CN102277378A - 提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的方法 - Google Patents
提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的方法 Download PDFInfo
- Publication number
- CN102277378A CN102277378A CN 201110236642 CN201110236642A CN102277378A CN 102277378 A CN102277378 A CN 102277378A CN 201110236642 CN201110236642 CN 201110236642 CN 201110236642 A CN201110236642 A CN 201110236642A CN 102277378 A CN102277378 A CN 102277378A
- Authority
- CN
- China
- Prior art keywords
- chlorella
- gene
- atlec1
- lec1
- acid
- 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
- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 25
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 title claims description 21
- 229960004232 linoleic acid Drugs 0.000 title claims description 19
- 235000020778 linoleic acid Nutrition 0.000 title 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 title 2
- 235000019197 fats Nutrition 0.000 title 1
- 235000019198 oils Nutrition 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 241000219195 Arabidopsis thaliana Species 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 21
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 15
- 239000013604 expression vector Substances 0.000 claims description 15
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 10
- 239000004519 grease Substances 0.000 claims description 9
- 229960004488 linolenic acid Drugs 0.000 claims description 8
- 241000195648 Pseudochlorella pringsheimii Species 0.000 claims description 7
- 230000009466 transformation Effects 0.000 claims description 5
- 240000009108 Chlorella vulgaris Species 0.000 claims description 4
- 235000007089 Chlorella vulgaris Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 102000007469 Actins Human genes 0.000 claims description 2
- 108010085238 Actins Proteins 0.000 claims description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 claims description 2
- 235000007091 Chlorella pyrenoidosa Nutrition 0.000 claims description 2
- 241000195652 Auxenochlorella pyrenoidosa Species 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 4
- 101710088564 Flagellar hook-associated protein 3 Proteins 0.000 abstract description 2
- 102100022201 Nuclear transcription factor Y subunit beta Human genes 0.000 abstract description 2
- 239000003225 biodiesel Substances 0.000 abstract description 2
- 230000006872 improvement Effects 0.000 abstract description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 abstract description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 abstract description 2
- 241000024188 Andala Species 0.000 abstract 1
- 241000195493 Cryptophyta Species 0.000 description 41
- 238000006243 chemical reaction Methods 0.000 description 20
- 230000009261 transgenic effect Effects 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 230000012010 growth Effects 0.000 description 13
- 238000009396 hybridization Methods 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 238000000605 extraction Methods 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 10
- 230000029087 digestion Effects 0.000 description 10
- 150000002632 lipids Chemical class 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 230000002441 reversible effect Effects 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 108010042407 Endonucleases Proteins 0.000 description 6
- 102000004533 Endonucleases Human genes 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000004677 Nylon Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 5
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 229920001778 nylon Polymers 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 101150080441 LEC1 gene Proteins 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000219194 Arabidopsis Species 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 101100342814 Medicago truncatula LEC11 gene Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- MBMBGCFOFBJSGT-KUBAVDMBSA-N docosahexaenoic acid Natural products CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 3
- 229960002733 gamolenic acid Drugs 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241001147477 Cyclotella cryptica Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 238000000944 Soxhlet extraction Methods 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 2
- 235000021342 arachidonic acid Nutrition 0.000 description 2
- 229940114079 arachidonic acid Drugs 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 235000004626 essential fatty acids Nutrition 0.000 description 2
- 230000008175 fetal development Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 102100028220 ABI gene family member 3 Human genes 0.000 description 1
- 102000005869 Activating Transcription Factors Human genes 0.000 description 1
- 108010005254 Activating Transcription Factors Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 244000178993 Brassica juncea Species 0.000 description 1
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 1
- 235000005855 Brassica juncea var. subintegrifolia Nutrition 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 101100268665 Caenorhabditis elegans acc-1 gene Proteins 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000195585 Chlamydomonas Species 0.000 description 1
- 244000249214 Chlorella pyrenoidosa Species 0.000 description 1
- 238000007400 DNA extraction Methods 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
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150032501 FUS3 gene Proteins 0.000 description 1
- 241000692361 Fistulifera saprophila Species 0.000 description 1
- 241001262084 Himanthalia Species 0.000 description 1
- 101000724234 Homo sapiens ABI gene family member 3 Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000195644 Neochloris Species 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 244000010815 Phlomis lychnitis Species 0.000 description 1
- 101100122755 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NPA3 gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000003639 Student–Newman–Keuls (SNK) method Methods 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- HXWJFEZDFPRLBG-UHFFFAOYSA-N Timnodonic acid Natural products CCCC=CC=CCC=CCC=CCC=CCCCC(O)=O HXWJFEZDFPRLBG-UHFFFAOYSA-N 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 244000275904 brauner Senf Species 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
- DVSZKTAMJJTWFG-UHFFFAOYSA-N docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCCC=CC=CC=CC=CC=CC=CC(O)=O DVSZKTAMJJTWFG-UHFFFAOYSA-N 0.000 description 1
- 231100000463 ecotoxicology Toxicity 0.000 description 1
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002309 gasification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 230000002015 leaf growth Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000008117 seed development Effects 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 125000005480 straight-chain fatty acid group Chemical group 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010023 transfer printing Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000006200 vaporizer Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明提供了AtLEC1基因在提高小球藻油脂含量中的应用。该基因来源于拟南芥(Arabidopsis thaliana),是CCAAT-Box结合因子HAP3家族的一个成员。AtLEC1基因转化小球藻可以提高亚油酸(LA)和α-亚麻酸(ALA)含量,同时提高藻株的总油脂含量。本发明可应用于采用基因工程技术以藻类表达体系生产多不饱和脂肪酸和生物柴油。
Description
技术领域
本发明涉及植物编码基因在小球藻中的应用。具体地,涉及将一个来源于拟南芥(Arabidopsis thaliana)的具有完整阅读框架的基因AtLEC1构建成小球藻表达载体,并转入小球藻,使小球藻的亚油酸(LA)和α-亚麻酸(ALA)含量,及总油脂含量显著提高。
技术背景
微藻的光合作用效率高,生长周期短,单位面积年产量是粮食的几十倍乃至上百倍,而且微藻脂类含量在10%~70%,这是陆地植物远远达不到的。它具有产能大、无污染、可再生、易培养、含有较多的脂类物质等优点。它生长和繁殖在水域中,不会引起与农业、牧业用地竞争的矛盾。由于以上多方面的优势,微藻被认为是当今最有开发前途的能源之一[1]。本发明使用的小球藻是一种单细胞真核藻类,繁殖速度快,以无性生殖方式进行繁殖,可以进行光自养培养和无光照的异养培养,可规模化生产,本实验室有成熟的培养、筛选和转化技术[2]。
基因工程技术已被广泛应用于作物改良,并取得了许多显著的成效,而通过基因工程技术提高真核藻类总的油脂含量的研究很少有报道[3]。Jarvis等曾将硅藻C.cryptica的acc1基因转入硅藻C.cryptica和Navicula saprophila过表达,在mRNA水平上均检测到该基因的过量表达,但两种转基因硅藻的油含量并没有显著增加,这是首次尝试利用基因工程技术提高硅藻含油量的研究,然而未达到理想结果[4]。目前提高藻类油脂含量的最有成效的手段,是通过调控培养过程、培养条件、营养成分等手段提高藻类的油含量[5-6]。然而在这些手段中都不可避免的产生一个问题,那就是提高含油量要以生物量的降低为代价,当生物量增加时,油脂的含量就会比较低[7-8]。而基因工程技术有望解决这个问题。目前尚未 检索到利用基因工程手段提高藻类油脂含量并且总生物量不减少的成功报道。
转录调控是许多生物学调控的关键步骤,转录因子可调控特定代谢途径中的一系列相关基因的表达,使得该代谢途径从整体水平上发生改变。LEC1(Leafy Cotyledon 1)是转录激活因子HAP3(CCAAT-Box结合因子)基因家族的一个成员,在胚胎发育早期LEC1维持胚柄细胞的特性以及子叶的特性,而在胚胎发育的后期LEC1基因的表达与储藏物质的积累、胚抗脱水性的获得等种子成熟过程相关[9]。Loton等在拟南芥中过量表达LEC1的幼苗体内发现了油体蛋白和贮藏蛋白等种子特异RNA的积累,但是过表达的种子发芽率很低,植株生长很弱,最终不育或者死亡[10]。Mu等的研究表明,发育早期诱导过量表达拟南芥LEC1基因可影响ABI3、FUS3和WRINKLED1等转录因子,提高脂肪酸合成相关基因表达的整体水平,叶片中的油脂含量得到显著提高[11]。Shen等将玉米ZwLEC1基因置于胚特异性的弱启动子EPA1之下,获得高含油量的转基因玉米,但是植株叶片减小到野生型的一半左右;将玉米ZwLEC1基因置于胚特异性的强启动子OLE之下,种子萌发和叶片生长发生了非常严重的缺陷[12]。Tan等将油菜来源的BnLEC1基因置于种子特异性的启动子napinA之下在芥菜中表达,结果转基因植株在发芽后出现严重异常,全部死亡或者不育;因此对启动子napinA进行了一系列的改造,当启动强度只有原来的18%时,植株生长繁殖正常并且油含量得到显著提高[13]。在高等植物中,最重要的事件是种子发育并成熟以及再次萌发,LEC1基因在强启动子下启动表达的后果都是致死的。小球藻是低等真核植物,不需要形成种子并且是无性繁殖,不存在LEC1基因在强启动子下过表达造成致死的因素。目前尚未检索到利用拟南芥编码的LEC1基因转化藻类并提高油脂含量的研究。
多不饱和脂肪酸(Polyunsaturated fatty acids,PUFAs)是指含有两个或两个以上双键且碳链长为18~22个碳原子的直链脂肪酸,主要包括亚油酸(Linoleic Acid,LA,18:2△9,12)、γ-亚麻酸(γ-Linolenic Acid,GLA,18:3△6,9,12)、花生四烯酸(Arachidonic Acid,AA,20:4△5,8,11,14)、二十碳五烯酸(Eicosapentaenoic Acid,EPA, 20:5△5,8,11,14,17)、二十二碳六烯酸(Decosahexaenoic Acid,DHA,20:5△4,7,10,13,16,19)等。其中,亚油酸及亚麻酸被公认为人体必需脂肪酸(Essential fatty acids,EFA),可进一步衍化成具有不同功能的高度不饱和脂肪酸,如AA、EPA、DHA等[14]。藻类含有丰富的多不饱和脂肪酸,主要包括亚油酸(Linoleic acid,LA,18:2△9,12)、γ-亚麻酸(γ-Llinolenic Acid,GLA,18:3△6,9,12)等。本发明发现AtLEC1使小球藻的亚油酸(LA)和α-亚麻酸(ALA)含量,及总油脂含量显著提高。
发明内容
本发明的一个目的是提供基因AtLEC1用于提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的应用。
脂肪酸的检测主要采用GC-MS(气相色谱-质谱联用技术)来完成。其原理是通过GC(气相色谱)将不同的脂肪酸组分分离,然后通过MS(质谱)检测器分别对每种组分进行质谱定性分析,从而确定每一种组分的成分及含量的比较成熟的联用分析技术。
索氏抽提法(Soxhlet extractor method)是公认测定油脂含量的经典方法,目前被国内外普遍采用。利用溶剂回流及虹吸原理,使固体物质连续不断地被纯溶剂萃取,最后萃取出的物质富集在烧瓶中。
具体地,在一个方面,本发明涉及AtLEC1基因在提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的应用。
在另一个方面,本发明提供一种提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的方法,其特征在于将AtLEC1基因转化到小球藻中。
在本发明的应用及方法的一个具体实施方案中,AtLEC1基因是拟南芥来源的。更优选地,AtLEC1基因的序列为SEQ ID NO:1所示的序列,其编码的蛋白序列为SEQ ID NO:2所示的序列。
在另一个具体的实施方案中,所述AtLEC1基因包含在植物表达载体中。所述表达载体可以是引导外源基因在植物中表达的任一种表达载体。优选地,所述表达载体可以是植物表达载体pBIN19、pBI121、pBI221,pCambia 1300,pGreen。优选地为pGreen0029。
本发明的载体也可含有适当的启动子。在本发明中可使用任何一种强 启动子。这些启动子包括但不限于花椰菜花叶病毒(CaMV 35S)、Ubiqutin、Actin启动子。它可单独使用或与其它的植物启动子结合使用。
本发明的表达载体可通过使用Ti质粒,Ri质粒,植物病毒载体,直接的DNA转化,微注射,电穿孔等方式导入植物细胞。
在又一个具体的实施方案中,所述小球藻选自椭圆小球藻(Chlorella ellipsoidea),普通小球藻(Chlorella vulgaris)和蛋白核小球藻(Chlorella pyrenoidosa)。
附件说明
图1含AtLEC1基因的载体pEB-AtLEC1。
图2含nos终止子的载体图构建过程。
图3空载(UN CK)植物表达载体图构建过程。
图4基因AtLEC1的植物表达载体图构建过程。
图5转基因藻株的PCR检测。M:Marker,1-3:阳性藻株,CK:阴性对照。
图6转基因藻株的RT-PCR检测。M:Marker,1-4:阳性藻株,CK:阴性对照。
图7转基因藻株的Southern检测。第四泳道为对照,其它均为阳性藻株。1:LEC1-1(EcoR I),2:LEC1-1(Xba I),3:LEC1-3(Xho I),4:UN CK(Xho I),5:LEC1-3(EcoR I),6:LEC1-8(Xba I),7:LEC1-8(Xho I),8:LEC1-9(EcoR I),9:LEC1-9(Xba I)。
图8转基因藻株和对照生长曲线。
图9纯化多代的转基因藻株脂肪酸GC-MS测定结果比较。
SEQ ID NO:1.AtLEC1核苷酸序列。
SEQ ID NO:2.AtLEC1氨基酸序列。
SEQ ID NO:3.nos终止子片段克隆所用正向引物。
SEQ ID NO:4.nos终止子片段克隆所用反向引物。
SEQ ID NO:5.ubi启动子片段的克隆所用正向引物。
SEQ ID NO:6.ubi启动子片段的克隆所用反向引物。
SEQ ID NO:7.从pEB-AtLEC1载体中扩增基因AtLEC1的正向引物。
SEQ ID NO:8.从pEB-AtLEC1载体中扩增基因AtLEC1的反向引物。
SEQ ID NO:9.从基因AtLEC1内部扩增的正向引物。
SEQ ID NO:10.从基因AtLEC1内部扩增的反向引物。
具体实施方式
下面参考实施例和附图详细描述本发明。本领域的普通技术人员可以理解的是,下述实施例是举例说明的目的,其不应以任何方式解释为对本发明的限制。本发明的保护范围由后附的权利要求所限定。
实施例1.AtLEC1基因小球藻载体的构建
1.按照公开的序列(NCBI Reference Sequence:NM 102046.4)合成基因AtLEC1,并连入T载体(购自北京全式金生物技术有限公司),构成含有基因AtLEC1的载体pEB-AtLEC1,测序验证基因AtLEC1序列为SEQ IDNO:1,所表达的蛋白序列为SEQ ID NO:2,该质粒载体图见图1。
2.以pGreen0029(购自the John Innes Centre)为基础载体,构建含Ubiquitin启动子和nos终止子的对照载体pG29-UN-CK。
用高保真酶Pfu(购自北京全式金生物技术有限公司)从载体pGreen0029上扩增nos终止子,用引物SEQ ID NO:3(正向引物)和SEQ ID NO:4(反向引物)。
PCR反应体系如下:
PCR反应条件如下:
1.95℃ 5min
2.94℃ 30s
3.55℃ 40s
4.72℃ 1min 回复至2,32cycles
5.72℃ 10min
6.4℃ pause
扩增产物直接使用限制性内切酶Not I和Sac I进行双酶切,与同样双酶切的pGreen0029连接,构建中间载体pGreen0029-nos(图2),测序验证。
用高保真酶Pfu(来源同上)从含Ubi启动子的载体(pC1303,购自Cambia公司)上扩增Ubi启动子,所用引物SEQ ID NO:5(正向引物)和SEQ ID NO:6(反向引物)。
PCR反应体系如下:
PCR反应条件如下:
1.95℃ 5m
2.94℃ 30s
3.55℃ 40s
4.72℃ 4min 回复至2,32cycles
5.72℃ 10min
6.4℃ pause
扩增产物直接使用限制性内切酶HindIII和BamHI进行双酶切,与同样双酶切的pGreen0029-nos连接,构建含Ubiquitin启动子和nos终止子的对照载体pGreen0029-Ubi-Nos(简写UN-CK),测序验证,其载体图见图3。
3.AtLEC1基因植物表达载体的构建
用高保真酶Pfu(来源同上)从含有基因AtLEC1的载体pEB-AtLEC1中扩增AtLEC1,使用引物为SEQ ID NO:7(正向引物)和SEQ ID NO:8(反向引物),反应体系和反应条件同Ubi启动子的克隆,利用Spe I、Not I酶切位点,双切后获得AtLEC1基因,定向克隆于经同样酶切的小球藻中间表达载体UN-CK,获得小球藻表达质粒p29-UN-LEC1,转化大肠杆菌DH-5α(购自北京全式金生物技术有限公司),随机挑取单克隆,使用引物为SEQ ID NO:9和SEQ ID NO:10进行PCR验证,反应体系和反应条件同Ubi启动子的克隆,证明获得了含有SEQ ID NO:1的AtLEC1基因的植物表达载体。载体构建程序见图4。
实施例2.转AtLEC1基因的小球藻的获得及检测
1.小球藻的转化
所用小球藻为椭圆小球藻(Chlorella ellipsoidea,来自中国科学院水生生物研究所),参照文献[2]培养小球藻至适宜的生长状态,离心收集藻细胞后进行高渗处理,采用上述文献[2]相同的电激转化条件对椭圆小球藻进行电激,将电激后小球藻细胞涂于含30mg/L G418固体培养基(参照文献[2])中培养,约4~6周后检测平板上长出的藻落。
2.对转基因藻株进行PCR检测
挑取平板上的单藻落细胞接种于15mg/L G418液体培养基(参照文献[2])中培养2周(浓度达到约为1×108个/mL),5000r/min离心10min收集藻细胞,在液氮中将细胞磨碎,参照文献[2]中的方法提取小球藻总DNA,得到的DNA溶于灭菌的双蒸水中放置-80℃保存。
用高保真酶Pfu(来源同上)扩增基因AtLEC1,所用引物SEQ ID NO: 7(正向引物)和SEQ ID NO:8(反向引物),反应体系和反应条件参见实施例一的Ubi启动子的克隆,图5为部分转基因藻株PCR检测结果,共获得了52个转基因阳性藻株系,编号为LEC1-1,-2,-3...直到LEC1-52。
3.对转基因藻株进行RT-PCR检测
挑取PCR检测阳性的藻株单藻落细胞,培养收集方法同上,取约100mg置于液氮中研磨至材料完全破碎,加入TRIzol(购自Invitrogen公司)室温放置5分钟。加入氯仿抽提两次,取上清,用异丙醇沉淀总RNA,空气干燥后,溶于DEPC H2O。以试剂盒(FSK-100,TOYOBO Co.,LTD.)自带的Random primer进行逆转录获得cDNA,并进行RT-PCR(具体操作参见FSK-100,TOYOBO Co.,LTD.)。
RT-PCR反应体系如下
进行下列操作
1.30℃ 10min
2.42℃ 30min
3.90℃ 5min
4.5℃ 5min
取适量上述逆转录产物以引物SEQ ID NO:9和SEQ ID NO:10进行PCR扩增,反应体系和反应条件参见实施例1的Ubi启动子的克隆,图6为部分转基因藻株RT-PCR检测结果。
4.对转基因藻株进行Southern检测
Southern杂交是分子生物学的经典实验方法,其基本原理是将待检测的DNA样品固定在固相载体上,与标记的核酸探针进行杂交,在与探针有同源序列的固相DNA的位置上显示出杂交信号。通过Southern杂交可 以判断被检测的DNA样品中是否有与探针同源的片段。本实验操作及所用试剂主要参考文献[15]。
4.1探针标记
使用引物SEQ ID NO:9和SEQ ID NO:10从载体pEB-AtLEC1(图1)PCR扩增基因AtLEC1,反应体系和反应条件参见实施例一,探针标记使用Random Primer DNA Labeling Kit ver.2.0(TAKARA BIOTECHNOLOGY(DALIAN)CO.,LTD.Code D6045)。PCR产物纯化回收后取10ng-1μg作为模板放于0.2mL离心管中,加Random Primer 2μL,ddH2O补齐16μL,95℃变性5min,立即置冰浴5min。
在另一个1.5mL离心管中加入试剂盒DNA Labeling Kit ver.2.0自带的其它组分:
将变性模板DNA加入到上管中,室温或37℃1hr。
4.2小球藻总DNA提取和酶切
挑取转AtLEC1单藻落细胞和对照(转UN-CK的藻落细胞)分别接种于15mg/L G418液体培养基(参照文献[2])中培养2周,5000r/min离心10min收集藻细胞,在液氮中将细胞磨碎,参照文献[2]中的方法提取小球藻总DNA,获得的DNA用限制性内切酶酶进行酶切。LEC1-1-2分别用EcoR I和Xba I单酶切;LEC1-3-3分别用EcoR I和Xho I单酶切;UN CK用Xho I单酶切;LEC1-8-4分别用Xba I和Xho I单酶切;LEC1-9-5分别用EcoR I和Xba I单酶切。限制性内切酶均购自Takara公司。
反应体系:100μL反应体系中包括DNA 10~20μg、buffer 10μL、限制性内切酶4μL,其余用ddH2O补平。
反应条件:37℃过夜,补加1μL酶继续酶切3-5小时,然后1%琼脂糖电泳检测酶切效果,DNA需切成弥散的一条带,没有主带。酶切体系加ddH2O至体积200μL,加等体积氯仿抽提后乙醇醇沉,70%乙醇洗,晾干后加水不超过50μL,65℃热10min备用。
4.3胶的制备及转膜
配制0.8%的琼脂糖凝胶,加入酶切回收产物50μL,在1×TAE电泳缓冲液中,30V电压下电泳至溴酚蓝到胶下端,用ddH2O将胶洗两遍,浸于数倍体积的碱性缓冲液中轻摇15min,换一次碱性缓冲液浸泡并轻轻摇荡20min。同时剪一张比凝胶大1mm的尼龙膜(CAT.No:RPN303B,Amersham Biosciences UK Limited),预先在双蒸水中浸泡使其湿透,浸润后的尼龙膜浸入碱性转移缓冲液中至少15min。在0.4M的NaOH和1M NaCl碱性转移缓冲液条件下,用真空转膜仪(美国伯乐Bio-rad 785型真空转印仪)将琼脂糖凝胶上的DNA转到带正电荷的尼龙膜上,转膜条件为真空度50m bar转1h-2h。转膜完成后将膜浸泡于中和缓冲液II中室温15min。置于滤纸上晾干,紫外交联(1.245J/cm)8min,80℃烘烤1h,最后将尼龙膜立即用于杂交或用保鲜膜包好放在-20℃冰箱待用。
4.4预杂交及杂交
在杂交瓶中加入杂交液10-20mL(0.5M Na3PO4(pH7.2),1mM EDTA(pH8.0),7%SDS),将膜的背面贴紧杂交瓶壁,正面朝向杂交液,注意不要产生气泡,放入杂交炉中,使杂交体系升到65℃。预杂交2-3h后,倒出预杂交的杂交液,换入5mL左右的杂交液。在上述制作的探针中加0.4N的NaOH使其变性,迅速加到杂交瓶中,65℃杂交过夜。
4.5洗膜与压片
取出尼龙膜在65℃依次使用下列条件洗膜:
2×SSC,0.5%SDS,15min
1×SSC,0.5%SDS,15min
0.5×SSC,0.1%SDS,15min
在洗膜的过程中,不断振摇,并保持膜一直是湿润的状态,不断用放射性检测仪探测膜上的放射强度。期间洗膜2次后用monitor检测杂交信号的强弱,降低到20μGy/h左右时即可。洗完的膜用滤纸吸干膜表面的水分,并用保鲜膜包裹。
将膜正面向上,放入暗盒中(加双侧增感屏),在暗室的红光下,在膜的正面向上贴覆一张X光片,合上暗盒,置于-80℃低温冰箱中曝光。 根据信号强弱决定曝光时间,一般时间在2天左右。
4.6显影及定影
从-70℃中取出的暗盒在显影室打开后,取出X光片,显影液中浸泡1min,期间不断晃动光片,取出沥掉一些水分,浸到定影液中1min,ddH2O冲洗两遍并照相,见附图7。点样顺序如下:1:LEC1-1(EcoR I),2:LEC1-1(Xba I),3:LEC1-3(Xho I),4:UN_CK(Xho I),5:LEC1-3(EcoR I),6:LEC1-8(Xba I),7:LEC1-8(Xho I),8:LEC1-9(EcoR I),9:LEC1-9(Xba I)。
4.7结果分析
从质粒载体图4的酶切位点可以分析出,在***小球藻基因组的片段上没有EcoR I、Xba I和Xho I的酶切位点,使用这三种酶分别进行单酶切。图7中的泳道4是对照藻株,没有条带说明没有杂交到内源的基因,其它泳道均各有一个条带,说明在各转基因藻株的基因组里面只***了一个拷贝的AtLEC1基因。
实施例3.小球藻的生长曲线
实施例2中分子检测为阳性藻株的后代LEC1-1和LEC1-3,以及对照载体转化藻株CK,参照文献[2]在15mg/L G418液体培养基中扩大培养,同时野生藻(WT)在不含抗生素的液体培养基中扩大培养。使用50mL三角瓶培养4-6天后,按干重约0.2g/L的起始浓度在150mL新培养基中扩大培养,每天同一时间取样测定生物量,连续8天记录,测定结果见表1。
依据测定结果,制作转基因藻和野生藻的生长曲线,见图8。
在图8中可以发现小球藻从第6天开始进入平台期(生长量达到动态平衡),到第8天藻的生物量开始下降。使用SPSS软件(IBM公司)对测定的藻生物量分别进行Student-Newman-Keuls检验。检验结果表明转基因藻和对照(野生藻及对照载体转化藻株CK)的生物量没有显著性差异(P>0.05)。
实施例4.AtLEC1基因提高小球藻不同脂肪酸含量的GC-MS分析
实施例2中分子检测为阳性的藻株,按参照文献[2]在30mg/L G418固体培养基上连续划板、点板进行纯化,在15mg/L G418液体培养基中扩大培养。使用50mL三角瓶培养4-6天后,按干重0.1g/L的起始浓度在150mL新培养基扩大培养,7天后离心收集藻细胞使用GC-MS测定各脂肪酸组分含量变化。
1.脂肪酸的提取
离心收集生长达到平台期的小球藻(OD540=16),用去离子水洗涤,-80℃冷冻干燥,冻干后在85℃烘箱过夜烘干,在研钵中研磨成均匀的细粉。称取50mg小球藻粉,加入80μL的十七烷酸(Heptadecanoic Acid,d17:0,购自Sigma公司,溶于甲醇浓度100μM/mL)作为脂肪酸含量测定的内参。5mL 5%KOH-CH3OH,70℃水浴5h后加入HCl酸化至其pH值达2.0。再加入4mL 14%BF3-CH3OH(购自Aldtich公司)溶液,70℃水浴1.5h。加入2mL 0.9%NaCl溶液,混匀后静止片刻。加入2mL氯仿:正己烷(V/V 1∶4)抽提,吸取抽提液,N2吹干。最后溶于150μL乙酸乙酯。
2.终产物GC-MS检测分析实验
所用GC-/MS仪为TurboMass(PerkinElmer公司),柱子:BPX-70,30m×0.25mm×0.25vm,柱温120℃,气化室温度230℃。取1μL终产物上样,分流比10∶1。
3.GC-MS结果分析
分析结果表明,在获得纯化多代藻落中,这两个藻株的C16:0(棕榈酸)均比对照提高,其中LEC1-1提高64%,LEC1-3提高27%;C18:1(油酸)均比对照提高,LEC1-1比对照提高94%,LEC1-3提高75%;LEC1-3的C18:2(亚油酸)比对照提高21%;LEC1-3的C18:3(亚麻酸)比对照提高36%(图9),以上这些差异均显著(P<0.01)。
实施例5.AtLEC1基因提高小球藻总油脂含量的分析
本实验使用藻株及培养方法同上实施例4,总油脂含量分析方法为索氏抽提法参见文献[16]。索氏提取器是由提取瓶、提取管、冷凝器三部分组成的,提取管两侧分别有虹吸管和连接管。提取时,将待测样品包在脱 脂滤纸包内,放入提取管内。提取瓶内加入适合该待测样品的低沸点有机溶剂,加热提取瓶,有机溶剂气化,由连接管上升进入冷凝器,凝成液体滴入提取管内,浸提样品中的脂类物质。待提取管内有机溶剂液面达到一定高度,溶有粗脂肪的有机溶剂经虹吸管流入提取瓶。流入提取瓶内的有机溶剂继续被加热气化、上升、冷凝,滴入提取管内,如此循环往复,使固体物质不断为纯的溶剂所苹取、将萃取出的物质富集在烧瓶中。
首先将收集的藻离心,置于玻璃平皿中90℃-110℃过夜烘干,加入液氮研磨成均匀的粉末。使用低沸点有机溶剂氯仿∶甲醇(V/V=3∶1)将藻粉末浸润而将油脂浸出来,烘干称重计算油脂含量。离心收集生长达到平台期的小球藻(OD540=16),用去离子水洗涤,-80℃冷冻干燥,冻干后在85℃烘箱过夜烘干,在研钵中研磨成均匀的细粉。称取0.5g小球藻粉,使用索氏提取器(货号:ST-80,购自北京赛福莱博科技有限公司)检测藻株油脂总含量。附表统计显示转基因藻株均比对照有较大提高。转AtLEC1藻株LEC1-1比对照提高31.36%,藻株LEC1-3总油脂含量比对照提高32.87%(表3),差异显著(P<0.01)。
表1转基因藻和对照组生长量测定
藻株 | 0天 | 1天 | 2天 | 3天 | 4天 | 5天 | 6天 | 7天 | 8天 |
LEC11 | 0.213692 | 0.543672 | 2.2115 | 4.72168 | 6.44912 | 8.68805 | 10.3567 | 10.9453 | 10.4367 |
LEC11 | 0.209552 | 0.520398 | 2.4546 | 4.62926 | 6.28534 | 9.20525 | 10.1257 | 10.3214 | 10.2376 |
LEC11 | 0.219896 | 0.547551 | 2.44598 | 4.47342 | 6.25948 | 9.6147 | 10.9746 | 10.6324 | 10.7456 |
LEC13 | 0.266452 | 0.539971 | 2.42254 | 4.69926 | 6.12156 | 9.1251 | 10.6746 | 10.68145 | 10.9939 |
LEC13 | 0.240584 | 0.563245 | 2.71978 | 4.9127 | 6.25948 | 9.63625 | 10.7734 | 10.89115 | 10.67065 |
LEC13 | 0.259548 | 0.571003 | 2.64426 | 4.70788 | 6.43567 | 8.94665 | 11.2645 | 10.48115 | 10.42755 |
CK | 0.227205 | 0.540232 | 2.1643 | 3.65794 | 5.30436 | 7.3473 | 10.51568 | 10.6111 | 10.58445 |
CK | 0.225481 | 0.502644 | 2.06689 | 4.05276 | 5.58298 | 7.18178 | 10.77428 | 10.8697 | 10.71375 |
CK | 0.229791 | 0.504368 | 2.17551 | 4.12172 | 5.24746 | 7.71522 | 10.11224 | 11.06365 | 10.88615 |
WT | 0.236944 | 0.577468 | 2.44014 | 4.3148 | 5.4078 | 7.68348 | 10.76048 | 10.9421 | 10.6939 |
WT | 0.236513 | 0.548504 | 2.61254 | 4.00032 | 5.73884 | 7.65418 | 10.89152 | 11.30845 | 10.77405 |
WT | 0.237375 | 0.533332 | 2.51341 | 4.05204 | 5.34228 | 8.13176 | 10.25016 | 10.70505 | 11.17575 |
注:单位为g/L
表2转LEC1藻株总含油量测定
参考文献:
1.Yusuf Chisti.Biodiesel from microalgae.(2007).Biotechnol Adv.25(3):294-306.
2.Ying Chen,et al.(2001).Highly efficiencies expression of rabbit neutrophil peptide 1gene in Chlorella ellipsoidea cells.Curr Genet.39:365-370.
3.Randor Radakovits,et al.(2010).Genetic engineering of algae for enhanced biofuel production.Eukaryotic cell.9(4):486-501.
4.Jarivs Eric,et al.(1991).Transient expression of firefly luciferase in protoplast of the green alga Chlorella ellipsoidea.Curr Genet.19(6):317-332.
5.Yanna Liang,et al.(2009).Biomass and lipid productivities of Chlorella vulgaris under autotrophic,heterotrophic and mixotrophic growth conditions.Biotechnol Lett.31:1043-1049.
6.Yantao Li,et al.(2010).Inhibition of Starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii.Biotechnology and Bioengineering.107(2):258-268.
7.Li Y.,et al.(2008).Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans.Applied Microbiology and Biotechnology.81:629-636.
8.Scragg A.H.,et al.(2002).Growth of microalgae with increased calorific values in a tubular bioreactor.Biomass and Bioenergy.23(1):67-73.
9.Marilyn A.L. West,et a1.(1994).LEAFY COTYLEDON 1 is an essential regulator of late embryogenesis and cotyledon identity in Arabidopsis.Plant Cell.6:1731-1745.
10.Lotan T.,et al.(1998).Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells.Cell.93(6):1195-1205
11.Jinye Mu,et al.(2008).LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis.Plant Physiol.148(10):1042-1054.
12.Bo Shen,et al.(2010).Expression of ZmLEC1 and ZmWRI1 Increases Seed Oil Production in Maize l.Plant Physiol.153:980-987.
13.Helin Tan,et al.(2011).Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds.Plant Physiol.156:1577-1588.
14.Zhiyou Wen,et al.(2003).Heterotrophic production of eicosapentaenoic acid by microalgae.Biotech Adv.21:273-94.
15.Joe Sambrook,et al.(2001).Molecular Cloning:A Laboratory Manual,3rd ed.Cold Spring Harbor Laboratory Press.502-509.
16.Punin Crespo,et al.(2004).Determination of aliphatic hydrocarbons in the alga Himanthalia elongata.Ecotoxicology and Environmental Safety.57:226-230 。
Claims (8)
1.AtLEC1基因在提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的应用。
2.提高小球藻中总油脂、亚油酸或α-亚麻酸的含量的方法,其特征在于将AtLEC1基因转化到小球藻中。
3.权利要求1的应用或权利要求2的方法,其中所述AtLEC1基因来自拟南芥(Arabidopsis thaliana)。
4.权利要求1的应用或权利要求2的方法,其中所述AtLEC1基因的序列为SEQ ID NO:1所示的序列,其编码的蛋白序列为SEQ ID NO:2所示的序列。
5.权利要求1的应用或权利要求2的方法,其中所述AtLEC1基因包含在植物表达载体中。
6.权利要求5的应用或方法,其中所述表达载体选自植物表达载体pBIN19、pBI121、pBI221,pCambia 1300,pGreen,优选地为pGreen0029。
7.权利要求6的应用或方法,其中所述载体包含启动子,所述启动子选自花椰菜花叶病毒启动子CaMV 35S、Ubiqutin启动子、或Actin启动子。
8.权利要求1的应用或权利要求2的方法,其中所述小球藻选自椭圆小球藻(Chlorella ellipsoidea),普通小球藻(Chlorella vulgaris)或蛋白核小球藻(Chlorella pyrenoidosa)。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 201110236642 CN102277378B (zh) | 2011-08-17 | 2011-08-17 | 提高小球藻中总油脂、亚油酸、油酸或α-亚麻酸的含量的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 201110236642 CN102277378B (zh) | 2011-08-17 | 2011-08-17 | 提高小球藻中总油脂、亚油酸、油酸或α-亚麻酸的含量的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102277378A true CN102277378A (zh) | 2011-12-14 |
CN102277378B CN102277378B (zh) | 2013-07-24 |
Family
ID=45103108
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 201110236642 Expired - Fee Related CN102277378B (zh) | 2011-08-17 | 2011-08-17 | 提高小球藻中总油脂、亚油酸、油酸或α-亚麻酸的含量的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102277378B (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103397007A (zh) * | 2013-07-25 | 2013-11-20 | 中国科学院遗传与发育生物学研究所 | CeDGAT1基因及其应用 |
CN104312991A (zh) * | 2014-10-16 | 2015-01-28 | 中国科学院遗传与发育生物学研究所 | CeDGAT1突变体及其应用 |
CN107936098A (zh) * | 2017-12-21 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | 椭圆小球藻nf‑ya基因及其应用 |
CN108101973A (zh) * | 2017-12-21 | 2018-06-01 | 中国科学院遗传与发育生物学研究所 | 椭圆小球藻nf-yc基因及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1948338A (zh) * | 2006-11-10 | 2007-04-18 | 中国科学院遗传与发育生物学研究所 | 一个调控植物脂肪酸代谢的转录因子及其编码基因与应用 |
-
2011
- 2011-08-17 CN CN 201110236642 patent/CN102277378B/zh not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1948338A (zh) * | 2006-11-10 | 2007-04-18 | 中国科学院遗传与发育生物学研究所 | 一个调控植物脂肪酸代谢的转录因子及其编码基因与应用 |
Non-Patent Citations (3)
Title |
---|
《GENBANK》 20110528 Theologis,A.et al NM_102046.4 1-3 4 , * |
《Plant Physiology》 20081031 Jinye Mu et al LEAFY COTYLEDON1 Is a Key Regulator of Fatty Acid Biosynthesis in Arabidopsis 1042-1054 1-8 第148卷, 第2期 * |
《Plant Physiology》 20110731 Helin Tan et al Enhanced Seed Oil Production in Canola by ConditionalExpression of Brassica napus LEAFY COTYLEDON1 andLEC1-LIKE in Developing Seeds 1577-1588 1-8 第156卷, 第3期 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103397007A (zh) * | 2013-07-25 | 2013-11-20 | 中国科学院遗传与发育生物学研究所 | CeDGAT1基因及其应用 |
CN104312991A (zh) * | 2014-10-16 | 2015-01-28 | 中国科学院遗传与发育生物学研究所 | CeDGAT1突变体及其应用 |
CN104312991B (zh) * | 2014-10-16 | 2017-06-20 | 中国科学院遗传与发育生物学研究所 | CeDGAT1突变体及其应用 |
CN107936098A (zh) * | 2017-12-21 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | 椭圆小球藻nf‑ya基因及其应用 |
CN108101973A (zh) * | 2017-12-21 | 2018-06-01 | 中国科学院遗传与发育生物学研究所 | 椭圆小球藻nf-yc基因及其应用 |
CN108101973B (zh) * | 2017-12-21 | 2020-07-14 | 中国科学院遗传与发育生物学研究所 | 椭圆小球藻nf-yc基因及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN102277378B (zh) | 2013-07-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7097805B2 (ja) | 脂質製造のための工程 | |
Chen et al. | Soybean (Glycine max) WRINKLED1 transcription factor, GmWRI1a, positively regulates seed oil accumulation | |
CN102782140B (zh) | 靶向参与脂肪酸生物合成的植物基因的工程改造锌指蛋白 | |
Yang et al. | Development of a stable genetic system for Chlorella vulgaris—A promising green alga for CO2 biomitigation | |
CN102277378B (zh) | 提高小球藻中总油脂、亚油酸、油酸或α-亚麻酸的含量的方法 | |
US10077450B2 (en) | Sugarcane bacilliform viral (SCBV) enhancer and its use in plant functional genomics | |
CN104651236A (zh) | 一种用于提高莱茵衣藻脂肪酸含量的转基因衣藻、构建方法及其用途 | |
Zhang et al. | Stearic acid desaturase gene negatively regulates the thermotolerance of Pinellia ternata by modifying the saturated levels of fatty acids | |
CN105647822B (zh) | 一株过表达来源于寄生疫霉的ω-3脱饱和酶的重组高山被孢霉、其构建方法及应用 | |
Hua et al. | Molecular regulation and genetic improvement of seed oil content in Brassica napus L | |
JP6818193B2 (ja) | 中性脂質の製造方法、トランスジェニック植物及びキット | |
CN105755034A (zh) | 莱茵衣藻Dof基因的重组表达载体及其构建方法和应用 | |
BR112013004948B1 (pt) | região reguladora de transcrição quimérica compreendendo intensificador viral baciliforme de cana-deaçúcar (scbv), constructo e processo de produção de uma planta transgênica | |
KR101679130B1 (ko) | Bass2 단백질 또는 이를 암호화하는 유전자를 포함하는 식물 종자의 크기 및 종자 내 저장 지방의 함량 증가용 조성물 | |
CN108101973B (zh) | 椭圆小球藻nf-yc基因及其应用 | |
CN102260686B (zh) | 提高小球藻中总油脂、亚油酸或油酸的含量的方法 | |
CN102191268A (zh) | 一种获得无选择标记基因转基因平邑甜茶植株的方法 | |
CN102533804B (zh) | 白沙蒿Δ12脂肪酸脱氢酶(As FAD2)基因及用途 | |
CN102295691B (zh) | Bccp2基因及其在提高植物及藻类油脂含量方面的应用 | |
CN103703135B (zh) | 微粒体ω6油酸去饱和酶 | |
CN103589737B (zh) | 一种微藻三酰甘油合成调控基因及其应用 | |
CN104877021A (zh) | 与植物脂肪酸和油脂代谢相关的油菜转录因子BnFUS3及其编码基因与应用 | |
CN102127540A (zh) | 一种提高大豆品质的方法及其专用dna分子 | |
CN104357415A (zh) | CemGPDH基因及其应用 | |
US9650644B2 (en) | Gene silencing of sugar-dependent 1 in Jatropha curcas |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
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: 20130724 Termination date: 20170817 |