CN112830963A - 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 - Google Patents
一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 Download PDFInfo
- Publication number
- CN112830963A CN112830963A CN202110270214.0A CN202110270214A CN112830963A CN 112830963 A CN112830963 A CN 112830963A CN 202110270214 A CN202110270214 A CN 202110270214A CN 112830963 A CN112830963 A CN 112830963A
- Authority
- CN
- China
- Prior art keywords
- gene
- cotton
- protein
- ghfla19
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 90
- 229920000742 Cotton Polymers 0.000 title claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 26
- 238000011161 development Methods 0.000 title claims abstract description 22
- 230000001850 reproductive effect Effects 0.000 title claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 12
- 230000001276 controlling effect Effects 0.000 title claims abstract description 10
- 230000009261 transgenic effect Effects 0.000 claims abstract description 26
- 108091033409 CRISPR Proteins 0.000 claims abstract description 25
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 14
- 206010021929 Infertility male Diseases 0.000 claims abstract description 6
- 208000007466 Male Infertility Diseases 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000219146 Gossypium Species 0.000 claims description 46
- 241000196324 Embryophyta Species 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 14
- 241000894006 Bacteria Species 0.000 claims description 7
- 244000299507 Gossypium hirsutum Species 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 7
- 230000001488 breeding effect Effects 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 235000009429 Gossypium barbadense Nutrition 0.000 claims description 5
- 240000002024 Gossypium herbaceum Species 0.000 claims description 4
- 235000004341 Gossypium herbaceum Nutrition 0.000 claims description 4
- 235000009432 Gossypium hirsutum Nutrition 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 3
- 238000010362 genome editing Methods 0.000 claims description 3
- 240000000047 Gossypium barbadense Species 0.000 claims description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims description 2
- 238000010459 TALEN Methods 0.000 claims description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- 238000003197 gene knockdown Methods 0.000 claims description 2
- 238000003209 gene knockout Methods 0.000 claims description 2
- 230000009368 gene silencing by RNA Effects 0.000 claims description 2
- 239000011701 zinc Substances 0.000 claims description 2
- 229910052725 zinc Inorganic materials 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 1
- 230000035899 viability Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 16
- 230000018109 developmental process Effects 0.000 description 16
- 238000001514 detection method Methods 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000003321 amplification Effects 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 238000010839 reverse transcription Methods 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 230000007152 anther development Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000021121 meiosis Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000008119 pollen development Effects 0.000 description 3
- 235000018322 upland cotton Nutrition 0.000 description 3
- XEPSCVXTCUUHDT-AVGNSLFASA-N Arg-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCN=C(N)N XEPSCVXTCUUHDT-AVGNSLFASA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DSDPLOODKXISDT-XUXIUFHCSA-N Ile-Leu-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O DSDPLOODKXISDT-XUXIUFHCSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 208000021267 infertility disease Diseases 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Inorganic materials [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WYPUMLRSQMKIJU-BPNCWPANSA-N Ala-Arg-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O WYPUMLRSQMKIJU-BPNCWPANSA-N 0.000 description 1
- XCVRVWZTXPCYJT-BIIVOSGPSA-N Ala-Asn-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N XCVRVWZTXPCYJT-BIIVOSGPSA-N 0.000 description 1
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-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
- AWNAEZICPNGAJK-FXQIFTODSA-N Ala-Met-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O AWNAEZICPNGAJK-FXQIFTODSA-N 0.000 description 1
- PEIBBAXIKUAYGN-UBHSHLNASA-N Ala-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 PEIBBAXIKUAYGN-UBHSHLNASA-N 0.000 description 1
- RUXQNKVQSKOOBS-JURCDPSOSA-N Ala-Phe-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RUXQNKVQSKOOBS-JURCDPSOSA-N 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- NTAZNGWBXRVEDJ-FXQIFTODSA-N Arg-Asp-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NTAZNGWBXRVEDJ-FXQIFTODSA-N 0.000 description 1
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 1
- DDBMKOCQWNFDBH-RHYQMDGZSA-N Arg-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O DDBMKOCQWNFDBH-RHYQMDGZSA-N 0.000 description 1
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 1
- LDGUZSIPGSPBJP-XVYDVKMFSA-N Asp-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)O)N LDGUZSIPGSPBJP-XVYDVKMFSA-N 0.000 description 1
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-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
- ZRUBWRCKIVDCFS-XPCJQDJLSA-N Asp-Leu-Thr-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZRUBWRCKIVDCFS-XPCJQDJLSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- 229920000018 Callose Polymers 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- MTCXQQINVAFZKW-MNXVOIDGSA-N Gln-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MTCXQQINVAFZKW-MNXVOIDGSA-N 0.000 description 1
- XKPACHRGOWQHFH-IRIUXVKKSA-N Gln-Thr-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XKPACHRGOWQHFH-IRIUXVKKSA-N 0.000 description 1
- DCBSZJJHOTXMHY-DCAQKATOSA-N Glu-Pro-Pro Chemical compound OC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DCBSZJJHOTXMHY-DCAQKATOSA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 101710091977 Hydrophobin Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- DFFTXLCCDFYRKD-MBLNEYKQSA-N Ile-Gly-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N DFFTXLCCDFYRKD-MBLNEYKQSA-N 0.000 description 1
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 1
- DLEBSGAVWRPTIX-PEDHHIEDSA-N Ile-Val-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)[C@@H](C)CC DLEBSGAVWRPTIX-PEDHHIEDSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101100288095 Klebsiella pneumoniae neo gene Proteins 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 1
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- ZDBMWELMUCLUPL-QEJZJMRPSA-N Leu-Phe-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ZDBMWELMUCLUPL-QEJZJMRPSA-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
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 1
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- FLCMXEFCTLXBTL-DCAQKATOSA-N Lys-Asp-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N FLCMXEFCTLXBTL-DCAQKATOSA-N 0.000 description 1
- DCRWPTBMWMGADO-AVGNSLFASA-N Lys-Glu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O DCRWPTBMWMGADO-AVGNSLFASA-N 0.000 description 1
- AEIIJFBQVGYVEV-YESZJQIVSA-N Lys-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCCCN)N)C(=O)O AEIIJFBQVGYVEV-YESZJQIVSA-N 0.000 description 1
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 1
- AETNZPKUUYYYEK-CIUDSAMLSA-N Met-Glu-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O AETNZPKUUYYYEK-CIUDSAMLSA-N 0.000 description 1
- IIHMNTBFPMRJCN-RCWTZXSCSA-N Met-Val-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IIHMNTBFPMRJCN-RCWTZXSCSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- YCCUXNNKXDGMAM-KKUMJFAQSA-N Phe-Leu-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YCCUXNNKXDGMAM-KKUMJFAQSA-N 0.000 description 1
- UNBFGVQVQGXXCK-KKUMJFAQSA-N Phe-Ser-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O UNBFGVQVQGXXCK-KKUMJFAQSA-N 0.000 description 1
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 1
- YUPRIZTWANWWHK-DZKIICNBSA-N Phe-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N YUPRIZTWANWWHK-DZKIICNBSA-N 0.000 description 1
- UVKNEILZSJMKSR-FXQIFTODSA-N Pro-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1 UVKNEILZSJMKSR-FXQIFTODSA-N 0.000 description 1
- UEHYFUCOGHWASA-HJGDQZAQSA-N Pro-Glu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 UEHYFUCOGHWASA-HJGDQZAQSA-N 0.000 description 1
- FHZJRBVMLGOHBX-GUBZILKMSA-N Pro-Pro-Asp Chemical compound OC(=O)C[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1)C(O)=O FHZJRBVMLGOHBX-GUBZILKMSA-N 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- OHKLFYXEOGGGCK-ZLUOBGJFSA-N Ser-Asp-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OHKLFYXEOGGGCK-ZLUOBGJFSA-N 0.000 description 1
- CDVFZMOFNJPUDD-ACZMJKKPSA-N Ser-Gln-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CDVFZMOFNJPUDD-ACZMJKKPSA-N 0.000 description 1
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- QPPYAWVLAVXISR-DCAQKATOSA-N Ser-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QPPYAWVLAVXISR-DCAQKATOSA-N 0.000 description 1
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 1
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- UKKROEYWYIHWBD-ZKWXMUAHSA-N Ser-Val-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UKKROEYWYIHWBD-ZKWXMUAHSA-N 0.000 description 1
- JMZKMSTYXHFYAK-VEVYYDQMSA-N Thr-Arg-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O JMZKMSTYXHFYAK-VEVYYDQMSA-N 0.000 description 1
- PRNGXSILMXSWQQ-OEAJRASXSA-N Thr-Leu-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PRNGXSILMXSWQQ-OEAJRASXSA-N 0.000 description 1
- CSNBWOJOEOPYIJ-UVOCVTCTSA-N Thr-Thr-Lys Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(O)=O CSNBWOJOEOPYIJ-UVOCVTCTSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- GBIUHAYJGWVNLN-AEJSXWLSSA-N Val-Ser-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N GBIUHAYJGWVNLN-AEJSXWLSSA-N 0.000 description 1
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010054251 arabinogalactan proteins Proteins 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010080488 arginyl-arginyl-leucine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 108010082795 phenylalanyl-arginyl-arginine Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002623 sporogenic effect Effects 0.000 description 1
- 210000003046 sporozoite Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种调控棉花雄性生殖发育的GhFLA19‑D蛋白及其编码基因与应用,涉及植物基因工程技术领域。本发明提供的调控棉花雄性生殖发育的GhFLA19‑D蛋白具有如SEQ ID No:1所示的氨基酸序列。所述GhFLA19‑D蛋白是一种类成束阿拉伯半乳聚糖蛋白,本发明实验发现GhFLA19‑D基因在花药中特异性表达,通过CRISPR/Cas9对该基因进行敲除,发现被完全编辑的转基因植株表现为完全的雄性不育,表现为花药干瘪、不开裂和花粉无活力等特点,其可用于培育棉花雄性不育系。
Description
技术领域
本发明涉及植物基因工程技术领域,尤其是涉及一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用。
背景技术
棉花是世界上重要的经济作物之一,其具有明显的杂种优势。目前,棉花杂种优势的利用仍然以人工去雄为主,需要消耗大量的劳动力,制种成本高。雄性不育系的出现为杂种优势利用指明了新的方向,但是在棉花中对于育性相关的基因的报道较少。因此对该类基因的克隆对于棉花雄性器官的发育机制和棉花的杂种优势利用具有重要意义。
花药作为花粉形成和发育的场所,是整个雄蕊的核心部分;花药发育过程主要经历了从雄蕊原基的出现到花粉粒的成熟释放过程。根据前人对模式植物拟南芥的研究,花药的发育依据其形态结构和细胞基础可以分为两大时期,即孢子体发生阶段和配子体发生阶段(Wallace等,2015)。第一个阶段,雄蕊原基经过细胞的分裂和分化形成完整的花药组织。花药形成典型的四药室结构,花药室通过相连的结缔组织横向连接在维管束上,每个花药室由四层体细胞构成,从外至内依次是表皮(Epidermis)、内层(Endothecium)、中层(Middle Layer)和绒毡层(Tapetum),他们包围着由造孢细胞发育而成的生殖细胞-花粉母细胞(Microspore Mother Cells,MMCs)。第二个阶段,花药进一步发育扩大,小孢子从四分体分裂,发育为成熟的花粉;与此同时,花药组织逐渐退化,花药开裂,花粉释放。花粉内壁(Intine)是花粉壁的重要组成部分,通常被认为是小孢子自身物质代谢而来,它的发育一般从花药发育的单核期开始。花粉内壁的结构与一般的植物细胞壁类似,其组成主要包括纤维素、半纤维素、果胶多聚物、水解酶和疏水蛋白等。花粉的发育涉及到很多与细胞壁相关的过程,如花粉母细胞的分化、胼胝质的形成与降解以及花粉发育后期花粉细胞内壁的形成。
目前,在棉花中,哪些涉及细胞壁相关的基因对花药和花粉的正常发育具有关键作用尚不清楚。此外,关于调控棉花雄性不育的基因也很少有研究报道。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用。本发明阐明了一种类成束阿拉伯半乳聚糖蛋白即GhFLA19-D蛋白在调控棉花雄性生殖发育中的应用,为培育棉花雄性不育系提供了育种资源。
本发明提供的技术方案如下:
一种调控棉花雄性生殖发育的GhFLA19-D蛋白,所述蛋白质具有如SEQ ID No:1所示的氨基酸序列。
在一个具体的实施方案中,所述氨基酸序列可以经过一个或多个氨基酸残基的取代、缺失或添加而衍生出具有同等功能的氨基酸序列。
一种调控棉花雄性生殖发育的基因,所述基因编码前述GhFLA19-D蛋白,所述基因的CDS序列如SEQ ID No:2所示。
在本发明中,编码GhFLA19-D蛋白的cDNA也在本发明的保护范围之内。本发明还包括与所述GhFLA19-D基因具有90%以上同源性,优选95%更优选99%以上同源性且编码类成束阿拉伯半乳聚糖蛋白的基因。
含有上述基因的重组载体也属于本发明旨在保护的内容。
所述GhFLA19-D蛋白或所述GhFLA19-D基因在控制棉花雄性生殖发育中的应用,特别地,所述蛋白或所述基因在棉花的花药和花粉发育中有作用。
所述GhFLA19-D蛋白或所述GhFLA19-D基因在培育雄性不育转基因棉花中的应用。
本发明经实验发现GhFLA19-D基因在棉花花药中特异性表达,通过荧光定量PCR发现该基因主要在花粉母细胞减数分裂时期到花粉双核时期表达较高。通过CRISPR/Cas9对该基因进行敲除,发现被完全编辑的转基因植株表现为完全的雄性不育。表现为花药干瘪、不开裂和花粉无活力等特点。因此,在培育雄性不育转基因棉花中可以利用所述蛋白或基因的功能。
所述棉花GhFLA19-D基因的突变基因在制备棉花雄性不育系中的应用。
在一个实施方案中,所述应用包括采用基因敲除、基因敲减或者基因编辑抑制所述基因的表达或使所述基因功能缺失,使突变后的棉花出现雄性不育性状。
在一个实施方案中,所述应用通过CRISPR/Cas9系统、TALEN系统、锌指酶系统、RNA干扰技术实现抑制所述基因的表达或使所述基因功能缺失。
在一个实施方案,所述CRISPR/Cas9系统在构建载体时,设计的CRISPR/Cas9载体的靶位点序列为:如SEQ ID No:3所示序列和如SEQ ID No:4所示序列。
在另一方面,本发明还提供了一种培育雄性不育系转基因棉花的方法,所述方法包括抑制棉花植物中GhFLA19-D基因的表达,得到雄性不育系转基因植物。
在一个具体的实施方案中,所述方法包括构建所述GhFLA19-D基因的CRISPR/Cas9载体,将表达载体导入宿主菌中,筛选表达所述基因的工程菌;将工程菌导入目标植物中,筛选转基因植株。
在一个实施方案中,所述棉花包括海岛棉和陆地棉,优选陆地棉。
有益效果:
(1)本发明研究了GhFLA19-D蛋白及其编码基因在花药花粉发育中的作用,发现该基因主要在花粉母细胞减数分裂时期到花粉双核时期表达较高,基因突变导致花药干瘪、不开裂和花粉无活力。GhFLA19-D基因完全编辑的单株表现为完全不育。阐明了该基因在棉花雄性发育中的功能,这在棉花杂交育种中有重要意义。
(2)本发明提供了GhFLA19-D蛋白及其编码基因在控制棉花雄性生殖发育中的应用,为棉花植物的遗传改造提供了有效的途径。
(3)本发明提供了一种通过敲除或突变GhFLA19-D基因从而培育棉花雄性不育材料的新方法。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为棉花GhFLA19-D基因的表达模式图(其中MC:花粉母细胞减数分裂时期;TTP:四分体时期;UNP:单核期;lUNP:单核后期;BNP:双核期;MP:成熟时期;Filament:花丝;Pistil:雌蕊;Petal:花瓣;Stem:茎;Leaf:叶;Root:根);
图2为本发明实施例提供的棉花转基因苗PCR检测结果(胶图中从左到右泳道内被检测样品株系编号依次是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17和18,图中B代表空白对照,N代表阴性对照,P代表阳性对照);
图3为完全编辑的转基因植株line3的编辑信息以及未编辑的转基因植株line12的编辑信息示意图以及GhFLA19-D基因CRISPR/Cas9敲除T2代植株表型图。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验材料:
本实验选取的棉花材料为陆地棉HM-1,种植于中国农业科学院棉花研究所棉花生物学国家重点实验室试验田(安阳白璧),管理措施为正常大田管理。
实验试剂与耗材:
酶及试剂盒:GXL DNA Polymerase高保真酶、荧光定量试剂盒、RNA反转录试剂盒、胶回收试剂盒、PCR产物纯化试剂盒均购自宝生物工程大连有限公司;Ultra One Step Cloning Kit试剂盒购自Vazyme公司;质粒少量提取试剂盒购自Magen公司;限制性内切酶购自NEB公司;DNA Marker、植物总RNA提取试剂盒购自TIANGEN公司。
其他药品:琼脂糖为西班牙原装产品,蛋白胨、酵母提取物、氯仿、异戊醇、乙醇、异丙醇、氯化钠等为国产分析纯,氨苄青霉素等购自宝生物工程大连有限公司,大肠杆菌感受态细胞购自北京天根生化科技公司。
培养基:LB液体培养基:胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeast extract)5g/L、氯化钠(NaCl)10g/L;LB固体培养基:胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeastextract)5g/L、氯化钠(NaCl)10g/L、琼脂粉15g/L,定容至1L;
LB选择培养基:在LB铺平板前,待培养基高压灭菌冷却至55度时加入相应浓度抗生素,摇匀后铺平板。
主要仪器:PCR扩增仪(BIO-RAD),高速离心机(Hettich MIKRO200R)、电泳设备(BIO-RAD)、凝胶成像系统(BIO-RAD)、荧光定量PCR仪(ABI7500)、电热恒温培养箱(上海森信)、恒温培养振荡器(上海智城)、人工气候室。
实施例
1.棉花GhFLA19-D基因的表达模式分析
对不同时期的花药进行取样并进行RNA的提取和cDNA的反转录,从Cottongen上获得GhFLA19-D的CDS序列,设计引物,进行荧光定量PCR的进行。图1为GhFLA19-D基因的表达模式。
克隆过程:
(1)取陆地棉HM-1材料不同发育时期的花药,迅速放入液氮中冷冻,在液氮中研磨,并保存于-80℃冰箱备用。
(2)植物总RNA提取:RNA的提取采用TIANGEN公司RNA提取试剂盒进行。
(3)cDNA的合成:将500ng RNA反转录为cDNA,采用Toyobo的反转录试剂盒FSQ-201,反转录体系为:
按下列组份配制RT反应液(反应液配制在冰上进行):
表1反转录体系
反转录反应条件如下:
37℃15min(反转录反应),
98℃5s(反转录酶的失活反应);
将反转录产物cDNA溶液稀释6倍作为PCR反应模板。
(4)荧光定量PCR
引物序列:
qGhFLA19-D-F:5’-CGCCATCTCCACATCGGATCTC-3’(SEQ ID No:5);
qGhFLA19-D-R:5’-TGTGGGGAGACGTGGAGAAAGA-3’(SEQ ID No:6)。
PCR反应体系:
表2荧光定量PCR反应体系
PCR反应程序:
表3荧光定量PCR反应程序
2.棉花GhFLA19-D基因的克隆
从Cottongen上获得GhFLA19-D的基因序列,设计引物,从上述步骤得到的花药cDNA中扩增GhFLA19-D的CDS序列,其开放阅读框为753bp,编码250个氨基酸,蛋白的相对分子量为27.55kDa,等电点为6.24。
GhFLA19-D基因编码的氨基酸序列如下SEQ ID No:1所示:MENFSSKPTIVILLLLTTVSTADLTSKELDAAILVLQSRGYTLFPNAISTSDLQVRLLSSQNSSIFTLFAPPDSLLFSLDLLSSARLYTFSLFLHVSPHFLSSSDLLALPRPAFIDTLLPNRRLFVEHAMSTRNGTALLTVSVDGVVVSVPDLFLGSNIVVHGLDGILVARYGSLVSEGSDNAIAEPPKFPYQTYVSPANPPETLPPTDLEMVTIGTQIKKDREAFRRDDDHATTKRTKHGTFFRFERVY;
GhFLA19-D基因的CDS序列为如下SEQ ID No:2所示:
ATGGAAAACTTTTCCTCCAAACCAACAATCGTAATCCTCCTCCTCCTCACCACCGTCAGCACCGCCGACTTAACTTCCAAAGAACTAGACGCAGCCATCTTAGTCCTTCAATCAAGAGGCTACACTCTCTTCCCCAACGCCATCTCCACATCGGATCTCCAAGTCCGCCTCCTCTCATCCCAAAACTCTTCCATATTCACTCTTTTTGCACCCCCGGACTCCCTCCTCTTCTCCCTCGACCTCCTCTCCTCCGCCCGCCTTTACACTTTCTCTCTCTTTCTCCACGTCTCCCCACATTTCCTCTCCTCCTCAGACCTCCTCGCCCTCCCTCGCCCCGCCTTCATCGACACCCTCCTCCCTAACCGTCGACTCTTCGTAGAACATGCTATGTCTACCCGCAACGGCACAGCCTTGCTAACTGTTTCCGTCGACGGGGTTGTCGTCTCCGTCCCGGATCTTTTCCTTGGATCCAACATTGTTGTCCACGGGCTTGATGGGATTCTTGTTGCAAGATACGGGTCCTTGGTTAGTGAAGGTAGTGACAATGCTATTGCTGAGCCACCCAAGTTCCCCTATCAAACCTATGTTTCGCCGGCCAACCCACCGGAGACTTTGCCCCCTACGGACCTGGAGATGGTCACAATCGGAACGCAAATCAAGAAAGATAGGGAGGCTTTCCGTCGTGATGATGACCATGCCACTACTAAGAGAACTAAACACGGTACATTTTTCCGGTTTGAACGCGTTTACTGA;
PCR扩增目的基因:
表4PCR扩增反应体系
PCR扩增程序为:98℃3min;98℃10s;56℃15s;68℃1min,35个循环;68℃10min。
引物序列:
GhFLA19-D-F:5’-ATGGAAAACTTTTCCTCCAAACC-3’(SEQ ID No:7);
GhFLA19-D-R:5’-TCAGTAAACGCGTTCAAACCGGAA-3’(SEQ ID No:8)。
反应结束后4℃保存。
(5)对目的片段使用胶回收试剂盒进行切胶回收。
(7)37℃过夜培养从抗性LB培养基上挑取单克隆后37℃摇菌培养。
(8)菌液PCR验证,挑取阳性克隆样品,送样至金唯智生物科技有限公司测序,测序正确的菌液中加入一定量的甘油,使甘油终浓度在20%左右,-70℃保存。
2.GhFLA19-D-CRISPR载体构建
2.1靶点序列确定和引物设计
根据提供的mRNA序列及对应的基因组序列信息,设计2个CRISPR靶位点,根据靶位点设计PCR扩增引物;将相应的引物加上in-fusion接头,进行合成后,用于后续连接实验。
表5靶位点序列
表6 PCR扩增引物
2.2目的片段扩增
采用重叠延伸PCR扩增出含有靶位点的片段,反应体系如下所示:
PCR体系:
表7目的片段扩增反应体系
PCR程序为:
2.3载体构建
利用BsaI酶切载体pRGEB32-GhU6.9-NPT II,酶切体系:
表8酶切体系
扩增完成后,将目的片段连接到酶切后的pRGEB32-GhU6.9-NPT II载体上
表9 In-fusion连接反应体系
37℃水浴30min,冰上放置5min,可-20℃保存。
2.4电击转化大肠杆菌
将构建的CRISPR载体电转入大肠杆菌TOP10,通过菌落PCR筛选阳性克隆子。检测引物为U6-7s:TGTGCCACTCCAAAGACATCAG(SEQ ID No:13),GhFLA19-D-inf-T2as:TTCTAGCTCTAAAACCGTCTCCCCACATTTCCTCT(SEQ ID No:12)。
阳性克隆子检测方法如下所示:
PCR体系:
表10阳性克隆子检测PCR体系
PCR程序:
CRISPR载体挑选1个阳性克隆子进行测序。
3.利用农杆菌介导棉花茎段的遗传转化
A.将阳性克隆转化到农杆菌感受态菌株GV3101,农杆菌扩大培养,离心弃上清,加入侵染液(MGL和AS),震荡使菌液悬浮,28℃摇床,200rpm/min活化至少30min;
B.将受体棉花(HM-1)种子用升汞杀菌,无菌水清洗后放入无菌苗培养基中,30℃培养6d;
C.将受体小苗下胚轴切成小茎段,用活化后的农杆菌侵染,并吹干;
D.将下胚轴平铺在放有滤纸的共培养培养基中,20℃暗培养1-2d;
E.下胚轴转入到2,4-D培养基中,放入光照培养室,20-30d左右继代一次;
F.愈伤组织长成米粒状颗粒,转入分化培养基中,进一步分化成胚状体;
G.将分化出的小苗继代到生根培养基中,直至长成生根良好健康的小苗;
H.将苗子转到水中,进行炼苗,一周左右后,种到温室。
4.转基因棉花植株基因编辑情况的检测
4.1转基因植株PCR检测
共得到18株转基因单株,剪取再生植株叶片,利用CTAB法抽提DNA,利用nptⅡ特异引物进行PCR检测。
表11转基因植株检测引物
PCR体系:
表12转基因植株检测反应体系
PCR反应程序:
图2示出了棉花转基因植株PCR检测的结果图,检测结果发现13株阳性单株,编号为3、4、5、6、7、8、10、12、13、14、15、17和18。
4.2二代测序检测转基因植株编辑情况
基于参考序列设计引物扩增包含两个靶点之间的序列,对13个单株的DNA进行扩增。扩增的引物序列如下:
表13待测序产物扩增引物
检测体系及程序:
扩增体系如下:擎科T3 Mix 27μl,Primer F(10μM)0.5μl,Primer R(10μM)0.5μl,DNA模板1.5μl,补水至30μl。反应程序为98℃2min;98℃10s,56℃10s,72℃10s,30cycles;72℃5min;25℃2min。
检测结果:利用上表的引物对这13个样品进行PCR扩增及建库。
对PCR扩增的文库样品进行混合,挖胶回收以后,测定样品浓度,上机进行高通量测序,然后以提供的序列为参考基因组序列对测序数据进行分析,获取每个样品中的靶位点区段的基因序列类型及其详细序列信息。
结果显示,3、4、5、6、7、8、10、13、14、15和18转基因植株的GhFLA19-D基因被完全编辑,12、17含有未编辑的GhFLA19-D野生型序列。
5.转基因植株的表型鉴定
对不同编辑类型的转基因单株进行表型观察,结果如图3所示,其中图3中A为完全编辑的转基因植株line3的编辑信息示意图,其中发现有两种编辑类型的序列,均与参考序列不一致;B为未完全编辑的转基因植株line12的编辑信息示意图,共发现两种类型的序列,其中一种与参考序列一致,另一种为编辑后的序列;C、E和G为WT的植株、花器官和花粉染色;D、F和H为T2代不育株的植株、花器官示意图和花粉染色结果图。
发现对GhFLA19-D完全编辑的单株表现为完全不育,具体表现为缩短的花丝和干瘪的花药,而且通过淀粉-碘化钾染色发现花药内无成熟的花粉粒。而对GhFLA19-D的杂交F2代单株进行调查发现,发现F2后代出现表型分离,其中有部分单株表现为雄性不育,其育性表型与T0代一致,表现为稳定不育。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 中国农业科学院棉花研究所
<120> 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用
<130> PA21000810
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 250
<212> PRT
<213> GhFLA19-D 氨基酸
<400> 1
Met Glu Asn Phe Ser Ser Lys Pro Thr Ile Val Ile Leu Leu Leu Leu
1 5 10 15
Thr Thr Val Ser Thr Ala Asp Leu Thr Ser Lys Glu Leu Asp Ala Ala
20 25 30
Ile Leu Val Leu Gln Ser Arg Gly Tyr Thr Leu Phe Pro Asn Ala Ile
35 40 45
Ser Thr Ser Asp Leu Gln Val Arg Leu Leu Ser Ser Gln Asn Ser Ser
50 55 60
Ile Phe Thr Leu Phe Ala Pro Pro Asp Ser Leu Leu Phe Ser Leu Asp
65 70 75 80
Leu Leu Ser Ser Ala Arg Leu Tyr Thr Phe Ser Leu Phe Leu His Val
85 90 95
Ser Pro His Phe Leu Ser Ser Ser Asp Leu Leu Ala Leu Pro Arg Pro
100 105 110
Ala Phe Ile Asp Thr Leu Leu Pro Asn Arg Arg Leu Phe Val Glu His
115 120 125
Ala Met Ser Thr Arg Asn Gly Thr Ala Leu Leu Thr Val Ser Val Asp
130 135 140
Gly Val Val Val Ser Val Pro Asp Leu Phe Leu Gly Ser Asn Ile Val
145 150 155 160
Val His Gly Leu Asp Gly Ile Leu Val Ala Arg Tyr Gly Ser Leu Val
165 170 175
Ser Glu Gly Ser Asp Asn Ala Ile Ala Glu Pro Pro Lys Phe Pro Tyr
180 185 190
Gln Thr Tyr Val Ser Pro Ala Asn Pro Pro Glu Thr Leu Pro Pro Thr
195 200 205
Asp Leu Glu Met Val Thr Ile Gly Thr Gln Ile Lys Lys Asp Arg Glu
210 215 220
Ala Phe Arg Arg Asp Asp Asp His Ala Thr Thr Lys Arg Thr Lys His
225 230 235 240
Gly Thr Phe Phe Arg Phe Glu Arg Val Tyr
245 250
<210> 2
<211> 753
<212> DNA
<213> GhFLA19-D CDS
<400> 2
atggaaaact tttcctccaa accaacaatc gtaatcctcc tcctcctcac caccgtcagc 60
accgccgact taacttccaa agaactagac gcagccatct tagtccttca atcaagaggc 120
tacactctct tccccaacgc catctccaca tcggatctcc aagtccgcct cctctcatcc 180
caaaactctt ccatattcac tctttttgca cccccggact ccctcctctt ctccctcgac 240
ctcctctcct ccgcccgcct ttacactttc tctctctttc tccacgtctc cccacatttc 300
ctctcctcct cagacctcct cgccctccct cgccccgcct tcatcgacac cctcctccct 360
aaccgtcgac tcttcgtaga acatgctatg tctacccgca acggcacagc cttgctaact 420
gtttccgtcg acggggttgt cgtctccgtc ccggatcttt tccttggatc caacattgtt 480
gtccacgggc ttgatgggat tcttgttgca agatacgggt ccttggttag tgaaggtagt 540
gacaatgcta ttgctgagcc acccaagttc ccctatcaaa cctatgtttc gccggccaac 600
ccaccggaga ctttgccccc tacggacctg gagatggtca caatcggaac gcaaatcaag 660
aaagataggg aggctttccg tcgtgatgat gaccatgcca ctactaagag aactaaacac 720
ggtacatttt tccggtttga acgcgtttac tga 753
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<400> 3
ccacatcgga tctccaagtc cgc 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列
<400> 4
ccacgtctcc ccacatttcc tct 23
<210> 5
<211> 22
<212> DNA
<213> 人工序列
<400> 5
cgccatctcc acatcggatc tc 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列
<400> 6
tgtggggaga cgtggagaaa ga 22
<210> 7
<211> 23
<212> DNA
<213> 人工序列
<400> 7
atggaaaact tttcctccaa acc 23
<210> 8
<211> 24
<212> DNA
<213> 人工序列
<400> 8
tcagtaaacg cgttcaaacc ggaa 24
<210> 9
<211> 36
<212> DNA
<213> 人工序列
<400> 9
catcggatct ccaagtccgc tgcaccagcc gggaat 36
<210> 10
<211> 38
<212> DNA
<213> 人工序列
<400> 10
gcggacttgg agatccgatg gttttagagc tagaaata 38
<210> 11
<211> 36
<212> DNA
<213> 人工序列
<400> 11
cgtctcccca catttcctct tgcaccagcc gggaat 36
<210> 12
<211> 35
<212> DNA
<213> 人工序列
<400> 12
ttctagctct aaaaccgtct ccccacattt cctct 35
<210> 13
<211> 22
<212> DNA
<213> 人工序列
<400> 13
tgtgccactc caaagacatc ag 22
<210> 14
<211> 21
<212> DNA
<213> 人工序列
<400> 14
actgggcaca acagacaatc g 21
<210> 15
<211> 23
<212> DNA
<213> 人工序列
<400> 15
gcatcagcca tgatggatac ttt 23
<210> 16
<211> 18
<212> DNA
<213> 人工序列
<400> 16
actagacgca gccatctt 18
<210> 17
<211> 18
<212> DNA
<213> 人工序列
<400> 17
agggcgagga ggtctgag 18
Claims (10)
1.一种调控棉花雄性生殖发育的GhFLA19-D蛋白,其特征在于,所述蛋白具有如SEQ IDNo:1所示的氨基酸序列。
2.一种调控棉花雄性生殖发育的基因,其特征在于,所述基因编码权利要求1的所述蛋白,所述基因的CDS序列如SEQ ID No:2所示。
3.权利要求1所述的蛋白或权利要求2所述的基因在控制棉花雄性生殖发育中的应用。
4.权利要求1所述的蛋白或权利要求2所述的基因在培育雄性不育转基因棉花中的应用。
5.根据权利要求4所述的应用,其特征在于,所述应用包括采用基因敲除、基因敲减或者基因编辑的方法抑制所述基因的表达或使所述基因功能缺失,使突变后的棉花出现雄性不育性状。
6.根据权利要求5所述的应用,其特征在于,通过CRISPR/Cas9系统、TALEN系统、锌指酶系统、RNA干扰技术实现抑制所述基因的表达或使所述基因功能缺失。
7.根据权利要求6所述的应用,其特征在于,在利用所述CRISPR/Cas9系统构建载体时,设计的CRISPR/Cas9载体的靶位点序列为:如SEQ ID No:3所示序列和如SEQ ID No:4所示序列。
8.一种培育雄性不育系转基因棉花的方法,其特征在于,所述方法包括抑制棉花植物中权利要求2所述基因的表达,得到雄性不育系转基因植物。
9.根据权利要求8所述的方法,其特征在于,所述方法包括构建所述基因的CRISPR/Cas9载体,将表达载体导入宿主菌中,筛选表达所述基因的工程菌;将工程菌导入目标植物中,筛选转基因植株。
10.根据权利要求8或权利要求9所述的方法,其特征在于,所述棉花包括海岛棉和陆地棉,优选陆地棉。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110270214.0A CN112830963B (zh) | 2021-03-12 | 2021-03-12 | 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110270214.0A CN112830963B (zh) | 2021-03-12 | 2021-03-12 | 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112830963A true CN112830963A (zh) | 2021-05-25 |
CN112830963B CN112830963B (zh) | 2022-05-31 |
Family
ID=75930039
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110270214.0A Expired - Fee Related CN112830963B (zh) | 2021-03-12 | 2021-03-12 | 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112830963B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113862278A (zh) * | 2021-08-24 | 2021-12-31 | 石河子大学 | 陆地棉GhMS20基因及其在创制棉花单显性雄性不育系的应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994025593A2 (en) * | 1993-05-03 | 1994-11-10 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) | Method for obtaining male-sterile plants |
WO2017121411A1 (zh) * | 2016-05-16 | 2017-07-20 | 中国科学院遗传与发育生物学研究所 | 一种与植物雄性育性相关的蛋白及其编码基因与应用 |
CN111574602A (zh) * | 2020-05-14 | 2020-08-25 | 中国科学院东北地理与农业生态研究所 | GmAMS1蛋白、编码基因及其抑制因子和创制植物细胞核雄性不育系的方法 |
-
2021
- 2021-03-12 CN CN202110270214.0A patent/CN112830963B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994025593A2 (en) * | 1993-05-03 | 1994-11-10 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) | Method for obtaining male-sterile plants |
WO2017121411A1 (zh) * | 2016-05-16 | 2017-07-20 | 中国科学院遗传与发育生物学研究所 | 一种与植物雄性育性相关的蛋白及其编码基因与应用 |
CN111574602A (zh) * | 2020-05-14 | 2020-08-25 | 中国科学院东北地理与农业生态研究所 | GmAMS1蛋白、编码基因及其抑制因子和创制植物细胞核雄性不育系的方法 |
Non-Patent Citations (4)
Title |
---|
ALICE Y. CHEUNG 等: "A Floral Transmitting Tissue-Specific Glycoprotein Attracts Pollen Tubes and Stimulates Their Growth", 《CELL》 * |
未披露: "Predicted:fasciclin-like arabinogalactan protein 19[Gossypium raimondii]", 《NCBI GENBANK蛋白序列数据库》 * |
杨杰 等: "植物亲和受精过程中花粉管的粘附和定向生长", 《植物生理学通讯》 * |
王雅琴: "棉花GhFLA4基因的克隆及表达分析", 《新疆农业科学》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113862278A (zh) * | 2021-08-24 | 2021-12-31 | 石河子大学 | 陆地棉GhMS20基因及其在创制棉花单显性雄性不育系的应用 |
CN113862278B (zh) * | 2021-08-24 | 2024-03-12 | 石河子大学 | 陆地棉GhMS20基因及其在创制棉花单显性雄性不育系的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN112830963B (zh) | 2022-05-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2019297209B2 (en) | Method of obtaining multi-leaf alfalfa material by means of MsPALM1 artificial site-directed mutant | |
CN112626080B (zh) | 一种控制大豆-根瘤菌匹配性的r基因及其蛋白质和应用 | |
CN107475210B (zh) | 一种水稻白叶枯病抗性相关基因OsABA2及其应用 | |
CN112961231A (zh) | 雄性不育基因ZmbHLH122及其在创制玉米雄性不育系中的应用 | |
CN112899247B (zh) | 雄性不育基因ZmTKPR1及其在创制玉米雄性不育系中的应用 | |
CN113637060B (zh) | 大豆GmSPA3a/3b蛋白及其相关生物材料在调控植物开花和株高中的应用 | |
CN113583099B (zh) | 培育苜蓿雄性不育系及相应保持系的方法及其相关生物材料 | |
CN112680461B (zh) | 雄性不育基因ZmPHD11及其在创制玉米雄性不育系中的应用 | |
CN113215172B (zh) | 雄性不育基因MsJMT及其应用 | |
CN112680459B (zh) | 雄性不育基因ZmTGA10及其在创制玉米雄性不育系中的应用 | |
CN113150098B (zh) | GmEID1蛋白在调控大豆开花和主茎节数中的应用 | |
US11365423B2 (en) | Method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants | |
CN112813098B (zh) | 利用人工突变创制玉米bhlh51雄性不育系 | |
CN112830963B (zh) | 一种调控棉花雄性生殖发育的GhFLA19-D蛋白及其编码基因与应用 | |
CN111662366A (zh) | 一种早花高产番茄材料的制备方法 | |
CN113005128A (zh) | 雄性不育基因ZmMYB84及其在创制玉米雄性不育系中的应用 | |
CN112980876B (zh) | GhGPAT12蛋白和GhGPAT25蛋白在调控棉花雄性生殖发育中的应用 | |
CN112680460B (zh) | 雄性不育基因ZmTGA9及其在创制玉米雄性不育系中的应用 | |
CN114921583A (zh) | 一种控制小麦株高的QTL及其候选基因TaDHL-7B和应用 | |
CN111499709B (zh) | 水稻穗粒数相关的rgn1蛋白及其编码基因与应用 | |
CN113046377A (zh) | 一种雄性不育基因MsGAL及其应用 | |
CN114231556B (zh) | GmECT2在调控植物高度方面的应用 | |
CN112680458B (zh) | 雄性不育基因ZmMYB33及其在创制玉米雄性不育系中的应用 | |
CN107338257B (zh) | 水稻谷氧还蛋白基因OsGrxC2在育种中的应用 | |
CN115925853A (zh) | 烟草控制腋芽起始的NtDA1蛋白质及其相关生物材料和应用 |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220531 |