CN116064436A - Loquat EjFADC1 gene, protein coded by same and application thereof - Google Patents
Loquat EjFADC1 gene, protein coded by same and application thereof Download PDFInfo
- Publication number
- CN116064436A CN116064436A CN202310130379.7A CN202310130379A CN116064436A CN 116064436 A CN116064436 A CN 116064436A CN 202310130379 A CN202310130379 A CN 202310130379A CN 116064436 A CN116064436 A CN 116064436A
- Authority
- CN
- China
- Prior art keywords
- gene
- ejfadc1
- loquat
- plant
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 70
- 235000009008 Eriobotrya japonica Nutrition 0.000 title claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 17
- 244000061508 Eriobotrya japonica Species 0.000 title description 12
- 241001092070 Eriobotrya Species 0.000 claims abstract description 39
- 241000196324 Embryophyta Species 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 12
- 230000002018 overexpression Effects 0.000 claims abstract description 9
- 239000013604 expression vector Substances 0.000 claims abstract description 8
- 241000589158 Agrobacterium Species 0.000 claims abstract description 4
- 238000009395 breeding Methods 0.000 claims abstract description 4
- 230000001488 breeding effect Effects 0.000 claims abstract description 4
- 230000001404 mediated effect Effects 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 230000009261 transgenic effect Effects 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 17
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 241000219194 Arabidopsis Species 0.000 abstract description 17
- 239000002299 complementary DNA Substances 0.000 abstract description 11
- 241000219195 Arabidopsis thaliana Species 0.000 abstract description 6
- 238000004043 dyeing Methods 0.000 abstract description 4
- 108091026890 Coding region Proteins 0.000 abstract description 3
- 101100438273 Arabidopsis thaliana CAN1 gene Proteins 0.000 abstract 1
- 101000952234 Homo sapiens Sphingolipid delta(4)-desaturase DES1 Proteins 0.000 abstract 1
- 238000003208 gene overexpression Methods 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 208000035199 Tetraploidy Diseases 0.000 description 3
- 208000026487 Triploidy Diseases 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000003792 electrolyte Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 101100390319 Arabidopsis thaliana FAD8 gene Proteins 0.000 description 2
- 101150072203 FAD8 gene Proteins 0.000 description 2
- 208000001034 Frostbite Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- -1 diene fatty acid Chemical class 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000012250 transgenic expression Methods 0.000 description 2
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010087894 Fatty acid desaturases Proteins 0.000 description 1
- 239000005561 Glufosinate Substances 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000220222 Rosaceae Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000009889 stress physiology Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
Images
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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
Abstract
The invention belongs to the field of plant molecular biology, and particularly relates to a loquat membrane lipid desaturase gene EjFADC1 and application thereof. The full length of the coding region sequence of the cDNA of the EjFADC1 gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The gene is induced to express at low temperature and is closely related to the cold resistance of loquat. The EjFADC1 gene over-expression vector is transferred into wild type Arabidopsis by an agrobacterium-mediated inflorescence dip-dyeing method. The result shows that the over-expression of EjFADC1 gene in wild type Arabidopsis thaliana can enhance the cold resistance of Arabidopsis thaliana. The gene can be used for breeding cold-resistant varieties of loquat and has good application prospect.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to a loquat EjFADC1 protein, and a coding gene and application thereof.
Background
Loquat (Eriobotrya japonica) is a subtropical evergreen tree of Eriobotrya genus of Rosaceae family; the fruit is rich in nutrition and has a health care function, so that the fruit has good economic value. Loquat flowers and fruits are easy to frost in autumn and winter, and frost often causes serious economic loss in major loquat producing areas, and even in some areas, the loquat flowers and fruits are out of order. Therefore, the cultivation of cold-resistant varieties has important value for promoting the development of industry.
Low temperatures often result in huge economic losses in agriculture. The fatty acid desaturase gene (FAD) gene enhances the fluidity of the film by increasing the content of unsaturated fatty acids, thereby enhancing the cold resistance of plants. The EjFADC1 gene is expressed by low temperature induction, and analysis of the evolutionary tree shows that the EjFADC1 gene and the FAD8 gene of arabidopsis thaliana belong to a large evolutionary branch, and the EjFADC1 gene and the FAD8 gene have similar functions, FAD8 is positioned on chloroplasts, belong to omega-3 type, and diene fatty acid on catalytic glyceride introduces the 3 rd double bond at omega-3 to form triene fatty acid. FAD8 is mainly induced by low temperature and is regulated at low temperature both at transcriptional and posttranslational levels. The cold resistance of the AtFAD8 can be improved by over-expressing the AtFAD8 in tobacco and rape; overexpression of OsFAD8 in rice can improve cold tolerance of rice. Functional research and application of the loquat EjFADC1 gene are not reported.
Disclosure of Invention
The invention aims to provide loquat EjFADC1 protein, and a coding gene and application thereof.
First, the present invention provides loquat EjFADC1 protein, which is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
The invention also provides a gene for encoding the loquat EjFADC1 protein.
Wherein the gene sequence is shown as SEQ ID No. 1.
The invention also provides a vector, a host cell and engineering bacteria containing the gene. Preferably, the vector is an over-expression vector.
The invention also provides application of the gene in plant cold-resistant breeding.
In one embodiment of the invention, the EjFADC1 gene is transferred into a plant genome and is overexpressed in a transgenic plant, so that the cold resistance of the plant is enhanced.
The invention also provides a construction method of the transgenic plant, which adopts an agrobacterium-mediated method to transfer the over-expression vector containing the EjFADC1 gene into a plant genome, and screens to obtain the transgenic plant.
Wherein, compared with the wild type, the transgenic plant has obviously enhanced cold resistance.
According to the invention, 1 EjFADC1 gene closely related to loquat cold resistance is cloned from loquat leaves. The real-time fluorescence quantitative PCR proves that the low-temperature induced EjFADC1 gene is expressed in a large quantity, and the expression quantity of the gene is higher than that of the loquat material with strong cold resistance. The over-expression vector of the EjFADC1 gene is constructed by utilizing a genetic engineering means, and is transferred into wild arabidopsis for over-expression, so that the cold resistance of the arabidopsis can be obviously enhanced. The invention provides a good application prospect for cold-resistant breeding of angiosperm.
Drawings
FIG. 1 is an electrophoresis photograph of the identification of the coding region sequence of the Eriobotrya japonica EjFADC1 gene. M is DL2000 DNA marker,1 is PCR product of EjFADC1 gene ORF.
FIG. 2 is a cDNA nucleotide sequence diagram of the coding region of the Eriobotrya japonica EjFADC1 gene.
FIG. 3 is a diagram showing the amino acid sequence and domain division of the Eriobotrya japonica EjFADC1 protein.
FIG. 4 shows that the Eriobotrya japonica EjFADC1 gene is expressed in Eriobotrya japonica leaves under low temperature induction.
FIG. 5 is a PCR identification of EjFADC1 transgenic Arabidopsis plants.
FIG. 6 is a plant morphological phenotype of overexpression of EjFADC1 in Arabidopsis. The low-temperature treated wild arabidopsis thaliana has a serious wilting and water loss state compared with the transgenic plant, most leaf edges begin to be in a dipping and curling state, the old leaf edges turn yellow, and the transgenic plant has little tender leaf edges frostbite and part of the old leaf edges turn yellow.
FIG. 7 is electrolyte leakage and MDA content of leaf blades of Arabidopsis overexpressing EjFADC1.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 cloning of the cDNA sequence of the Eriobotrya japonica EjFADC1 Gene
Extraction of total RNA of loquat leaf
Collecting low-temperature treated loquat leaves, rapidly placing the loquat leaves into a freezing tube, quickly freezing the loquat leaves in liquid nitrogen for 2 hours, and placing the loquat leaves into an ultralow temperature refrigerator at-80 ℃ for later use. And extracting total RNA in the loquat leaves by adopting an RNA extraction kit according to instructions, and detecting the concentration and quality of the RNA by using a trace nucleic acid concentration detector.
