隆菌液60个果,不注射任何(作为对照2)40个果;具体是在成熟前3周在树上,用一次性灭菌注射器吸取1mL重悬液注射果实阴面和阳面各一针。树体和果实进行正常田间栽培管理,每隔3天调查一次果肉色泽,共三次。如图11其中OE1和OE2分别代表2个白肉桃瞬时过表达PpHSP20-like1的果肉色泽表型,CK1代表不含PpHSP20-like1基因的空载体对照的果肉色泽表型,CK2代表不注射任何液体对照的果肉色泽表型。结果表明:与空载体对照1和不注射任何对照2相比,PpHSP20-like1瞬时过表达导致注射部位白肉产生红肉色泽,PpHSP20-like1促进了果肉红色色泽的产生。本实施例对白肉桃品种‘小红花’进行了重组表达质粒瞬时转化,同时其具有广谱性,对其它种类的白肉桃也具有类似效果。
序列表
<110> 江苏省农业科学院
<120> 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1179
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggaagatg accctcctaa aatgtccgtc aatccatcgt ttgccgacca gctttttcta 60
ctgaacttcg taatgggaaa ttatttgggc cctgatgtca cgtttgataa cctcgaatgt 120
tcagcatttc aaaggatagc tgaaggttca cctccgtata tgtcaagtta tttggggcct 180
tcttatgtta gtgtgtctct tttggagggt ttatattact atctcttgag aaatgttcac 240
cctagtctca ttttgagacc agaaatgttg cataagtatt taaagggcag cctacctttg 300
ccaaattcag ggctgacgaa agacagttgg cagttcacaa attttttccc tttggatctt 360
catgaacaga tatggtaccc agaaagcttc agaattgtta aggggattct ttttattgat 420
gatcctgtta catcatgtat gaaagaggac gatctggaaa agttcaaatc tttatcaggt 480
ataaataaca tgaaaattga tatagatgaa gccatacgat atcaacacaa gtacctggat 540
aatggggaaa gtaagacgaa ttgcttgaat gatgattgca atttcacaaa gacagaattt 600
ttttccaatg gaaatggaaa ttggtcggaa agatttcagc aaaaatataa gagaaggtgc 660
ttctctcatt ctccatcaaa gccagcattt ccccaagttg ttactacaaa acatttaagc 720
aaatatggag cttcatggaa gacctgcaaa ccagatggac ctgtgttcat gccccttatc 780
tctgttccta atttggaaga atgtacttca gattcttctg ttgttttgtc tgggactgcc 840
aggaaagggg tagttgggcc acctgttggt gttgtggaca ttggtgttag caaggctgca 900
tattacttcc gggttgctct accaggggtc aggaaggatt tttgtcaatt caattgtgag 960
attgaatcta atggcaagat tcatctccaa ggcgtcacaa gtggtggaaa tcccataagg 1020
aaacgttcgc gtgtattcca aatgaaattg cagcaattat gcccacccgg accattcacg 1080
ctctcgttca gccttccagg acctgttgat ccaaggctct ttgcaccaaa ttttggacct 1140
gatggcattt ttgaaggagt tatcataaaa gacgagtga 1179
<210> 2
<211> 392
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Glu Asp Asp Pro Pro Lys Met Ser Val Asn Pro Ser Phe Ala Asp
1 5 10 15
Gln Leu Phe Leu Leu Asn Phe Val Met Gly Asn Tyr Leu Gly Pro Asp
20 25 30
Val Thr Phe Asp Asn Leu Glu Cys Ser Ala Phe Gln Arg Ile Ala Glu
35 40 45
Gly Ser Pro Pro Tyr Met Ser Ser Tyr Leu Gly Pro Ser Tyr Val Ser
50 55 60
Val Ser Leu Leu Glu Gly Leu Tyr Tyr Tyr Leu Leu Arg Asn Val His
65 70 75 80
Pro Ser Leu Ile Leu Arg Pro Glu Met Leu His Lys Tyr Leu Lys Gly
85 90 95
Ser Leu Pro Leu Pro Asn Ser Gly Leu Thr Lys Asp Ser Trp Gln Phe
100 105 110
Thr Asn Phe Phe Pro Leu Asp Leu His Glu Gln Ile Trp Tyr Pro Glu
115 120 125
Ser Phe Arg Ile Val Lys Gly Ile Leu Phe Ile Asp Asp Pro Val Thr
130 135 140
Ser Cys Met Lys Glu Asp Asp Leu Glu Lys Phe Lys Ser Leu Ser Gly
145 150 155 160
Ile Asn Asn Met Lys Ile Asp Ile Asp Glu Ala Ile Arg Tyr Gln His
165 170 175
Lys Tyr Leu Asp Asn Gly Glu Ser Lys Thr Asn Cys Leu Asn Asp Asp
180 185 190
Cys Asn Phe Thr Lys Thr Glu Phe Phe Ser Asn Gly Asn Gly Asn Trp
195 200 205
Ser Glu Arg Phe Gln Gln Lys Tyr Lys Arg Arg Cys Phe Ser His Ser
210 215 220
Pro Ser Lys Pro Ala Phe Pro Gln Val Val Thr Thr Lys His Leu Ser
225 230 235 240
Lys Tyr Gly Ala Ser Trp Lys Thr Cys Lys Pro Asp Gly Pro Val Phe
245 250 255
Met Pro Leu Ile Ser Val Pro Asn Leu Glu Glu Cys Thr Ser Asp Ser
260 265 270
Ser Val Val Leu Ser Gly Thr Ala Arg Lys Gly Val Val Gly Pro Pro
275 280 285
Val Gly Val Val Asp Ile Gly Val Ser Lys Ala Ala Tyr Tyr Phe Arg
290 295 300
Val Ala Leu Pro Gly Val Arg Lys Asp Phe Cys Gln Phe Asn Cys Glu
305 310 315 320
Ile Glu Ser Asn Gly Lys Ile His Leu Gln Gly Val Thr Ser Gly Gly
325 330 335
Asn Pro Ile Arg Lys Arg Ser Arg Val Phe Gln Met Lys Leu Gln Gln
340 345 350
Leu Cys Pro Pro Gly Pro Phe Thr Leu Ser Phe Ser Leu Pro Gly Pro
355 360 365
Val Asp Pro Arg Leu Phe Ala Pro Asn Phe Gly Pro Asp Gly Ile Phe
370 375 380
Glu Gly Val Ile Ile Lys Asp Glu
385 390
Claims (7)
1.一种基因PpHSP20-like1在促进植物器官产生红色色泽中的应用,所述植物器官为拟南芥叶、拟南芥花萼或者桃果肉,所述基因PpHSP20-like1的核苷酸序列为SEQ ID NO.1,所述基因PpHSP20-like1在植物中过表达。
2.一种含有权利要求1所述的基因PpHSP20-like1的表达载体在促进植物器官产生红色色泽中的应用,所述植物器官为拟南芥叶、拟南芥花萼或者桃果肉,所述基因PpHSP20- like1的核苷酸序列为SEQ ID NO.1,所述基因PpHSP20-like1在植物中过表达。
3.根据权利要求2所述的应用,其特征在于,所述的表达载体的构建包括如下步骤:
以红肉桃品种‘野鸡红’的cDNA为模板,设计引物,扩增得PpHSP20-like1基因开放阅读框全长片段,PCR产物纯化后连接到pENTR/D-TOPO载体,构建的质粒命名为pENTR/D-TOPO:PpHSP20-like1;通过GATEWAY方法用LR酶将pENTR/D-TOPO: like1PpHSP20-like1连接到R05载体上,构建的质粒命名为R05: PpHSP20-like1。
4.根据权利要求3所述的应用,其特征在于,所述引物为:
P1: 5’- CACCATGGAAGATGACCCTCCTAAAATGTCCGTC -3’,
P2: 5’- CTCGTCTTTTATGATAACTCCTTCAAAAAT-3’。
5.一种含有权利要求1所述的基因PpHSP20-like1或者权利要求2所述的表达载体的宿主菌在促进植物器官产生红色色泽中的应用,所述植物器官为拟南芥叶、拟南芥花萼或者桃果肉,所述基因PpHSP20-like1的核苷酸序列为SEQ ID NO.1,所述基因PpHSP20-like1在植物中过表达。
6.根据权利要求5所述的应用,其特征在于,所述宿主菌以农杆菌GV3101为出发菌株。
7.基因PpHSP20-like1或者含有基因PpHSP20-like1的表达载体在产生改良桃果实红肉色泽的转基因桃中的应用,所述基因PpHSP20-like1的核苷酸序列为SEQ ID NO.1,所述基因PpHSP20-like1在桃中过表达。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111177712.7A CN113881685B (zh) | 2021-10-09 | 2021-10-09 | 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111177712.7A CN113881685B (zh) | 2021-10-09 | 2021-10-09 | 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113881685A CN113881685A (zh) | 2022-01-04 |
CN113881685B true CN113881685B (zh) | 2024-03-29 |
Family
ID=79005647
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111177712.7A Active CN113881685B (zh) | 2021-10-09 | 2021-10-09 | 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113881685B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115011612B (zh) * | 2022-06-28 | 2023-07-07 | 洛阳师范学院 | 促进植物器官产生花青素的色泽基因ZjFAS2及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103966210A (zh) * | 2014-05-29 | 2014-08-06 | 江苏省农业科学院 | 桃ssap分子标记引物组合、分子标记组合及其在桃品种遗传多样性分析上的应用 |
WO2015165425A1 (en) * | 2014-04-29 | 2015-11-05 | Institute Of Botany, The Chinese Academy Of Sciences | Methods and materials for improving plant stress resistance |
CN109679966A (zh) * | 2019-01-16 | 2019-04-26 | 安徽农业大学 | AcMYB123和AcbHLH42基因在调控猕猴桃果肉花青素合成中的应用 |
CN110408650A (zh) * | 2019-07-25 | 2019-11-05 | 中国农业大学 | NOR-like1基因及其编码的蛋白质在调控番茄果实产量中的应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1618193B1 (en) * | 2003-04-16 | 2008-05-07 | BASF Plant Science GmbH | Use of genes for increasing the oil content in plants |
US9085774B2 (en) * | 2005-04-19 | 2015-07-21 | Basf Plant Science Gmbh | Methods controlling gene expression |
-
2021
- 2021-10-09 CN CN202111177712.7A patent/CN113881685B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015165425A1 (en) * | 2014-04-29 | 2015-11-05 | Institute Of Botany, The Chinese Academy Of Sciences | Methods and materials for improving plant stress resistance |
CN103966210A (zh) * | 2014-05-29 | 2014-08-06 | 江苏省农业科学院 | 桃ssap分子标记引物组合、分子标记组合及其在桃品种遗传多样性分析上的应用 |
CN109679966A (zh) * | 2019-01-16 | 2019-04-26 | 安徽农业大学 | AcMYB123和AcbHLH42基因在调控猕猴桃果肉花青素合成中的应用 |
CN110408650A (zh) * | 2019-07-25 | 2019-11-05 | 中国农业大学 | NOR-like1基因及其编码的蛋白质在调控番茄果实产量中的应用 |
Non-Patent Citations (3)
Title |
---|
Characterization and genetic map** of a new blood-flesh trait controlled by the single dominant locus DBF in peach;Shen, Z.等;Tree Genetics & Genomes;20130821;1435-1446 * |
Integrated analysis of HSP20 genes in the developing flesh of peach: identification, expression profiling, and subcellular localization;Chunhua Zhang等;BMC Plant Biology;20231221;1-17 * |
新疆红肉苹果转录因子MsMYB10基因的克隆、序列分析及原核表达;王延玲等;中国农业科学;20101231;第43卷(第3期);2735-2743 * |
Also Published As
Publication number | Publication date |
---|---|
CN113881685A (zh) | 2022-01-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107435047B (zh) | 一种植物磷信号网络中耐低磷关键基因GmPHR25及其与应用 | |
CN108588092B (zh) | 一种梨花色素苷合成转录因子PbMYB109及其应用 | |
CN108841841B (zh) | 一种番茄转录因子SlbZIP6的克隆及其在抗高温胁迫中的应用 | |
CN107056911B (zh) | 一种促进植物提前开花的草莓转录因子及其应用 | |
CN114014917B (zh) | 一种FvbHLH36蛋白及其编码基因和用途 | |
CN113881685B (zh) | 促进植物器官产生红色色泽的基因PpHSP20-like1及其应用 | |
CN112724213B (zh) | 甘薯花青苷合成和抗逆相关蛋白IbMYB4及其编码基因与应用 | |
CN110257401B (zh) | 毛果杨PtrMYB119基因在提高烟草耐旱性中的应用 | |
CN114703199B (zh) | 一种植物抗旱性相关的基因TaCML46及应用 | |
CN107557384B (zh) | 一种诱导植株矮化的遗传转化体系及其构建和应用 | |
CN116083445A (zh) | 一种CrBZR1基因及其应用 | |
CN115976039A (zh) | 软枣猕猴桃光响应基因AaHY5like9及其应用 | |
CN114480414A (zh) | 一种增强植物耐寒性或培育高耐寒性植物的方法 | |
WO2007061146A1 (en) | A method for producing chinese cabbage transformant using tissues of flower stalk and a transformant with promoted soft rot resistance obtained from the method | |
CN106480069B (zh) | 黄瓜CsERF025基因及其在促进黄瓜果实顺直发育中的应用 | |
CN106947770B (zh) | 苤蓝基因及其在培育紫色微型番茄中的应用 | |
CN116515857B (zh) | 一种仁用杏PaPIP1-2基因及其在提高植物抗寒性中的应用 | |
CN117210490B (zh) | 一种调控苹果属植物自花结实的pchr基因及其应用 | |
CN117126865B (zh) | 一种促进类胡萝卜素含量积累的LbaMYB44基因及应用 | |
CN113564182B (zh) | 蝴蝶花SVP-like基因的应用及获得蝴蝶花基因沉默或敲除植株的方法 | |
CN115948454B (zh) | 一种葡萄VvDREB2c基因在提高植物耐热能力中的应用 | |
CN110229801B (zh) | 一种控制水稻叶片衰老的基因及其编码的蛋白质 | |
CN117987424A (zh) | 茶树CsAPL1基因在调控植物开花中的应用 | |
CN117802150A (zh) | 水稻每穗粒数和粒重相关mog1蛋白及其编码基因与应用 | |
CN116606359A (zh) | 负向调控植物抗旱性的蛋白质及其编码基因与应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |