CN117070529A - Sweet potato transcription factor IbDREB1d and application thereof - Google Patents
Sweet potato transcription factor IbDREB1d and application thereof Download PDFInfo
- Publication number
- CN117070529A CN117070529A CN202311096573.4A CN202311096573A CN117070529A CN 117070529 A CN117070529 A CN 117070529A CN 202311096573 A CN202311096573 A CN 202311096573A CN 117070529 A CN117070529 A CN 117070529A
- Authority
- CN
- China
- Prior art keywords
- sweet potato
- ibdreb1d
- transcription factor
- seq
- ibfls
- 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
- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 58
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 56
- 102000040945 Transcription factor Human genes 0.000 title claims description 24
- 108091023040 Transcription factor Proteins 0.000 title claims description 24
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000011957 flavonols Nutrition 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 230000009261 transgenic effect Effects 0.000 claims abstract description 11
- 150000007946 flavonol Chemical class 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- 238000009825 accumulation Methods 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 4
- 230000035897 transcription Effects 0.000 claims abstract 6
- 238000013518 transcription Methods 0.000 claims abstract 6
- 235000009508 confectionery Nutrition 0.000 claims abstract 3
- 238000000034 method Methods 0.000 claims description 8
- -1 flavonol compound Chemical class 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 4
- 230000003993 interaction Effects 0.000 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 abstract description 7
- 235000002595 Solanum tuberosum Nutrition 0.000 abstract description 7
- 230000002018 overexpression Effects 0.000 abstract description 4
- 241001275118 Phytolacca heptandra Species 0.000 abstract description 3
- 150000002216 flavonol derivatives Chemical class 0.000 abstract description 2
- 230000006872 improvement Effects 0.000 abstract description 2
- 239000013598 vector Substances 0.000 description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 4
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010397 one-hybrid screening Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 description 2
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010076511 Flavonol synthase Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 description 2
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 description 2
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 2
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- GXMWXESSGGEWEM-UHFFFAOYSA-N isoquercitrin Natural products OCC(O)C1OC(OC2C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C1O GXMWXESSGGEWEM-UHFFFAOYSA-N 0.000 description 2
- 235000008777 kaempferol Nutrition 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000005875 quercetin Nutrition 0.000 description 2
- 229960001285 quercetin Drugs 0.000 description 2
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 description 2
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 2
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 description 2
- 235000005493 rutin Nutrition 0.000 description 2
- 229960004555 rutoside Drugs 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 102000004726 Connectin Human genes 0.000 description 1
- 108010002947 Connectin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- IKMDFBPHZNJCSN-UHFFFAOYSA-N Myricetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC(O)=C(O)C(O)=C1 IKMDFBPHZNJCSN-UHFFFAOYSA-N 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 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
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000021384 green leafy vegetables Nutrition 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 description 1
- 235000007743 myricetin Nutrition 0.000 description 1
- 229940116852 myricetin Drugs 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—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)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of biotechnology, and in particular relates to a sweet potato transcription factorIbDREB1dAnd applications thereof. The sweet potato transcription factorIbDREB1dThe nucleotide sequence is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3. It was found that overexpression compared to wild sweet potato plantsIbDREB1dThe accumulation of flavonols in the transgenic sweet potato plants increased, indicatingIbDREB1dForward regulation of sweet potato flavonol synthesis for sweet potatoThe variety improvement of the potato has important significance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sweet potato transcription factor IbDREB1d and application thereof.
Background
Sweet potato is an important grain, feed, industrial raw material and energy crop in China, and leaf vegetable type sweet potato is a sweet potato variety which uses fresh and tender stems and leaves as vegetables, and compared with common sweet potato, the yield of the stems and leaves is higher. The sweet potato is a asexual propagation crop, the cross incompatibility between seeds and in-seeds severely limits the resource utilization and parent free combination in the sweet potato breeding, and the breeding practice shows that the conventional cross breeding method is difficult to quickly breed a new sweet potato variety with high quality and high yield. The genetic engineering technology is utilized to cultivate new varieties of sweet potatoes, so that the degradation of sweet potato germplasm can be reduced, the yield and quality of sweet potatoes can be improved, and better environmental benefit and social benefit can be realized.
The flavonol is a unique flavonoid compound with antiallergic, anti-inflammatory and anticancer properties, and mainly comprises substances such as quercetin, kaempferol, myricetin, isoquercitrin, rutin and the like, and the flavonol synthase is the first key enzyme for flavonol synthesis. The flavonol synthase gene (IbFLS) is the first key enzyme gene for flavonol synthesis, no gene for regulating and controlling IbFLS is reported at present, the transcription factor IbDREB1d for regulating and controlling the IbFLS is screened by a yeast single hybridization technology, and the forward regulation and control of the flavonol synthesis of the sweet potato by the IbDREB1d is verified by transgenic sweet potato.
Disclosure of Invention
The invention aims to provide a sweet potato transcription factor IbDREB1d and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the nucleotide sequence of the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO. 2.
The amino acid sequence of the protein coded by the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO. 3.
A vector containing the sweet potato transcription factor IbDREB1d.
An engineering bacterium containing sweet potato transcription factor IbDREB1d.
The primer pair for amplifying the sweet potato transcription factor IbDREB1d has the sequence as follows: 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3'.
The application of the sweet potato transcription factor IbDREB1d in promoting the synthesis and accumulation of sweet potato flavonols.
The sweet potato transcription factor IbDREB1d, the sweet potato IbFLS gene promoter or the interaction of the sweet potato transcription factor IbDREB1d and the sweet potato IbFLS gene promoter is applied to cultivation of transgenic sweet potato with altered flavonol compound content, wherein the nucleotide sequence of the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO.2, and the nucleotide sequence of the sweet potato IbFLS gene promoter is shown as SEQ ID NO. 1.
The invention has the remarkable advantages that:
compared with wild sweet potato plants, the research of the invention discovers that the accumulation amount of flavonols compounds of transgenic sweet potato plants which overexpress IbDREB1d is increased, which indicates that IbDREB1d positively regulates the synthesis of the flavonols compounds of sweet potato, and has important significance for variety improvement of sweet potato.
Drawings
Fig. 1: agarose gel electrophoresis of sweetpotato IbFLS gene promoter.
Fig. 2: self-activation detection of recombinant bait vector IbFLSpro-pAbAi.
Fig. 3: interaction verification of IbFLS gene promoter and IbDREB1d.
Fig. 4: plasmid map of the overexpression vector pEGOEP-35S-DREB1D (namely pEGOEP35S-G418-DREB 1D).
Fig. 5: the IbDREB1d over-expression transgenic sweetpotato is obtained through rooting transformation.
Fig. 6: and (3) carrying out PCR verification on the IbDREB1d over-expressed transgenic sweet potato.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
Extracting total RNA of leaf tissue of 'Fucai potato No. 18', reversely transcribing into cDNA as a template, and amplifying by a PCR method with 5'-GGCAGCTCTTCCAATAGCGGCG-3' and 5'-TTTTTAGCAAGATAATGGAG-3' as primers to obtain the IbFLS gene promoter with the base sequence of 2746bp and the nucleotide sequence shown as SEQ ID No. 1.
Example 2
The cloned gene promoter sequence of the 'Fu potato No. 18' IbFLS is constructed in a bait vector pAbAi (Clontech) by a homologous recombination method, and the recombinant bait vector is named IbFLSpro-pAbAi. According toThe recombinant bait vector IbFLSpro-pAbAi was integrated into the Yeast Y1H Gold genome, resulting in a bait strain, coated with the following media, as described in Gold Yeast One-Hybrid Library Screening System (Clontech): SD-Ura/AbA (0 ng/mL), SD-Ura/AbA (100 ng/mL), SD-Ura/AbA (150 ng/mL), SD-Ura/AbA (200 ng/mL), SD-Ura/AbA (250 ng/mL), and culturing at 28℃for 3d. The results show that the recombinant bait vector IbFLSpro-pAbAi is not self-activated in the presence of 200ng/mLAbA, and that the vector can be used for the next single-hybrid yeast screening library experiment.
Extracting total RNA of leaf tissue of' Fucai potato No. 18The Gold Yeast One-Hybrid Library Screening System (Clontech) constructed a cDNA library and loaded into the prey vector pGADT7-Rec to obtain a sweetpotato cDNA Yeast library plasmid. According to->Yeast single hetero screening library experiments were performed by the method described in Gold Yeast One-Hybrid Library Screening System (Clontech), the sweet potato cDNA library plasmid and recombinant bait vector IbFLSpro-pAbAi were co-transformed into Yeast strain Y1 HGold, and the transformed resuspended bacteria were plated on SD-Leu solid plates containing AbA at the corresponding concentration and cultured for 3d at 28 ℃. Candidate transcription factor IbDREB1d was obtained by PCR detection, sequencing analysis and sweetpotato genome database alignment annotation. The nucleotide sequence of the transcription factor IbDREB1d is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3.
To further verify the interaction of the IbFLS gene promoter with IbDREB1d, the following media were applied after dilution by constructing the transcription factor IbDREB1d in the prey vector pGADT7-AD (Clontech), the IbFLS promoter in the decoy vector pAbAi, and then co-transforming the yeast strain Y1 HGold: SD-Leu/AbA (0 ng/mL), SD-Leu/AbA (400 ng/mL), and incubation at 28℃for 3d. The verification procedure used pGADT7-AD empty vector as control. The results show that IbDREB1d can bind to IbFLS promoter to develop AbA resistance, indicating that IbDREB1d can interact with IbFLS promoter.
Example 3
Extracting total RNA of leaf tissue of 'Fucai potato No. 18', reversely transcribing the total RNA into cDNA as a template, and amplifying the cDNA by a PCR method by using 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3' as primers to obtain a transcription factor IbDREB1d. The transcription factor IbDREB1d sequence was ligated with the super-expression vector pEGOEP-35S (available from Ai Dijing Biotechnology Co., ltd.) to obtain the super-expression vector pEGOEP-35S-DREB1d. The transgenic sweet potato is obtained by a rooting transformation method, and the specific steps are as follows:
1) Single colony of Agrobacterium rhizogenes K599 was picked up and cultured in 3ml LB liquid medium containing antibiotics (50 mg/L kanamycin+50 mg/Lstreptomycin) at 28℃with shaking for 1 d%OD 600 >0.4)。
2) 0.5ml of the bacterial liquid is taken, 30ml of LB liquid medium containing antibiotics (50 mg/L kanamycin+50mg/L streptomycin) and 0.5% glucose is added, and the culture is carried out at 28 ℃ for 16-18h in a shaking way.
3) The bacterial liquid was centrifuged at 5000Xg for 10min (4 ℃ C.), the supernatant was discarded, 30ml of a medium (1/2MS+0.5% glucose+0.1 mM AS) was added to the pellet, and the pellet was subjected to shaking culture at 28℃for 6 hours to activate the infectivity of the bacteria (OD was adjusted) 600 =0.2)。
4) The stem node of 'Fucai potato No. 18' is put into activated bacterial liquid, and is infected by shaking at 125rpm for 10-20min, then is washed with sterile water for multiple times, and then is sucked dry by filter paper.
5) The pedicles (cut next to medium) were placed in medium (1/2MS+3% sucrose+0.7% agar+0.01 mM AS) and incubated for 3d in the dark.
6) The stem nodes were transferred to medium (1/2MS+125ppm Timetin+125mg/L cefotaxin+50 ppm kana+3% sucrose+0.7% agar) and rooting was performed.
7) After rooting, the root system is transferred to a 1/2MS (medium containing 200ppm Titin, 200mg/L cefotaxine) +3% sucrose+0.7% agar to induce sprouting, and plantlets are formed.
8) Transgene validation using PCR, PCR reaction procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 45s, extension at 72℃for 1min,35 cycles, and extension at 72℃for 10min. Obtaining IbDREB1d over-expression transgene positive plants.
And (3) taking transgenic plants and wild plants ('Fucai potato No. 18') of 30d years old, and measuring the flavonol content of stems and leaves.
TABLE 1 comparison of IbDREB1d over-expressed transgenic sweetpotato and wild type control flavonol content
Sequential order | Substance (B) | IbDREB1d overexpressing transgenic sweetpotato (mg/kg) | Wild sweet potato (mg/kg) |
1 | Rutin | 12.21 | 2.01 |
2 | Isoquercitrin | 83.46 | 30.97 |
3 | Quercetin | 3.15 | 0.49 |
4 | Kaempferol | 0.31 | 0.05 |
Total flavonol content | 99.13 | 33.52 |
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. Sweet potato transcription factorIbDREB1dWhich is provided withIs characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.
2. The sweet potato transcription factor of claim 1IbDREB1dA protein encoded therein, characterized in that: the amino acid sequence is shown as SEQ ID NO. 3.
3. A transcription factor comprising sweet potato as claimed in claim 1IbDREB1dIs a carrier of (a).
4. A transcription factor comprising sweet potato as claimed in claim 1IbDREB1dIs an engineering bacterium.
5. A method for amplifying the sweet potato transcription factor of claim 1IbDREB1dIs characterized in that: the sequence is as follows: 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3'.
6. The sweet potato transcription factor of claim 1IbDREB1dThe application of the sweet potato flavonol compound in promoting the synthesis and accumulation of sweet potato flavonol compounds.
7. Sweet potato transcription factorIbDREB1dSweet potatoIbFLSGene promoter, or sweet potato transcription factorIbDREB1dWith sweet potatoIbFLSUse of gene promoter interactions in the cultivation of transgenic sweetpotato with altered flavonol content, characterized in that: sweet potato transcription factorIbDREB1dThe nucleotide sequence is shown as SEQ ID NO.2, sweet potatoIbFLSThe nucleotide sequence of the gene promoter is shown as SEQ ID NO. 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311096573.4A CN117070529A (en) | 2023-08-29 | 2023-08-29 | Sweet potato transcription factor IbDREB1d and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311096573.4A CN117070529A (en) | 2023-08-29 | 2023-08-29 | Sweet potato transcription factor IbDREB1d and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117070529A true CN117070529A (en) | 2023-11-17 |
Family
ID=88719344
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311096573.4A Pending CN117070529A (en) | 2023-08-29 | 2023-08-29 | Sweet potato transcription factor IbDREB1d and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117070529A (en) |
-
2023
- 2023-08-29 CN CN202311096573.4A patent/CN117070529A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107338230B (en) | The application of OsMPK11 albumen and its encoding gene in regulation plant drought resistance | |
CN107629121A (en) | One from transcription factor ZmNLP9 of corn and application thereof | |
CN117384927A (en) | Application of tomato LecRK26 gene in regulation and control of plant gray mold resistance | |
US20090205073A1 (en) | Transgenic plants and methods for controlling bolting in sugar beet | |
CN115851821B (en) | Application of BBX16 gene in improving plant salt tolerance | |
CN103183731B (en) | Dendrobe DnMYB type transcription factor, coding gene, carrier and engineering bacteria and application thereof | |
CN103014035A (en) | Tumorous stem mustard stress-resistant gene, plant expression vector, construction method and application thereof | |
CN117511975A (en) | Application of LecRK27 gene in improving fruit uniformity and yield | |
CN116694661A (en) | ShN/AINV5-4D gene for regulating plant germination rate and application thereof | |
WO2020093128A1 (en) | Method for producing castor oil plant seeds lacking ricin/rca, castor oil plants lacking ricin/rca, method for identifying castor oil plants lacking ricin/rca, polynucleotides, constructs and uses thereof | |
CN113481176B (en) | Application of GA3ox1 protein in regulating alfalfa plant type | |
CN117070529A (en) | Sweet potato transcription factor IbDREB1d and application thereof | |
CN114921473A (en) | Gene for negatively regulating synthesis of cassava endogenous salicylic acid and application thereof | |
CN104805100B (en) | Paddy gene OsS μ 2 applications in plant leaf blade aging is delayed of BP | |
CN114644701B (en) | Use of proteins derived from corn and related biomaterials | |
KR102573952B1 (en) | Gene editing system for simultaneous gene editing of E2 and its homolog genes and uses thereof | |
Ilori et al. | Transgene expression in cowpea (Vigna unguiculata (L.) Walp.) through Agrobacterium transformation of pollen in flower buds | |
CN114644702B (en) | Tango protein, related biological material and plant breeding method | |
CN116121298B (en) | Application of inhibiting expression of HSRP1 gene in improving heat resistance of plants | |
CN116024232B (en) | Application of HvBGlu3 gene in regulation and control of barley grain beta-glucan content | |
CN110229801B (en) | Gene for controlling rice leaf senescence and protein coded by same | |
KR101272473B1 (en) | Method for producing transgenic Miscanthus plant and the plant thereof | |
CN117551689A (en) | Protein related to soybean plant height, single plant pod number and single plant grain number, biological material and application thereof | |
CN106674340B (en) | Hybrid paper mulberry transcription factor BpSEM and coding gene and application thereof | |
CN114717243A (en) | GRMZM2G063882 gene, encoding protein, biological material and application thereof in plant drought resistance |
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 |