Reverse transcription of total RNA of loquat leaf into cDNA
Carrying out denaturation treatment on total RNA of loquat leaves, wherein the reaction system is as follows: the total RNA amount was 2000ng, wherein Oligo DT15 was 1. Mu.L (50 nmol/L), N6 was 0.5. Mu.L (50 nmol/L), RNase free water was added and the total volume was 14.5. Mu.L. The reaction procedure was 70℃for 10min and 4℃for 2min, and after the completion of the reaction, the reaction was rapidly taken out from the PCR apparatus.
Reverse transcription of total RNA of loquat leaves: 5 XM-MLV buffer 4. Mu.L, 10mM dNTP 1. Mu.L, RNase Inhibitor 0.25. Mu.L, and M-MLV 0.25. Mu.L were added to the denatured reaction solution, the reaction procedure was 60min at 42℃and 10min at 70℃and 30. Mu.LddH 2O was added to dilute the solution after the completion of the reaction, and the solution was stored at-20℃for further use.
Cloning of EjFADC1 Gene
The complete cDNA sequence of EjFADC1 was found by sequence alignment of transcriptome data and loquat genome, and clone primers of Oligo 7 designed gene were used, FLEjFADC1-F:5'-ATGGCGAGTTGGGTCCTCTCTGAAT-3', FLEjFADC1-R:5'-TTAATTTGATGGAGGAAAGCCACC-3'. The gene was amplified using a Phanta Max Super-Fidelity DNA Polymerase (P505-d 1/d2/d 3) kit, the EjFADC1 gene was amplified in a system of 2X Phanta Max Buffer at 25. Mu.L, dNTP Mix (10 mM) at 1.0. Mu.L, F (10. Mu.M) at 2.0. Mu.L, R (10. Mu.M) at 2.0. Mu.L, phanta Max Super-Fidelity DNA polymerase at 1.0. Mu.L, cDNA at 1.0. Mu.L, and ddH was added 2 The amplification procedure was 95℃for 4min, followed by 35 cycles of 95℃for 30sec,56℃for 30sec, and 72℃for 40sec, with a total volume of 50. Mu.L O, and 10min after the end of the cycle. After the PCR amplification is finished, electrophoresis detection is carried out, the agarose DNA gel recovery kit is used for recovery, the concentration and the quality are measured, and the sample is preserved at the temperature of minus 20 ℃. The recovered target fragment is connected with a Vector pTOPO-Blunt Vector and then transferred into competent cells of escherichia coli, monoclonal is selected, sequencing is carried out, sequencing results are analyzed and spliced, and the full length (SEQ ID No. 1) of the cDNA sequence of the loquat EjFADC1 gene is obtained, and the sequence picture is shown in figure 2.
Using BioEdit software, the protein sequence (SEQ ID No. 2) was translated through the full length of the cDNA sequence of the Eriobotrya japonica EjFADC1 gene, and the division of the transmembrane domains was performed according to the characteristics of the protein, and found that the EjFADC1 protein had four transmembrane domains (FIG. 3).
EXAMPLE 2 analysis of expression level of EjFADC1 Gene of Eriobotrya japonica
Selecting tetraploid loquat B431 (2n=4x=68) with different cold resistance, and triploid loquat B431×GZ23 (2n=3x=51); the cold resistance is as follows: b431×gz23 (LT 50 ＝-9.419)>B431(LT 50 ＝-7.195)。Taking branches with consistent thickness of B431 and B431 XGZ 23, taking common loquat as a stock, stressing the uniformly grafted seedlings at the temperature of minus 3 ℃ for 72 hours, each processing 4 biological repetitions, taking leaves with the same leaf position, extracting RNA, and reversely transcribing into cDNA as a template.
According to the full length of the cDNA sequence of the loquat EjFADC1 gene, designing a real-time fluorescence quantitative primer qEjFADC1F by using oligo 7.0 software: 5'-TTTGGAACCGACCCAGAACC-3' and qEjFADC1R:5'-CTACACAACCCACCTGCGAT-3'. The specificity of the PCR is detected by PCR, and the real-time fluorescent quantitative PCR can be performed on the premise of ensuring the specific amplification of the PCR. The loquat actin gene is taken as an internal reference gene, and the primer is qEjacin-F: 5'-AATGGAACTGGAATGGTCAAGGC-3' and qEjacin-R: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3'. Amplification was performed using a three-step procedure, with 3 biological replicates per reaction. The PCR reaction procedure was: pre-denaturation at 95℃for 1min;95℃20s,58℃20s,72℃30s,45 cycles; the dissolution profile was collected. Based on the Ct values obtained, the expression amounts of the EjFADC1 gene in triploid and tetraploid loquat under low temperature stress were calculated by using a 2-DeltaDeltaCT method, respectively (FIG. 4). The result shows that the expression level of the EjFADC1 gene in triploid loquat with strong cold resistance is higher than that of tetraploid with weak cold resistance.
EXAMPLE 3 construction of plant transgenic vector pFGC5941-EjFADC1 of Eriobotrya japonica EjFADC1 Gene
PCR amplification is adopted, and enzyme cutting sites are introduced at two ends of CDS region of loquat EjFADC1 gene. The cDNA of the total RNA reverse transcription of the loquat leaf is used as a template, and EjFADC1-F is used as a template: 5'-GGCGCGCCATGGCGAGTTGGGTCCTCTCTGAAT-3' (introduction of AscI cleavage site) and EjFADC1-R:5'-CGGGATCCTTAATTTGATGGAGGAAAGCCACC-3' (BamHI cleavage site was introduced) was used as a primer and PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase. The PCR products were subjected to 1% agarose gel electrophoresis and recovered using agarose gel DNA recovery kit. The recovered PCR product is connected with pTOPO-Blunt vector, transferred into competent cells of escherichia coli, and subjected to sample transfer and sequencing after monoclonal selection. According to the vector sequence, primers are designed at the junction of the vector and the target fragment for sequencing, plasmids are extracted, and plasmids of a transgenic vector pFGC5941 are extracted at the same time. Double-enzyme digestion of pTOPO-Blunt-EjFADC1 recombinant plasmid and pFGC5941 vector are respectively carried out by using AscI and BamHI restriction enzymes, and after the double-enzyme digestion of EjFADC1 gene and pFGC5941 are connected by using T4-DNA ligase, the mixture is transferred into competent cells of escherichia coli to obtain plant transgenic expression vector pFGC5941-EjFADC1. And (3) carrying out sample feeding and sequencing on the obtained expression vector, designing a primer at the joint of the vector and the target fragment according to the vector sequence, sequencing by bidirectional sequencing, and carrying out sequencing result to obtain the sequence of the vector sequence and the sequence of the EjFADC1 gene at the same time, so as to prove that the EjFADC1 gene is successfully connected with the pFGC5941 vector.
EXAMPLE 4 transfer of the transgenic expression vector pFGC5941-EjFADC1 into wild type Arabidopsis thaliana
Taking 2 mu g of pFGC5941-EjFADC1 plasmid, adding 100 mu L of agrobacteria competent cells, sucking and beating, and mixing uniformly; ice-bath for 30min, transferring into liquid nitrogen, rapidly freezing for 1min, water-bathing at 37deg.C for 5min, ice-bathing for 10min, adding 800 μl of LB liquid medium, and shaking at 28deg.C at 250rpm for 5h; transferring the bacterial liquid into LB (50 mL LB+50. Mu.g/mL Kan+50. Mu.g/mL Rif) solid selective medium, uniformly coating, and inversely culturing at 28 ℃ for 48 hours; selecting a monoclonal, and inoculating the monoclonal into 10mL of liquid LB culture medium (containing 10 mug/mL Kan+10 mug/mL Rif); the culture was shaken overnight at 28℃and 200rpm to OD=0.8. 2mL of the bacterial liquid was taken to 100mL of LB liquid medium (50. Mu.g/mL Kan and 50. Mu.g/mL Rif) and cultured until OD600 = 0.7-0.8, the bacterial liquid was transferred to 250 mL centrifuge tubes (sterilized), centrifuged at room temperature and 5000rpm for 5min, the supernatant was removed, 2mL of infection buffer (0.5% Silwet L-77,5% sucrose, 1/2MS medium) was added to each tube, and the bacterial cells were gently suspended, and 48mL of infection buffer (100 mL of infection buffer: 5g sucrose, 50. Mu.LSwet L-77,1/2MS medium) was further added.
Placing Arabidopsis seeds on wet filter paper, placing at 4deg.C for 48 hr, sowing into nutrient soil, and culturing under the conditions of temperature 22 deg.C, humidity 75%, and darkness for 14 hr/10 hr; watering the arabidopsis plants thoroughly the day before the transgenic; shearing off the horns on the Arabidopsis plants before dip-dyeing, and immersing inflorescences in pFGC5941-EjFADC1 Agrobacterium dip-dyeing liquid for about 60s for dip-dyeing conversion; covering a black sealing film, keeping the high-temperature and high-humidity environment in the film, and after dark culture for 2 days, uncovering the film and putting the film back into the normal environment for continuous growth. The method is used for infecting 3-4 times with the interval time of 7d.
Example 5 transgenic Arabidopsis screening and phenotypic characterization of the Eriobotrya japonica EjFADC1 Gene
Collecting EjFADC1 transgenic mature seeds of Arabidopsis thaliana, oven drying at 37deg.C, and vernalizing at 4deg.C. The vernalized seeds are sown on peatmoss, 20mg/L glufosinate (carrier pFGC5941 resistant to glufosinate) is sprayed once every three days for about ten days, and the surviving plant seeds are collected after spraying 3-4 times.
EjFADC1 transgenic Arabidopsis DNA was extracted. Taking 1 small piece of Arabidopsis leaves, placing the leaves in a 1.5mL eppendorf tube, placing the eppendorf tube into liquid nitrogen for quick freezing, and grinding; adding 600 mu L of extraction buffer, vortex shaking, and placing on ice; after all samples are treated, placing the samples in a water bath at 65 ℃ for 25min; taking out the sample from the water bath, standing to room temperature, cooling to room temperature, adding 340 mu L of potassium acetate solution, and carrying out vortex vibration for 20min in an ice bath; 12000rpm, high speed centrifugation for 10min, transferring the supernatant to a new eppendorf tube; adding equal volume of isopropanol, centrifuging at 4deg.C and 12000rpm for 20min, discarding supernatant, and rinsing with ice absolute ethanol (absolute ethanol is put into a refrigerator at-20deg.C for 2 hr in advance); rinsing the precipitate with 70% ethanol and 100% ethanol in sequence; after drying the precipitate, it was dissolved in 50. Mu.L of sterile water.
The DNA of wild type Arabidopsis is used as a control, an identification primer (35S-F: TGAGACTTTTCAACAAAGG ATAATT,35S-R: TGTCCTCTCCAAATGAAATGAAC) is designed according to a 35S promoter on a pFGC5941 vector to carry out PCR identification on the survival seedlings, and meanwhile, a cloning primer of a target gene is used for carrying out PCR identification to carry out screening identification on positive plant DNA of transgenic Arabidopsis. PCR amplification procedure: 95 ℃ for 4min; 25 cycles were performed at 95℃for 30s,56℃for 30s, and 72℃for 40 s; and at 72℃for 10min. After the reaction was completed, 1% agarose gel electrophoresis was performed, and detection was performed in a gel imaging system (fig. 5), and the result showed that 21 arabidopsis plants were obtained in total, of which 15 were positive plants.
Planting wild type (Wt) and T3 generation homozygous transgenic plants in turfy soil, culturing, wherein each 3 plants is a basin, each basin is a biological repetition, 3 biological repetitions are added, the culture temperature is about 23+ -0.5deg.C, the humidity is about 60%, and the luminous flux of illumination is denseDegree (PPFD) 50+ -5 mu mol.m -2 ·s -1 Photoperiod was 14/10 (day/light) and incubated for 6 weeks until rosette leaves were formed. Wild type plants and transgenic plants growing for about 6 weeks are placed in a low-temperature treatment box, the temperature reduction speed is 1 ℃/h until the temperature reaches 0 ℃, the cultured illumination is weak light, the culture is maintained for 96 hours at low temperature, and the relative conductivities of the leaves and the MDA content of 96 hours and 96 hours are measured after the treatment is finished. The result shows that the wilting and water loss states of the wild arabidopsis thaliana treated at low temperature are more serious than those of the transgenic plant, most of leaf edges are in a dipping and curling state, the old leaf edges are yellow, and the transgenic plant has little tender leaf edges frostbite and even the old leaf edges are yellow; after 6d of growth recovery at 23 ℃, most of leaf edges of wild plants turn yellow, a small part of plants extract reproductive branches, a small number of old leaf edges of transgenic plants turn yellow, and a large number of reproductive branches are extracted from the plants, so that the growth speed of the reproductive branches is faster than that of the wild plants (figure 6). Electrolyte leakage and MDA are important evaluation indexes of plant stress physiology, and reflect the damage of cells and the peroxidation degree of membrane lipid; the test results show that the heterologous over-expression EjFADC1 remarkably reduces the electrolyte leakage and MDA content of the arabidopsis leaves and improves the cold resistance of the arabidopsis (figure 7).
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. Loquat EjFADC1 protein, which is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
2. A gene encoding the loquats EjFADC1 protein of claim 1.
3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.
4. A vector comprising the gene of claim 2 or 3.
5. A host cell comprising the vector of claim 4.
6. An engineered bacterium comprising the gene of claim 2 or 3.
7. Use of the gene according to claim 2 or 3 in cold tolerant breeding of plants.
8. The use according to claim 7, wherein the gene according to claim 2 or 3 is transferred into the genome of a plant and overexpressed in the transgenic plant, enhancing the cold tolerance of the plant.
9. A method for constructing a transgenic plant, which is characterized in that an agrobacterium-mediated method is adopted to transfer an over-expression vector containing the gene as set forth in claim 2 or 3 into a plant genome, and the transgenic plant is obtained by screening.
10. The method of claim 9, wherein the transgenic plant has significantly increased cold tolerance compared to wild type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310130379.7A CN116064436A (en) | 2023-02-17 | 2023-02-17 | Loquat EjFADC1 gene, protein coded by same and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310130379.7A CN116064436A (en) | 2023-02-17 | 2023-02-17 | Loquat EjFADC1 gene, protein coded by same and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116064436A true CN116064436A (en) | 2023-05-05 |
Family
ID=86171382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310130379.7A Pending CN116064436A (en) | 2023-02-17 | 2023-02-17 | Loquat EjFADC1 gene, protein coded by same and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116064436A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640198A (en) * | 2023-06-06 | 2023-08-25 | 西南大学 | Cold-resistant gene EjGLK1 of loquat, protein encoded by gene and application of gene |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001085968A2 (en) * | 2000-05-09 | 2001-11-15 | Bioriginal Food & Science Corp. | Production of conjugated linoleic and linolenic acids in plants |
| CN103740739A (en) * | 2014-01-20 | 2014-04-23 | 中国科学院寒区旱区环境与工程研究所 | Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof |
| CN105400805A (en) * | 2015-12-24 | 2016-03-16 | 浙江大学 | Gene participating in peach fruit fatty acid forming and application thereof |
| CN108410829A (en) * | 2018-03-14 | 2018-08-17 | 内蒙古农业大学 | Mongolian Ammopiptanthus mongolicus chloroplaset omega-fatty acid desaturase AmFAD7 encoding genes and application |
| CN110862998A (en) * | 2019-12-11 | 2020-03-06 | 重庆师范大学 | Purple perilla FAD8 gene and application thereof in improving unsaturated fatty acid content and cold resistance of plants |
| US20220183296A1 (en) * | 2019-03-20 | 2022-06-16 | Sumitomo Chemical Company, Limited | Herbicidal composition and method for controlling weeds |
-
2023
- 2023-02-17 CN CN202310130379.7A patent/CN116064436A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001085968A2 (en) * | 2000-05-09 | 2001-11-15 | Bioriginal Food & Science Corp. | Production of conjugated linoleic and linolenic acids in plants |
| CN103740739A (en) * | 2014-01-20 | 2014-04-23 | 中国科学院寒区旱区环境与工程研究所 | Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof |
| CN105400805A (en) * | 2015-12-24 | 2016-03-16 | 浙江大学 | Gene participating in peach fruit fatty acid forming and application thereof |
| CN108410829A (en) * | 2018-03-14 | 2018-08-17 | 内蒙古农业大学 | Mongolian Ammopiptanthus mongolicus chloroplaset omega-fatty acid desaturase AmFAD7 encoding genes and application |
| US20220183296A1 (en) * | 2019-03-20 | 2022-06-16 | Sumitomo Chemical Company, Limited | Herbicidal composition and method for controlling weeds |
| CN110862998A (en) * | 2019-12-11 | 2020-03-06 | 重庆师范大学 | Purple perilla FAD8 gene and application thereof in improving unsaturated fatty acid content and cold resistance of plants |
Non-Patent Citations (3)
| Title |
|---|
| MIHO TAKEMURA等: "Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L", BIOSCI BIOTECHNOL BIOCHEM, 7 April 2012 (2012-04-07) * |
| NCBI: "Predicted: Pyrus x bretschneideri omega-3 fatty acid desaturase, chloroplastic (LOC103930542), mRNA", GENBANK DATABASE, 9 June 2022 (2022-06-09), pages 009339892 * |
| 刘训言等: "植物ω-3脂肪酸去饱和酶的研究进展", 细胞生物学杂志, no. 1, 20 February 2004 (2004-02-20), pages 2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640198A (en) * | 2023-06-06 | 2023-08-25 | 西南大学 | Cold-resistant gene EjGLK1 of loquat, protein encoded by gene and application of gene |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845590B (en) | Wild grape VyPPR gene and application of encoding protein thereof in drought stress | |
| CN105254726B (en) | ERF class transcription factor relevant to plant stress-resistance and its encoding gene and application | |
| CN111235165B (en) | Lily susceptible fungal gene LrWRKY-S1 and application thereof | |
| CN112779234A (en) | Phyllostachys pubescens PeAPX5 gene and application thereof | |
| CN116064436A (en) | Loquat EjFADC1 gene, protein coded by same and application thereof | |
| CN110452917B (en) | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress | |
| CN102477435A (en) | Method for improving plant drought resistance using Poncirus trifoliata transcription factor gene PtrABF | |
| CN115161332B (en) | Vitis spinosa VdERF2 gene and encoding protein and application thereof | |
| CN115838404B (en) | EjMYB44 gene for regulating and controlling flowering phase of loquat, and encoding protein and application thereof | |
| CN113234720B (en) | Wheat long-chain non-coding RNAlncR156 and application thereof in regulation and control of wheat response to drought stress | |
| CN110184279A (en) | New a promotion branch development gene SrDREB2A and its expression vector and application in stevia rebaudianum | |
| CN115992153A (en) | Wheat stem rot resistance gene TaP CS, encoding protein and application thereof | |
| CN114807165A (en) | Application of corn ZmNAC78 gene | |
| CN105175522B (en) | Crowtoe AP2/ERF transcription factors and its encoding gene and application | |
| CN113088526A (en) | Heat shock related gene ZmHsf11 and application thereof in regulation and control of plant heat resistance | |
| CN116355867A (en) | Loquat EjFAD2 gene, protein coded by same and application thereof | |
| CN115948417B (en) | Barley HvFRF1 gene, protein, expression vector and application | |
| CN115960190B (en) | Loquat EjGASA6 gene, protein coded by same and application thereof | |
| CN108410829A (en) | Mongolian Ammopiptanthus mongolicus chloroplaset omega-fatty acid desaturase AmFAD7 encoding genes and application | |
| CN114478730B (en) | Wheat TaVQ14 protein, and coding gene and application thereof | |
| CN116515857B (en) | Apricot PaPIP1-2 gene for kernel and application thereof in improving cold resistance of plants | |
| CN115011631B (en) | Protein for regulating drought resistance of corn at seedling stage, and coding gene and application thereof | |
| CN116200401B (en) | Application of feather needle grass sugar transporter gene SpSWEET13 in promotion of plant root sticky soil | |
| CN114875025B (en) | Drought and ABA inducible promoter P SCBV-YZ2060 And applications thereof | |
| CN115838738B (en) | Paeonia ostii PoWRKY71 gene and application thereof in drought tolerance of plants |
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 |