CN112481277A - Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector - Google Patents
Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector Download PDFInfo
- Publication number
- CN112481277A CN112481277A CN202011574583.0A CN202011574583A CN112481277A CN 112481277 A CN112481277 A CN 112481277A CN 202011574583 A CN202011574583 A CN 202011574583A CN 112481277 A CN112481277 A CN 112481277A
- Authority
- CN
- China
- Prior art keywords
- cnfls
- antisense
- gene
- double
- synthesis
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—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
- C12N15/825—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 involving pigment biosynthesis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Virology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Golden camellia teaCnFLS+ antisenseF3’HA method for promoting plant flavonol synthesis by constructing a double-gene vector belongs to the technical field of genetic engineering. The method comprises the following steps: 1) cloned golden camelliaCnFLSGene, synthetic tobacco antisense expressionF3’HAn interfering sequence of a gene; 2) construction ofCnFLS+ antisenseF3’HA double gene vector; 3) will be provided withCnFLS+ antisenseF3’HTransferring the double-gene vector into escherichia coli, extracting plasmids after correct sequencing, transferring the plasmids into agrobacterium, and transforming the Nicotiana benthamiana by adopting an agrobacterium-mediated leaf disc method; 4) by PCRIdentifying positive strain of transgenic tobacco, and determining the content of flavonol and polyphenol in the positive strain. The invention over-expresses golden camelliaCnFLSGenes, Simultaneous design interferenceF3’HThe expression of the gene(s) is,CnFLSgene overexpression promotes synthesis of flavonol, antisense interferenceF3’HThe expression of the gene can inhibit the synthesis of polyphenol and anthocyanin, and further can yellow the flower color of the plant.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to golden camelliaCnFLS+ antisenseF3’HA method for promoting plant flavonol synthesis by using double-gene vector construction.
Background
The flavonoid is widely present in most plants, is a decisive pigment group formed by most plant colors, can improve the stress resistance of the plants, is a natural organic antioxidant, and has higher nutritional and health-care values. Flavonoids are a general term for a large class of secondary metabolites, and are mainly present in the vacuole of the epidermal tissue cells of petals, wherein flavones (flavanone), flavonols (flavanol) and the like belong to a yellow system, anthocyanidins (anthocyanins) belong to a red system, and polyphenols (polyphenols) are colorless.
Camellia nitidissima (A) and (B)Camellia nitidissima) The flower color is golden, which not only has extremely high ornamental value, but also is a rare genetic resource for yellow camellia breeding. The previous researches found that the main reason for flower color formation of golden camellia is caused by high flavonol content in petalsFLSThe gene (flavonol synthase gene) is a key gene for synthesizing flavonol, but the predecessor only overexpresses the camellia nitidissima aloneCnFLSThe gene is difficult to obviously increase the content of flavonol in the model plant to form golden flowers.FLSGenes andF3’Hthe gene has substrate competition effect, and is found in a plurality of researchesFLSThe expression level of the gene is increased,F3’Hthe expression level of the gene is reduced, and the advantage is increasedIn the synthesis of flavonol, thereby forming yellow flowers.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to design and provide golden camelliaCnFLS+ antisenseF3’HA method for promoting plant flavonol synthesis by using double-gene vector construction. The invention obtains golden camellia by PCR amplificationCnFLSArtificially synthesized tobaccoF3’HGene antisense sequence, enzyme digestion construction of over-expression by using OSCR cloning technologyCnFLS+ antisense expressionF3’HThe obtained double-gene expression vector is used for transforming the Nicotiana benthamiana, so that the flavonol content of a positive strain is obviously improved.
Golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized by comprising the following steps of:
1) cloned golden camelliaCnFLSGene, synthetic tobacco antisense expressionF3’HAn interfering sequence of a gene;
2) construction ofCnFLS+ antisenseF3’HA double gene vector;
3) will be provided withCnFLS+ antisenseF3’HTransferring the double-gene vector into escherichia coli, after the sequencing is correct, extracting plasmids and transferring the plasmids into agrobacterium, and transforming the Nicotiana benthamiana by adopting an agrobacterium-mediated leaf disc method;
4) and (3) identifying the positive strain of the transgenic tobacco by adopting PCR, determining the contents of a flavone component, a polyphenol component and an anthocyanin component of the flower of the positive strain by using an HPLC method, and calculating the total amount of the flavone, the total amount of the polyphenol and the total amount of the anthocyanin.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting the synthesis of plant flavonol by constructing a double-gene vector is characterized in that the clone in the step 1)CnFLSThe gene is specifically as follows: based on Camellia ChysanthaCnFLSDesigning primers pFLS-F and pFLS-R according to a gene sequence, carrying out PCR amplification by using ExTaq enzyme, carrying out gel electrophoresis analysis and detection on an amplification product, and then recovering and purifying the amplification product, wherein the nucleotide sequence of the PFLS-F is shown as SEQ ID No.1, the nucleotide sequence of the pFLS-R is shown as SEQ ID No.2, and the PCR amplification reaction program is as follows: 98Pre-denaturation at deg.C for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ for 5 min.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the method for synthesizing the interference sequence in the step 1) is specifically as follows: according to tobaccoF3’HSequence of Gene, design and Synthesis of antisenseF3’HGene interference sequence, antisense expressionF3’HThe nucleotide sequence of the interference sequence of the gene is shown as SEQ ID NO. 3.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the step 2) specifically comprises the following steps: subjecting the product obtained in step 1)CnFLSGene, antisenseF3’HThe gene interference sequences are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-antiF3’HIntermediate vector is subjected to enzyme digestion connection by using OSCR cloning technology, and a product is recovered to obtainCnFLS+ antisenseF3’HA double gene vector.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized in that the enzyme digestion system is as follows: buffer 2 muL, BsmBI/Esp3I: 1 muL, T4_ ligase 1 muL, pBWA (V) KS-FLS 4 muL, pBWD (LA)1C-antiF3’H: 4µL，H 28 muL of O, 20 muL of Total, wherein the enzyme digestion ligation reaction program is as follows: 20 min at 37 ℃; 10min at 37 ℃, 10min at 20 ℃ and 5 cycles; 20 min at 37 ℃; 5min at 80 ℃.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting the synthesis of plant flavonol by constructing a double-gene vector is characterized in that the step 3) is implementedCnFLS+ antisenseF3’HThe method comprises the following steps of transferring the double-gene vector into escherichia coli, extracting plasmids and transferring the plasmids into agrobacterium after correct sequencing:CnFLS+ antisenseF3’HTransforming the competence of escherichia coli by using the double-gene expression vector, coating a resistant LB plate, performing inverted culture at 37 ℃ for 12h, selecting bacteria, performing PCR identification and sequencing, correctly extracting plasmids by corresponding sequencing, and performing heat shock on the plasmidsCnFLS+ antisenseF3’HDouble baseThe vector is ligated into Agrobacterium.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the method for transforming the Nicotiana benthamiana by the leaf disc method in the step 3) specifically comprises the following steps: will containCnFLS+ antisenseF3’HCulturing Agrobacterium with double gene vectors in LB liquid culture medium containing 50mg/L Kan under shaking at 28 deg.C and 200rpm to OD600 of 0.4-0.5, centrifuging to collect thallus, resuspending cells in liquid culture medium containing 100 μ M/L acetosyringone to obtain infection solution, collecting strong tobacco sterile seedling leaves with age of 4-6 weeks, cutting into leaf discs, blowing, inoculating in co-culture medium, culturing at 28 deg.C for 3 days, washing the leaves with sterile water for 10 times, blowing, inoculating in screening culture medium, selecting and culturing at illumination period of 16h/8h and 25 deg.C, subculturing once every 2 weeks, inoculating fresh untreated leaves in screening culture medium as control, when adventitious bud grows to 1-2cm, cutting, transferring into rooting culture medium, rooting for 2-3 weeks, and rooting for 2-3 weeks, Removing the strong Nicotiana benthamiana tissue culture seedlings from the tissue culture bottle, and transferring the tissue culture bottles to outdoor culture.
The golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized in that the step 4) specifically comprises the following steps: the kit is used for positive identification, and the PCR program comprises the following steps: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 s, annealing at 65 ℃ for 10 s, extension at 72 ℃ for 1min for 30s, 30 cycles; extending at 72 deg.C for 5min, detecting by 1% agarose gel electrophoresis, collecting flowers after positive plants bloom, freezing with liquid nitrogen, storing at-80 deg.C, and determining by real-time fluorescent quantitative PCRCnFLS+ antisenseF3’HRelative expression of the two genes in positive flowers.
Golden camellia obtained by the methodCnFLS+ antisenseF3’HA double gene vector.
The golden camellia teaCnFLS+ antisenseF3’HApplication of double-gene vector in promoting plant flavonol synthesis.
The invention relates to a method for preparing golden camellia through overexpressionCnFLSGenes, co-interferenceF3’HExpression of genesThe design is reasonable, and the design is reasonable,CnFLSthe over-expression of the gene promotes the synthesis of flavonol, reducesF3’HGene pairFLSThe substrate competition effect of the gene reduces the synthesis of polyphenol and anthocyanin, increases the synthesis of flavonol, improves the content of the flavonol in the plant and can achieve the aim of yellowing the flower color of the plant.
Drawings
FIG. 1 shows the total content of polyphenols and total content of flavonoids in transgenic positive lines;
FIG. 2 shows the contents of polyphenol and flavone components in transgenic positive lines, wherein EGC: epigallocatechin, GC: gallocatechin, CG: catechin gallate, ECG: epicatechin gallate is a polyphenol component. Qu 3R: quercetin 3-rutinoside, Ru: rutin, Qu 7G: quercetin 7-O- β -D-glucoside, Qu 3G: quercetin 3-O-glucose is flavone component.
Detailed Description
The invention will be further explained with reference to the drawings and examples.
Example 1:
1. golden camellia teaCnFLSCloning of Gene, antisense expression of tobaccoF3’HInterfering sequence synthesis of genes:
(1) prepared from Camellia nitidissimaCnFLSGene sequence (Genbank ID: JF 343560.1) the nucleotide sequences of the designed primers are shown in SEQ ID NO.1 and 2, and shown in Table 1, ExTaq enzyme (RR 902, TaKaRa) is used for PCR amplification to obtain the full-length CDS sequence of the target gene. The PCR reaction procedure was as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ for 5 min.
(2) The PCR products were analyzed by 1% agarose gel electrophoresis (110 v, 45 min). The amplification product was recovered and purified using Axygen gel recovery kit (AP-MN-P-250, Axygen).
(3) According to tobaccoF3’HThe nucleotide sequence of the antisense expression interference sequence is designed as shown in SEQ ID NO.3, and the interference sequence is artificially synthesized.
2. CnFLS+ antisenseF3’HConstruction of a double-gene vector:
(1) will be provided withCnFLSGene, antisenseF3’HThe gene interference sequences are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-anti F3' H intermediate vectors.
(2) Carrying out enzyme digestion and linkage by using an OSCR cloning technology, wherein an enzyme digestion system is as follows:
Buffer: 2µL BsmBI/Esp3I: 1µL T4_ligase: 1µL pBWA(V)KS-FLS: 4µL pBWD(LA)1C-antiF3’H: 4µL
H2a connection reaction process of 8 muL Total:20 muL: 20 min at 37 ℃; 10min at 37 ℃, 10min at 20 ℃ and 5 cycles; 20 min at 37 ℃; 5min at 80 ℃.
(3) After the reaction is completed, products are recovered, 5 muL of the ligation products are transformed into escherichia coli competence, the escherichia coli competence is transformed and coated on a (kanamycin) resistant LB plate, inverted culture is carried out for 12 hours at 37 ℃, and single colonies are selected for PCR identification and sequencing. After the sequencing result is obtained, correspondingly sequencing the correctly extracted plasmid to obtainCnFLS+ antisenseF3’HA double gene vector. By using a heat shock methodCnFLS+ antisenseF3’HThe double gene vector was ligated into the GV3101 agrobacterium.
3. Leaf disc method for transforming Nicotiana benthamiana:
(1) will containCnFLS+ antisenseF3’HGV3101 Agrobacterium with a double-gene vector was shake-cultured in LB liquid medium containing 50mg/L Kan at 200rpm at 28 ℃ to OD 600: 0.4-0.5, and collecting the thalli by centrifugation at 8000rpm and 4 ℃. Resuspend cells in a liquid culture medium containing 100 μ M/L Acetosyringone (AS) to prepare an infection solution.
(2) Taking strong tobacco sterile seedling leaves with the seedling age of 4-6 weeks, shearing the strong tobacco sterile seedling leaves into leaf discs, and infecting for 5-10 min. Blow-drying, inoculating on co-culture medium, and co-culturing at 28 deg.C in dark for 3 days.
(3) The co-cultured leaves are washed 10 times by sterile water, dried, inoculated on a screening culture medium, selectively cultured under the conditions of illumination period of 16h/8h and 25 ℃, and subcultured once every 2 weeks. Fresh untreated leaves were inoculated onto screening media as controls. When the adventitious bud grows to about 1-2cm, cutting off and transferring into a rooting culture medium. And (4) rooting 2-3 weeks (the root length is 2-3 cm), taking out the strong growing Nicotiana benthamiana tissue culture seedling from the tissue culture bottle, and transferring the tissue culture seedling to the outdoor for culture.
4. Positive identification and index determination of transgenic tobacco:
(1) positive identification of the root plants was carried out using the T5 Direct PCR Kit (Plant) with the primers shown in Table 1, PCR program: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 s, annealing at 65 ℃ for 10 s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension is carried out for 5min at 72 ℃ and detection is carried out by 1% agarose gel electrophoresis.
(2) After the positive plants blossomed, the flowers were collected and frozen in liquid nitrogen and stored at-80 ℃. The relative expression of the target gene in the flowers of the positive lines was determined by using real-time fluorescent quantitative PCR (qPCR) primers shown in Table 1.
(3) Measuring the content of flavone component, polyphenol component and anthocyanin component in the positive plant flower by HPLC method, and calculating total flavone amount (TF), total polyphenol amount (TP) and total anthocyanin amount (TA), and performing separate overexpression with wild tobaccoCnFLSGene and single over-expression antisenseF3’HPositive lines of the genes were compared.
TABLE 1 primer sequences
Comparative experiment 1:
and (4) transplanting the non-transformed wild type Nicotiana benthamiana, and planting in the same environment. Measuring the contents of flavone component, polyphenol component and anthocyanin component in plant flower by HPLC method, and calculating total flavone amount (TF), total polyphenol amount (TP) and total anthocyanin amount (TA), andCnFLS+ antisenseF3’HPositive lines of the double gene vectors were compared.
Comparative experiment 2:
obtained by cloning in step 1CnFLSThe gene was purified by homologous recombination according to the EXclone kit instructions (exv 09,baige Gene science and technology (Jiangsu) Ltd), primers were designed (see Table 1), and PCR was performed according to the following procedure: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 10 s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ for 10 min. After recovery of the correct product, it was ligated into the pCambia1300 vector. The pCambia1300 vector was ligated into Agrobacterium GV3101 using a heat shock method.
Transforming Nicotiana benthamiana according to the steps 3 and 4, measuring contents of flavone component, polyphenol component and anthocyanin component of plant flowers by using an HPLC method, and calculating total flavone amount (TF), total polyphenol amount (TP) and total anthocyanin amount (TA) of the plant flowers, andCnFLS+ antisenseF3’HPositive lines of the double gene vectors were compared.
Comparative experiment 3:
prepared from Camellia nitidissimaF3’HGene sequence (Genbank ID: HQ 290518.1) primers were designed (see Table 1) and cloned to obtainF3’HA gene sequence. Using the homologous recombination method, primers (see Table 1) were designed according to the EXclone kit instructions (exv 09, Baige Gene science, Inc.; Jiangsu Co., Ltd.) and PCR was performed as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 10 s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ for 10 min. After recovery of the correct product, it was ligated into the pCambia1300 vector. The pCambia1300 vector was ligated into Agrobacterium GV3101 using a heat shock method.
Transforming Nicotiana benthamiana according to the steps 3 and 4, measuring contents of flavone component, polyphenol component and anthocyanin component of plant flowers by using an HPLC method, and calculating total flavone amount (TF), total polyphenol amount (TP) and total anthocyanin amount (TA) of the plant flowers, andCnFLS+ antisenseF3’HPositive lines of the double gene vectors were compared. The test results are shown in fig. 1 and 2.
As is clear from the test results, as shown in Table 2, expression of example 1 by the method provided by the present inventionCnFLS+ antisenseF3’HThe total amount of polyphenols (TP) and the contents of EGC and GC in the positive lines of the double gene vectors are significantly lower than those in comparative experiment 3 (over-expression alone)F3’HGenes), CG and ECG were also lower in content than comparative experiment 3, but poor in contentThe difference is not significant; the total flavone content and the flavone component content were significantly higher in example 1 than in comparative experiment 1 (wild-type tobacco) and comparative experiment 2 (over-expression alone)FLSA gene). Show thatCnFLS+ antisenseF3’HDouble gene vector inhibitsF3’HThe promotion effect of the gene on polyphenol synthesis is greater than that of single over-expression, and the promotion effect of the gene on flavonol synthesis is promotedCnFLSA gene. The invention relates to a method for preparing golden camellia through overexpressionCnFLSGene, simultaneous antisense inhibitionF3’HReasonable design of gene expression, reduceF3’HGene pairFLSThe substrate competition effect of the gene reduces the synthesis of polyphenol, increases the synthesis of flavonol, improves the content of the flavonol in the plant and can achieve the aim of yellowing the flower color of the plant.
TABLE 2 content (mg/g) of polyphenol fraction and flavone fraction in comparative experiments 1-3 and examples
Sequence listing
<110> subtropical forestry research institute of China forestry science research institute
<120> method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> primer PFLS-F (primer PFLS-F)
<400> 1
aggagtgaga aggggaaaa 19
<210> 2
<211> 21
<212> DNA
<213> primer PFLS-R (primer PFLS-R)
<400> 2
cgagtgagtg aagaaggctc a 21
<210> 3
<211> 200
<212> DNA
<213> Antisense F3 'H gene interference sequence (Antisense F3' H gene interference sequence)
<400> 3
catcaacccc actctctctc ccccggatgg catctgacag ttgtgaaatc aatggctact 60
tcattccaaa gggctcaact cttcttgtca atgtatggcc atagcccgtg atccagatgc 120
gtgggccgag ccattagagt ttcggccaga acgattccta cctggtggcg aaaagcccaa 180
tgttgatgtt agggaaatga 200
Claims (10)
1. Golden camellia teaCnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized by comprising the following steps of:
1) cloned golden camelliaCnFLSGene, synthetic tobacco antisense expressionF3’HAn interfering sequence of a gene;
2) construction ofCnFLS+ antisenseF3’HA double gene vector;
3) will be provided withCnFLS+ antisenseF3’HTransferring the double-gene vector into escherichia coli, after the sequencing is correct, extracting plasmids and transferring the plasmids into agrobacterium, and transforming the Nicotiana benthamiana by adopting an agrobacterium-mediated leaf disc method;
4) and (3) identifying the positive strain of the transgenic tobacco by adopting PCR, determining the contents of a flavone component, a polyphenol component and an anthocyanin component of the flower of the positive strain by using an HPLC method, and calculating the total amount of the flavone, the total amount of the polyphenol and the total amount of the anthocyanin.
2. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting the synthesis of plant flavonol by constructing a double-gene vector is characterized in that the clone in the step 1)CnFLSThe gene is specifically as follows: based on Camellia ChysanthaCnFLSGene sequence design primers pFLS-F and pFLS-R, using ExTaq enzymePerforming PCR amplification, performing gel electrophoresis analysis and detection on an amplification product, and then recovering and purifying, wherein the nucleotide sequence of PFLS-F is shown as SEQ ID NO.1, the nucleotide sequence of pFLS-R is shown as SEQ ID NO.2, and the PCR amplification reaction program is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ for 5 min.
3. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the method for synthesizing the interference sequence in the step 1) is specifically as follows: according to tobaccoF3’HSequence of Gene, design and Synthesis of antisenseF3’HGene interference sequence, antisense expressionF3’HThe nucleotide sequence of the interference sequence of the gene is shown as SEQ ID NO. 3.
4. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the step 2) specifically comprises the following steps: subjecting the product obtained in step 1)CnFLSGene, antisenseF3’HThe gene interference sequences are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-antiF3’HIntermediate vector is subjected to enzyme digestion connection by using OSCR cloning technology, and a product is recovered to obtainCnFLS+ antisenseF3’HA double gene vector.
5. The Camellia chrysantha as set forth in claim 4CnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized in that the enzyme digestion system is as follows: buffer 2 muL, BsmBI/Esp3I: 1 muL, T4_ ligase 1 muL, pBWA (V) KS-FLS 4 muL, pBWD (LA)1C-antiF3’H: 4µL，H28 muL of O, 20 muL of Total, wherein the enzyme digestion ligation reaction program is as follows: 20 min at 37 ℃; 10min at 37 ℃, 10min at 20 ℃ and 5 cycles; 20 min at 37 ℃; 5min at 80 ℃.
6. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting the synthesis of plant flavonol by constructing a double-gene vector is characterized in that the step 3) is implementedCnFLS+ antisenseF3’HThe method comprises the following steps of transferring the double-gene vector into escherichia coli, extracting plasmids and transferring the plasmids into agrobacterium after correct sequencing:CnFLS+ antisenseF3’HTransforming the competence of escherichia coli by using the double-gene expression vector, coating a resistant LB plate, performing inverted culture at 37 ℃ for 12h, selecting bacteria, performing PCR identification and sequencing, correctly extracting plasmids by corresponding sequencing, and performing heat shock on the plasmidsCnFLS+ antisenseF3’HThe double gene vector is ligated into Agrobacterium.
7. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by constructing the double-gene vector is characterized in that the method for transforming the Nicotiana benthamiana by the leaf disc method in the step 3) specifically comprises the following steps: will containCnFLS+ antisenseF3’ HCulturing Agrobacterium with double gene vectors in LB liquid culture medium containing 50mg/L Kan under shaking at 28 deg.C and 200rpm to OD600 of 0.4-0.5, centrifuging to collect thallus, resuspending cells in liquid culture medium containing 100 μ M/L acetosyringone to obtain infection solution, collecting strong tobacco sterile seedling leaves with age of 4-6 weeks, cutting into leaf discs, blowing, inoculating in co-culture medium, culturing at 28 deg.C for 3 days, washing the leaves with sterile water for 10 times, blowing, inoculating in screening culture medium, selecting and culturing at illumination period of 16h/8h and 25 deg.C, subculturing once every 2 weeks, inoculating fresh untreated leaves in screening culture medium as control, when adventitious bud grows to 1-2cm, cutting, transferring into rooting culture medium, rooting for 2-3 weeks, and rooting for 2-3 weeks, Removing the strong Nicotiana benthamiana tissue culture seedlings from the tissue culture bottle, and transferring the tissue culture bottles to outdoor culture.
8. The Camellia chrysantha as set forth in claim 1CnFLS+ antisenseF3’HThe method for promoting plant flavonol synthesis by double-gene vector construction is characterized in that the step 4) specifically comprises the following steps: the kit is used for positive identification, and the PCR program comprises the following steps: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 s, annealing at 65 ℃ for 10 s, extension at 72 ℃ for 1min for 30s, 30 cycles; extension at 72 ℃ of 5mDetecting in, 1% agarose gel electrophoresis, collecting flowers after positive plants blossom, freezing with liquid nitrogen, storing at-80 deg.C, and determining by real-time fluorescent quantitative PCRCnFLS+ antisenseF3’HRelative expression of the two genes in positive flowers.
9. Camellia chrysantha obtained by the method of any one of claims 1 to 8CnFLS+ antisenseF3’HA double gene vector.
10. The camellia nitidissima of claim 9CnFLS+ antisenseF3’HApplication of double-gene vector in promoting plant flavonol synthesis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011574583.0A CN112481277B (en) | 2020-12-28 | 2020-12-28 | Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011574583.0A CN112481277B (en) | 2020-12-28 | 2020-12-28 | Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112481277A true CN112481277A (en) | 2021-03-12 |
| CN112481277B CN112481277B (en) | 2022-06-24 |
Family
ID=74915725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011574583.0A Active CN112481277B (en) | 2020-12-28 | 2020-12-28 | Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112481277B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117867011A (en) * | 2024-03-11 | 2024-04-12 | 四川省农业科学院作物研究所 | Application of FLS1 gene in control of formation of purple-leaf rape |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1254960A1 (en) * | 2001-05-02 | 2002-11-06 | Unilever Plc | A process for increasing the flavonoid content of a plant and plants obtainable thereby |
-
2020
- 2020-12-28 CN CN202011574583.0A patent/CN112481277B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1254960A1 (en) * | 2001-05-02 | 2002-11-06 | Unilever Plc | A process for increasing the flavonoid content of a plant and plants obtainable thereby |
Non-Patent Citations (4)
| Title |
|---|
| M. MAHAJAN: "Increase in flavan-3-ols by silencing flavonol synthase mRNA affects the transcript expression and activity levels of antioxidant enzymes in tobacco", 《PLANT BIOLOGY》 * |
| 周兴文等: "金花茶FLS基因的克隆及其植物表达载体的构建", 《植物研究》 * |
| 周兴文等: "金花茶黄烷酮3-羟化酶基因CnF3H的克隆及表达分析", 《分子植物育种》 * |
| 杜灵娟等: "葡萄风信子FLS1基因克隆及其表达与花色性状之间的关联性分析", 《西北林学院学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117867011A (en) * | 2024-03-11 | 2024-04-12 | 四川省农业科学院作物研究所 | Application of FLS1 gene in control of formation of purple-leaf rape |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112481277B (en) | 2022-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cutanda-Perez et al. | Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport | |
| CN107936104B (en) | Peony PsMYB12 transcription factor and coding gene and application thereof | |
| CN116004656B (en) | Banana maturation associated gene MabHLH130 and application thereof | |
| CN113717983A (en) | Longan gene DlGRAS34, protein and application thereof in regulating and controlling plant flowering | |
| CN112481277B (en) | Method for promoting plant flavonol synthesis by constructing camellia chrysantha CnFLS + antisense F3' H double-gene vector | |
| CN110819639A (en) | Tobacco low-temperature early-flowering related gene NtDUF599 and application thereof | |
| CN107177604B (en) | NtWRKY69 gene influencing tobacco pigment content and application thereof | |
| Chen et al. | Overexpression of Doritaenopsis hybrid EARLY FLOWERING 4-like4 gene, DhEFL4, postpones flowering in transgenic Arabidopsis | |
| CN115851765B (en) | Musa paradisiaca maturation-related gene MaMYC2-10 and application thereof | |
| CN115197951B (en) | Tea tree flavonol synthesis candidate gene CsNAC086 and application thereof | |
| CN112553249B (en) | Method for promoting synthesis of plant flavonol glycosides by constructing camellia chrysantha CnFLS + CnUFGT14 double-gene vector | |
| CN110819603A (en) | Application of RAP gene and breeding method for changing color of strawberry fruit | |
| US20060277631A1 (en) | Genes upregulated in a tomato plant having an increased anthocyanin content phenotype | |
| Gatica-Arias et al. | Over-expression of the transcription factor HlMYB3 in transgenic hop (Humulus lupulus L. cv. Tettnanger) modulates the expression of genes involved in the biosynthesis of flavonoids and phloroglucinols | |
| JP5403206B2 (en) | Method for modifying plant morphology | |
| KR101351265B1 (en) | Method for preparing transgenic alfalfa plant with increased anthocyanin content and the plant thereof | |
| Ghag et al. | Silencing of MusaANR1 gene reduces proanthocyanidin content in transgenic banana plants | |
| CN116855531B (en) | Application of peony PsASMT gene or vector thereof in changing plant flower color | |
| Kumar et al. | Overexpression of CsDFR and CsANR enhanced flavonoids accumulation and antioxidant potential of roots in tobacco | |
| CN104560906B (en) | Specifically expressed protein C YP734A1 like 1 and its application in fibrocyte | |
| CN116640200B (en) | Application of MfERF086 gene in alfalfa growth and development and/or cold resistance regulation | |
| CN110878310B (en) | Transcription factor for regulating plant flower color, recombinant eukaryotic expression vector and application | |
| CN110904110B (en) | Application of OsHAP3C gene expression reduction in rice variety with shortened heading period and prolonged growth period | |
| CN105950620B (en) | It is a kind of it is comprehensive lower cotton lycopene cyclase gene expression vector and application | |
| CN116445509A (en) | Application and application method of azalea camellia CaFT gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |