CN105695506A - 一种改良棉籽营养品质的方法及其用途 - Google Patents
一种改良棉籽营养品质的方法及其用途 Download PDFInfo
- Publication number
- CN105695506A CN105695506A CN201610255002.4A CN201610255002A CN105695506A CN 105695506 A CN105695506 A CN 105695506A CN 201610255002 A CN201610255002 A CN 201610255002A CN 105695506 A CN105695506 A CN 105695506A
- Authority
- CN
- China
- Prior art keywords
- gene
- cottonseed
- cotton
- ghpsy2
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 235000016709 nutrition Nutrition 0.000 title claims abstract description 11
- 235000012343 cottonseed oil Nutrition 0.000 title abstract description 41
- 235000021466 carotenoid Nutrition 0.000 claims abstract description 43
- 150000001747 carotenoids Chemical class 0.000 claims abstract description 43
- 229920000742 Cotton Polymers 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 101710173432 Phytoene synthase Proteins 0.000 claims abstract description 15
- 239000013604 expression vector Substances 0.000 claims abstract description 14
- 101150010975 PSY gene Proteins 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims description 19
- 241000196324 Embryophyta Species 0.000 claims description 18
- 230000009466 transformation Effects 0.000 claims description 9
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 5
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 2
- 230000031787 nutrient reservoir activity Effects 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 244000299507 Gossypium hirsutum Species 0.000 description 28
- 230000009261 transgenic effect Effects 0.000 description 17
- 239000012634 fragment Substances 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- 239000002385 cottonseed oil Substances 0.000 description 10
- 238000000605 extraction Methods 0.000 description 10
- 239000013598 vector Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 241000701489 Cauliflower mosaic virus Species 0.000 description 6
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 6
- 240000003768 Solanum lycopersicum Species 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 5
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 5
- YVLPJIGOMTXXLP-UHFFFAOYSA-N all-trans-Phytoen Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 235000013734 beta-carotene Nutrition 0.000 description 5
- 239000011648 beta-carotene Substances 0.000 description 5
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 5
- 229960002747 betacarotene Drugs 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000035764 nutrition Effects 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 description 3
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 description 3
- 241000219194 Arabidopsis Species 0.000 description 3
- 108010037139 Cryptochromes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 235000011765 phytoene Nutrition 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 2
- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 2
- 108010025815 Kanamycin Kinase Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000012877 elongation medium Substances 0.000 description 2
- 230000000408 embryogenic effect Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000008117 seed development Effects 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 235000019155 vitamin A Nutrition 0.000 description 2
- 239000011719 vitamin A Substances 0.000 description 2
- 229940045997 vitamin a Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 101710197635 Actin-4 Proteins 0.000 description 1
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108010066605 Geranylgeranyl-Diphosphate Geranylgeranyltransferase Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 235000009429 Gossypium barbadense Nutrition 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 1
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 1
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 238000000944 Soxhlet extraction Methods 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000013793 astaxanthin Nutrition 0.000 description 1
- 239000001168 astaxanthin Substances 0.000 description 1
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 1
- 229940022405 astaxanthin Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 201000011529 cardiovascular cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012869 germination medium Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- -1 isoprenoid compounds Chemical class 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 235000012661 lycopene Nutrition 0.000 description 1
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 1
- 239000001751 lycopene Substances 0.000 description 1
- 229960004999 lycopene Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 235000018322 upland cotton Nutrition 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明属于植物基因工程技术领域，具体涉及一种改良棉籽营养品质的方法及其用途。本发明要解决的技术问题是为提高棉籽类胡萝卜素含量改良棉籽营养品质提供一种新选择。本发明的技术方案是构建种子特异启动子驱动八氢番茄红素合成酶基因PSY的植物表达载体，并转化棉花，提高棉籽中类胡萝卜素含量，所述PSY基因为棉花GhPSY2基因。经本发明对目标基因进行调控后，成熟棉籽中类胡萝卜素含量显著提高。本发明方法简便易行，效果显著，有产生巨大经济效益的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种改良棉籽营养品质的方法及其用途。
背景技术
类胡萝卜素属于类异戊二烯化合物，是一类重要的植物天然色素。类胡萝卜素是一类脂溶性色素，其色彩亮丽，主要呈现黄色、橙红色及红色。对于动物和人体来说，类胡萝卜素是重要的健康保护物质。作为人和动物体内唯一的维生素A源，类胡萝卜素转化形成的维生素A是人体和动物不可或缺的营养物质，能够有效地起到视觉和皮肤系统的保健作用。此外，类胡萝卜素具有抗氧化作用，它的存在能够延缓衰老，降低心血管类疾病和癌症的发病率。但动物体自身不能直接合成类胡萝卜素，所需的类胡萝卜素均需通过食物链从植物或微生物中摄取。通过微生物发酵只能获得β-胡萝卜素、虾青素等少数几种类胡萝卜素，远不能满足需求。植物中直接提取的类胡萝卜素安全可靠，生理药理活性明显。然而植物体中类胡萝卜素含量有限，培育类胡萝卜素含量丰富的农作物具有重要应用价值。
棉花是世界上最重要的农作物之一，是人类获取天然纤维的主要来源。作为棉花生产过程中最大的副产物，棉籽具有巨大的可利用价值。棉籽中含有丰富的蛋白质及油脂等营养物质，是榨油和制取饲料的重要原料。棉籽中类胡萝卜素含量约15μg/g。培育棉籽中高类胡萝卜素的棉花品种，加大棉籽的综合利用，将有益于提升棉花种植的经济效益。
近年来，植物中类胡萝卜素代谢工程的主要目标是调控合成途径中结构基因的表达。目前，关于利用类胡萝卜素合成酶基因的表达来调控类胡萝卜素合成在诸多植物上已有较多成功的报道。例如，“黄金大米”，将Lcyb、Psy、CrtI(Pds)基因共转化入一个粳稻品种，得到金黄色的胚乳。分析表明，该胚乳中包含多种类胡萝卜素，且含量丰富。专利公开号为CN101979601A的中国专利申请公开一种提高番茄类胡萝卜素含量的方法，是利用基因工程手段构建隐花色素基因1，隐花色素基因1的羧基端和隐花色素基因2的羧基端的过量表达载体，转化番茄，获得转基因番茄，实现了转基因番茄果实中番茄红素及总类胡萝卜素含量较大幅的提高。
八氢番茄红素合成酶(PSY)是类胡萝卜素生物合成途径中所涉及的重要限速酶，它催化2分子的牻牛儿牻牛儿焦磷酸(GGPP)缩合形成第一个类胡萝卜素——八氢番茄红素。在番茄、油菜、拟南芥和马铃薯中过量表达八氢番茄红素合成酶，相应组织中积累类胡萝卜素的水平显著提高。可见，改变八氢番茄红素合成酶表达水平将深刻影响植物体中类胡萝卜素含量。但到目前为止，尚未见有关于棉籽类胡萝卜素含量改良的报道。
发明内容
本发明要解决的技术问题是为棉籽营养改良提供一种新选择。
本发明的技术方案是一种改良棉籽营养品质的方法，构建种子特异启动子驱动八氢番茄红素合成酶基因PSY的植物表达载体，并转化棉花，提高棉籽中类胡萝卜素含量，所述PSY基因为棉花GhPSY2基因。
具体的，所述的GhPSY2基因具有如SEQIDNo.1所示的核苷酸序列。
具体的，调控PSY基因表达的启动子为菜豆种子特异启动子。
优选的，所述的种子特异启动子为菜豆储存蛋白β-菜豆蛋白β-phaseolin基因的启动子pV，具有如SEQIDNo.2所示的核苷酸序列。
具体的，所述植物表达载体如SEQIDNo.3所示的核苷酸序列。
具体的，所述的转化为根癌农杆菌介导的遗传转化。
本发明还提供了所述的方法在提高棉籽类胡萝卜素含量中的用途。
本发明的有益效果：本发明通过研究和分析，采用特异启动子在种子发育时期特异表达棉花八氢番茄红素合成酶基因GhPSY2，获得了棉籽中类胡萝卜素含量显著提高的转基因棉花。试验结果证明，经本发明对目标基因进行调控后，改良的棉籽中类胡萝卜素含量显著提高，棉籽营养得到强化。本发明方法简便易行，效果显著，有产生巨大经济效益的潜力。
附图说明
图1pV:GhPSY2的表达载体T-DNA区的基因结构图
CaMV35S-P，花椰菜花叶病毒35S启动子；2×CaMV35S-P，串联两个花椰菜花叶病毒35S启动子；CaMV35S-T，花椰菜花叶病毒35S终止子；GhPSY2，棉花八氢番茄红素合成酶基因；GUS:NPTII，β-葡萄糖酸苷酶与新霉素磷酸转移酶融合基因；LB，T-DNA左边界；Nos-T，农杆菌冠瘿碱合成酶基因终止子；pV，菜豆种子特异启动子；RB，T-DNA右边界。
图2pLGN-pV:GhPSY2表达载体构建流程
具体实验方法见实施例2。CaMV35S-P，花椰菜花叶病毒35S启动子；2×CaMV35S-P，串联两个花椰菜花叶病毒35S启动子；CaMV35S-T，花椰菜花叶病毒35S终止子；GhPSY2，棉花八氢番茄红素合成酶基因；GUS:NPTII，β-葡萄糖酸苷酶与新霉素磷酸转移酶融合基因；LB，T-DNA左边界；Nos-T，农杆菌冠瘿碱合成酶基因终止子；pV，菜豆种子特异启动子；RB，T-DNA右边界。相关酶切位点及位置在各个载体上标出。
图3野生型(WT)棉花及pV:GhPSY2转基因棉花
A：GhPSY2表达水平(Relativeexpression)检测；B：野生型或转基因型棉花成熟种子；C：野生型或转基因型棉花粉碎后的成熟种子；D：用乙醚稀释20倍的棉籽油；E：棉籽油中类胡萝卜素含量。WT：野生型样品；#1、#2：pV:GhPSY2转基因棉花的两个株系；*和**显示与野生型对照间呈显著额极显著差异(t检验，n＝3)。
具体实施方式
下述实施例中所用到的常规实验操作：
1.DNA的提取
基因组DNA采用植物基因组DNA快速提取试剂盒(Aidlab)提取，详细步骤见说明书。
2.RNA的提取
RNA采用EASYspin植物RNA快速提取试剂盒(Aidlab)提取，详细步骤见说明书。
3.DNA片段的PCR扩增
扩增体系如下：10×ExPCRbuffer(Mg2+free)2.5μL，2.5mmol/LdNTPs2μL，25mmol/LMgCl22μL，引物1(5μmol/L)1μL，引物2(5μmol/L)1μL，ExTaqDNA聚合酶1U，基因组DNA约60ng，加入ddH2O至25μL。
扩增程序为：94℃，5min；94℃，30sec，56℃，30sec，72℃，1.5min，35个循环；72℃延伸10min。
4.DNA片段的回收、连接和克隆
使用BioFlux胶回收试剂盒回收DNA片段。使用T4DNAligase进行DNA片段连接。回收的片段与pUCm-T(上海生工)载体建立如下连接体系：10×T4DNA连接缓冲液1μL，载体DNA片段1μL，外源连接产物DNA片段1μL，T4DNA连接酶1μL，用ddH2O补足体积至10μL。载体DNA片段与外源连接产物DNA片段摩尔比为1︰3，16℃连接12h。之后将连接产物转化大肠杆菌DH5α。获得的抗性克隆经液体培养过夜，用BioFlux质粒提取试剂盒提取质粒，酶切验证后，在Invitrogen公司测序。
5.GUS组织化学染色
由于实验室采用的表达载体具有GUS报告基因，一般用GUS组织化学染色检测跟踪转基因。具体方法：取少量转基因棉花叶片(有伤口)置于96孔板中，加入GUS染液[0.1mol/LK3Fe(CN)6，0.1mol/LK4Fe(CN)6，0.01mol/LNa2EDTA，500mg/LX-Gluc，1％TritonX-100(v/v)，0.14mol/L磷酸钠缓冲液(pH7.0)]，在37℃恒温条件下放置2h左右，充分染液后再用75％乙醇脱色。植株叶片可被GUS染液染成特异蓝色的植株为转基因阳性。
6.棉籽油的抽提及其类胡萝卜素含量测定
棉籽油提取采用索氏抽提法：
1)将折叠好的滤纸包放入玻璃皿中在105℃烘箱中干燥2h，取出放入干燥器冷却至室温，准确称重记W1。
2)取约3g种子用粉碎机粉碎，过40目筛网，取约1g样品装入上述已称重的滤纸包中，放入105℃烘箱中干燥3h，取出放入干燥器冷却至室温，准确称重记W2。
3)上述装有样品并称重的滤纸包放入脂肪提取仪(BUCHIB-811抽提系统)中，足量乙醚浸泡过夜。
4)过夜后，70～80℃抽提8h，抽提完成后，收集底部油脂(即棉籽油)，纸包放入105℃烘箱中干燥3h，取出放入干燥器冷却至室温，准确称重记W3。
5)种子中粗脂肪含量(％)＝(W2-W3)/(W2-W1)×100％
进一步，将上述抽提获得的棉籽油用乙醚稀释n倍至酶标仪可检测范围，取200μL于酶标板，酶标仪检测445nm处的吸光值，将吸光值代入标准曲线(参考β-胡萝卜素标准曲线)，计算类胡萝卜素含量。
7.β-胡萝卜素标准曲线的制作
1)准确称取1mgβ-胡萝卜素标准品，用乙醚定容于10ml棕色容量瓶，β-胡萝卜素浓度为100μg/ml；
2)继续稀释至7个浓度梯度：0、0.1、1、3、6、9、12、15μg/mL；
3)取200μL于酶标板，酶标仪检测445nm处的吸光值；
4)线性回归，制作标准曲线。
表1引物一览表
| 引物 | 序列5’-3’ | 描述 |
| GhPSY2-U | GGATCCATGGCTGGTGTTCTTCTTTGGGTG | 基因扩增上游引物 |
| GhPSY2-D | GAATTCACTAGTGATCTTCAGAACAATTTG | 基因扩增下游引物 |
| pV-F | AAGCTTGAATTCATTGTACTCCCAGTATC | 启动子扩增上游引物 |
| pV-R | GGATCCCTCGAGTCTAGAAGTAGTATTG | 启动子扩增下游引物 |
| GhPSY2-F(RT) | ATGAATTTTGATGAGCTCTAT | RT-PCR上游引物 |
| GhPSY2-R(RT) | ATTAGCAATTCCGAGGGCTA | RT-PCR下游引物 |
| GhACT4-F(RT) | TTGCAGACCGTATGAGCAAG | RT-PCR上游引物 |
| GhACT4-R(RT) | ATCCTCCGATCCAGACACTG | RT-PCR下游引物 |
实施例1棉花八氢番茄红素合成酶基因GhPSY2获得
在Phytozome公共数据库(https://phytozome.jgi.doe.gov)上，以拟南芥八氢番茄红素合成酶基因AtPSY(At5g17230)碱基序列为诱饵，通过BlastN搜索雷蒙德氏棉中的同源基因。结果显示雷蒙德氏棉中编号为Gorai.006G009400的基因与At5g17230具有高度同源性。以Gorai.006G009400的CDS序列为参考设计引物(表1，GhPSY2-U&GhPSY2-D)，以陆地棉cDNA为模板，扩增获得片段，并在片段两端加上BamHⅠ和EcoRⅠ位点，测序结果如SEQIDNO.1。聚类分析表明，该序列与来着其它物种(如拟南芥、番茄、大豆等)的已知八氢番茄红素合成酶基因具有高度同源性，因此该序列被认为是棉花八氢番茄红素合成酶编码基因(GhPSY2)。
实施例2种子发育时期特异的GhPSY2表达载体的构建
GhPSY2构建入植物表达载体pLGN-Nos的流程见图2。pLGN-Nos载体是由传统的植物表达载体pBI121改造而来的一个双元植物表达载体。其T-DNA区段(RB和LB之间区域，图2)替换为了组成型的CaMV35S启动子(CaMV35S-P)控制报告基因GUS和标记基因NPTII的融合基因表达盒，以及另一个由CaMV35S-P控制的表达盒。
用引物(表1，pV-F和pV-R)从菜豆基因组中扩增克隆启动子pV，并在启动子两端加上HindⅢ和BamHⅠ位点。用BamHⅠ和EcoRⅠ将GhPSY2从克隆载体上切下，用HindⅢ和BamHⅠ将启动子pV从克隆载体上切下。上述两个片段同时加入到含有经HindⅢ和EcoRⅠ酶切的pLGN-Nos载体片段的连接反应体系中，连接获得最终的表达载体pLGN-pV:GhPSY2。所有限制性内切酶购自Roche公司，按照使用说明书操作。DNA片段的回收、连接和大肠杆菌转化按前述常规操作方法进行。参考Bio-RADMicroPulser用户说明书,将上述载体通过电激转化法导入农杆菌LB4404。
实施例3棉花的遗传转化
通过根癌农杆菌介导的方法进行上述表达载体的棉花遗传转化，所用培养基配方见表2。具体方法如下：选取颗粒饱满的野生型冀棉14号棉花种子(脱壳)，置于无菌三角瓶中，75％酒精润洗1分钟后0.1％HgCl2灭菌约10分钟(摇动三角瓶)，无菌水充分漂洗6-8次，置于30℃摇床(100-110rpm)，待胚根长出1cm左右(约24-36h，每隔8小时换一次水)，取出，将胚根插入种子萌发培养基中。30℃暗室培养至下胚轴伸长到2-3cm(约48h)。将携带表达框载体的农杆菌单菌落接种于含有50mg/LKm和125mg/LSm的液体YEB培养基中，置于28℃摇床(200rpm)。约20h后每隔一个小时测量菌液的OD值(OD600)，OD600在0.8-1.0适宜。收集农杆菌液，离心，8000rpm，1min，弃上清。用含有AS(乙酰丁香酮，100μmo1/L)的共培养液体培养基按1:1(v/v)重悬沉淀后，收集重悬液于100mL三角瓶，置于30℃摇床(100-110rpm)培养约30min。将下胚轴切成长约0.8-1cm的小段，置于重悬液中，30℃摇床上侵染40min，弃液体，将下胚段铺在共培养基表面，于暗室培养48h左右，转入筛选培养基中，30℃培养(16h光照/8h黑暗)，每隔15天左右继代一次至愈伤形成。待愈伤增多后，转到愈伤培养基上培养。挑取状态良好的胚性愈伤接入悬浮培养基中，置于30℃(100-110rpm)摇床上悬浮培养一周左右，经30目筛网过滤，将过滤后的细小胚性颗粒铺于体胚伸长培养基中，至绿色成熟体胚长出。转移至伸长培养基中继续培养体胚至1cm左右，转入生根培养基中直至幼苗长成(约10cm)，移栽至营养钵，继续培养。以上操作均须在无菌条件下完成。
将筛选出的GUS+且生长健壮的再生棉花幼苗移栽至种植钵，在温室中常规管理至棉花纤维和种子成熟。收获T1代转基因棉花种子，继续种植T1代并收获T2代种子，播种T2代种子后对萌发的T2代幼苗进行GUS组织染色(见常规操作方法)，筛选出纯合的转基因T2代株系(全部为GUS阳性或GUS阴性植株)，移栽T2代纯合株系，并检测靶标基因GhPSY2的表达水平和考察种子性状变化。
表2根癌农杆菌介导的棉花遗传转化用培养基
MS：Murashige&Skoog，1962；B5：Gamborg，1986；Gelrite：Sigma，货号：G1910；SH：Schenk&Hildebrandt，1972。
实施例4GhPSY2基因转录水平的检测
提取对照与转基因棉花开花后36天胚的RNA，逆转录合成cDNA一链，以此为模板进行定量PCR检测。具体操作步骤为：用cDNA一链合成试剂盒(TaKaRa公司)合成各种RNA的一链cDNA，操作均按试剂盒说明书进行。定量PCR在CFX96定量PCR检测系统(Bio-Rad)上进行，25μL的反应体系包括12.5μL2×SuperMix(Bio-Rad)，上下游引物各0.2μmol/L和1μL一链cDNA。温度循环参数为95℃预变性2min；95℃，10sec，57℃，20sec，扩增40循环。用棉花Actin4基因作内标。用定量PCR仪自带的分析软件Bio-RadCFXManager2.0计算各个基因的相对表达量。所用定量PCR引物序列见表1。
基因表达分析发现：GhPSY2在转基因样品中转录水平显著高于野生型样品，表明pV启动子可在胚中启动GhPSY2的表达(图3A)。
实施例5转基因棉花种子性状的考察
类胡萝卜素是一类重要萜类天然色素，呈现黄色、橙色或红色等。观察棉花成熟种子：野生型成熟种子呈白色，而pV:GhPSY2转基因棉花种子呈橙色(图3B)。进一步将成熟种子粉碎，野生型样品呈浅黄色，pV:GhPSY2转基因样品呈橙色(图3C)。以乙醚作为溶剂，抽提种子中的棉籽油，抽提获得的棉籽油20倍稀释于乙醚，野生型样品接近无色，而转基因样品仍然保持明显的黄色(图3D)。类胡萝卜素含量检测显示(图3E)：野生型样品的棉籽油中类胡萝卜素含量为38.63±4.02μg/mL，两个转基因样品的棉籽油中类胡萝卜素含量分别为269.05±4.47μg/mL和245.22±6.36μg/mL，较野生型样品大幅提高。综上所述，启动子pV启动八氢番茄红素合成酶基因GhPSY2的表达能促进棉花种子中类胡萝卜素的大量合成与积累。
上述实例表明，本发明改良棉籽营养的方法，能够实现在胚中上调GhPSY2的表达，促进类胡萝卜素合成，达到强化棉籽营养的目的。本发明，通过基因序列同源比对，发现并克隆获得一个棉花八氢番茄红素合成酶基因(GhPSY2)。利用菜豆种子特异启动子pV指导GhPSY2在棉籽中的特异表达，提高棉籽中八氢番茄红素合成酶水平，以达到提升棉籽中类胡萝卜素含量的目的，是强化棉籽营养的一种简单可行的方法。
参考文献：
Doyle,J.J.,Schuler,M.A.,Godette,W.D.,Zenger,V.,Beachy,R.N.,&Slightom,J.L.(1986).TheglycosylatedseedstorageproteinsofGlycinemaxandPhaseolusvulgaris.Structuralhomologiesofgenesandproteins.JournalofBiologicalChemistry,261(20),9228-9238。
Claims (7)
1.一种改良棉籽营养品质的方法，其特征在于：构建种子特异启动子驱动八氢番茄红素合成酶基因PSY的植物表达载体，并转化棉花，提高棉籽中类胡萝卜素含量，所述PSY基因为棉花GhPSY2基因。
2.如权利要求1所述的方法，其特征在于：所述的GhPSY2基因具有如SEQIDNo.1所示的核苷酸序列。
3.如权利要求1或2所述的方法，其特征在于：调控PSY基因表达的启动子为菜豆种子特异启动子。
4.如权利要求3所述的方法，其特征在于：所述的种子特异启动子为菜豆储存蛋白β-菜豆蛋白β-phaseolin基因的启动子pV，具有如SEQIDNo.2所示的核苷酸序列。
5.如权利要求1～4任一项所述的方法，其特征在于：所述植物表达载体如SEQIDNo.3所示的核苷酸序列。
6.如权利要求1～5任一项所述的方法，其特征在于：所述的转化为根癌农杆菌介导的遗传转化。
7.权利要求1～6任一项所述的方法在提高棉籽类胡萝卜素含量中的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610255002.4A CN105695506A (zh) | 2016-04-20 | 2016-04-20 | 一种改良棉籽营养品质的方法及其用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610255002.4A CN105695506A (zh) | 2016-04-20 | 2016-04-20 | 一种改良棉籽营养品质的方法及其用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105695506A true CN105695506A (zh) | 2016-06-22 |
Family
ID=56217521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610255002.4A Pending CN105695506A (zh) | 2016-04-20 | 2016-04-20 | 一种改良棉籽营养品质的方法及其用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105695506A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109811004A (zh) * | 2019-02-26 | 2019-05-28 | 西南大学 | 表达载体在棉花的次生壁发育期特异表达GhPSY2基因生产棕黄色纤维中的应用 |
| CN109852626A (zh) * | 2019-02-26 | 2019-06-07 | 西南大学 | 一种GhOR基因及其编码的蛋白、表达载体、转化植株和应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1227609A (zh) * | 1996-08-09 | 1999-09-01 | 卡尔金公司 | 在植物种子中生产类胡萝卜素化合物和特产油类的方法 |
| CN1275166A (zh) * | 1997-08-08 | 2000-11-29 | 卡尔金有限责任公司 | 在植物种子中产生类胡萝卜素化合物及特制油的方法 |
| CN1347454A (zh) * | 1999-03-05 | 2002-05-01 | 格林诺瓦森植物生物技术股份有限公司 | 改善植物的农艺和营养价值的方法 |
| WO2008042338A2 (en) * | 2006-09-28 | 2008-04-10 | Microbia, Inc. | Production of carotenoids in oleaginous yeast and fungi |
-
2016
- 2016-04-20 CN CN201610255002.4A patent/CN105695506A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1227609A (zh) * | 1996-08-09 | 1999-09-01 | 卡尔金公司 | 在植物种子中生产类胡萝卜素化合物和特产油类的方法 |
| CN1275166A (zh) * | 1997-08-08 | 2000-11-29 | 卡尔金有限责任公司 | 在植物种子中产生类胡萝卜素化合物及特制油的方法 |
| CN1347454A (zh) * | 1999-03-05 | 2002-05-01 | 格林诺瓦森植物生物技术股份有限公司 | 改善植物的农艺和营养价值的方法 |
| WO2008042338A2 (en) * | 2006-09-28 | 2008-04-10 | Microbia, Inc. | Production of carotenoids in oleaginous yeast and fungi |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTINE K. SHEWMAKER等: "Seed‐specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects", 《THE PLANT J》 * |
| 无: "Accession No.：XM_012628021.1，PREDICTED: Gossypium raimondii phytoene synthase 2, chloroplasticlike(LOC105798105),mRNA", 《GENBANK DATABASE》 * |
| 龚学臣等: "八氢番茄红素合成酶基因(PSY)对大豆的遗传转化", 《大豆科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109811004A (zh) * | 2019-02-26 | 2019-05-28 | 西南大学 | 表达载体在棉花的次生壁发育期特异表达GhPSY2基因生产棕黄色纤维中的应用 |
| CN109852626A (zh) * | 2019-02-26 | 2019-06-07 | 西南大学 | 一种GhOR基因及其编码的蛋白、表达载体、转化植株和应用 |
| CN109852626B (zh) * | 2019-02-26 | 2020-08-04 | 西南大学 | 一种GhOR基因及其编码的蛋白、表达载体、转化植株和应用 |
| CN109811004B (zh) * | 2019-02-26 | 2020-10-13 | 西南大学 | 表达载体在棉花的次生壁发育期特异表达GhPSY2基因生产棕黄色纤维中的应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6990653B2 (ja) | 迅速な植物形質転換のための方法および組成物 | |
| Yang et al. | Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.) | |
| CN102888425B (zh) | 利用转基因植物生产虾青素的方法 | |
| CN103826439A (zh) | 植物转化方法 | |
| CN107002030B (zh) | 使用土壤杆菌来转化植物细胞或植物组织的方法、转基因植物、转基因细胞或转基因组织、培养基及转化植物细胞或组织的方法的应用 | |
| CN102469769A (zh) | 使用土壤杆菌对小麦属的植物进行基因导入的方法及小麦属的植物的转化植物的制作方法 | |
| Naeem et al. | Feasible regeneration and agro bacterium-mediated transformation of Brassica juncea with Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene | |
| CN102321649A (zh) | 枸杞番茄红素β-环化酶基因及包括该基因的重组载体和宿主细胞及应用 | |
| CN116891859B (zh) | CaRAP2-12基因及在调控辣椒果实中类胡萝卜素合成中的应用 | |
| Sankari et al. | Identifying a carotenoid cleavage dioxygenase 4a gene and its efficient agrobacterium-mediated genetic transformation in Bixa orellana L. | |
| CN102250943A (zh) | 一种农杆菌介导的大豆离体组织培养方法 | |
| Tsai et al. | Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra | |
| Semiarti et al. | Agrobacterium-mediated transformation of Indonesian orchids for micropropagation | |
| Kshetry et al. | Shoot at Site: Advancing in planta transformation, regeneration and gene-editing through a cascade of wounding-mediated developmental regulators | |
| CN105695506A (zh) | 一种改良棉籽营养品质的方法及其用途 | |
| KR100402513B1 (ko) | 유전자 조작을 통한 식물체의 효과적인 형질전환 방법 | |
| CN105018520B (zh) | 调控4对棉花DELLA蛋白基因GhGAIs表达的植物表达载体及其用途 | |
| CN103820475B (zh) | 枸杞牦牛儿基牦牛儿焦磷酸合成酶基因及其编码的蛋白质和应用 | |
| US7026529B2 (en) | Methods for Agrobacterium-mediated transformation of dandelion | |
| Wang et al. | Transformation of LRP gene into Brassica napus mediated by Agrobacterium tumefaciens to enhance lysine content in seeds | |
| Akashi et al. | High-frequency embryogenesis from cotyledons of bird's-foot trefoil (Lotus corniculatus) and its effective utilization in Agrobacterium tumefaciens-mediated transformation | |
| KR101424123B1 (ko) | 캡산틴을 생성하는 형질전환 컬러쌀 및 이의 제조방법 | |
| Lei et al. | Agrobacterium-mediated transformation of cotton shoot apex with SNC1 gene and resistance to cotton Fusarium wilt in T1 generation | |
| CN102524064B (zh) | 麻疯树遗传转化苗的复壮及生根的培养方法 | |
| CN102533762B (zh) | 一种获得脱致敏转基因大豆新材料的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160622 |