CN112553249A - Method for promoting synthesis of plant flavonol glycosides by constructing camellia chrysantha CnFLS + CnUFGT14 double-gene vector - Google Patents
Method for promoting synthesis of plant flavonol glycosides by constructing camellia chrysantha CnFLS + CnUFGT14 double-gene vector Download PDFInfo
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Abstract
Golden camellia teaCnFLS+CnUFGT14A method for promoting plant flavonol glycoside synthesis by double-gene vector construction belongs to the technical field of genetic engineering. The method comprises the following steps: 1) cloned golden camelliaCnFLSAndCnUFGT14a gene; 2) constructing a double-gene vector; 3) transferring the double-gene vector into escherichia coli, extracting plasmids after correct sequencing, transferring the plasmids into agrobacterium, and transforming the Nicotiana benthamiana by a leaf disc method; 4) PCR identifies the positive strain of the transgenic tobacco, and the contents of flavonol glycosides and polyphenol in the positive strain are determined. The invention relates to a method for preparing golden camellia through overexpressionCnFLSGenes, simultaneously over-expressedCnUFGT14Reasonable design of gene willCnFLSFlavonols synthesized by gene promotion, and thenCnUFGT14The gene glycosylation forms stable flavonol glycoside, and increases flavonol glycoside in plantThe content of (A) can inhibit the synthesis of polyphenol and anthocyanin to a certain extent, 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+CnUFGT14A method for promoting plant flavonol glycoside synthesis by 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 content of flavonol glycoside 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 the flavonol glycosides of the model plant, and golden yellow flowers are formed.
Due to the fact thatFLSFlavonols synthesized by gene promotion are unstable and easily decomposed, so that it is necessary to useUFGTThe gene (the flavonoid transferase gene) glycosidates it to form stable flavonol glycosides.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide golden camelliaCnFLS+CnUFGT14A method for promoting plant flavonol glycoside synthesis by double-gene vector construction. Obtaining golden camellia by PCR amplificationCnFLS、CnUFGT14The gene is constructed by enzyme digestion using the OSCR (one step clone reaction) cloning techniqueCnFLS+CnUFGT14The obtained double-gene over-expression vector is used for transforming the Nicotiana benthamiana, so that the content of the flavonol glycosides of the positive strains is obviously improved.
Golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized by comprising the following steps of:
1) cloned golden camelliaCnFLSAndCnUFGT14a gene;
2) construction ofCnFLS+CnUFGT14A double gene vector;
3) will be provided withCnFLS+CnUFGT14Transferring 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 strains of the transgenic tobacco by adopting PCR, determining the contents of flavone components, polyphenol components and anthocyanin components in the flowers of the positive strains by using an HPLC method, and calculating the total content of the flavone, the total content of the polyphenol and the total content of the anthocyanin.
The golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing a double-gene vector is characterized in that the step 1) specifically comprises the following steps: prepared from Camellia nitidissimaCnFLSAndCnUFGT14primers pFLS-F/pFLS-R and pUFGT 14-F/pUFGT 14-R are respectively designed for the gene sequence, ExTaq enzyme is used for PCR amplification to obtain the full-length CDS sequence of the target gene, gel electrophoresis analysis and detection are carried out, recovery and purification are carried out to obtainCnFLSAndCnUFGT14the nucleotide sequence of the primer pFLS-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer pFLS-R is shown as SEQ ID NO.2, the nucleotide sequence of the primer pUFGT14-F is shown as SEQ ID NO.3, and the nucleotide sequence of the primer pUFGT14-R is shown as SEQ ID NO. 4.
The golden camellia teaCnFLS+CnUFGT14A method for promoting the synthesis of plant flavonol glycosides by constructing a double-gene vector,the method is characterized in that the PCR 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.
The golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized in that the step 2) specifically comprises the following steps: will be provided withCnFLSAndCnUFGT14the genes are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-UFGT14 intermediate vectors, and the intermediate vectors are obtained by enzyme cutting connection by adopting an OSCR cloning technology and then recoveredCnFLS+CnUFGT14A double gene vector.
The golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized in that the enzyme cutting system is as follows: 2 muL Buffer, BsmBI/Esp3I: 1 muL T4_ ligase: 1 muL, 4 muL pBWA (V) KS-FLS, 4 muL pBWD (LA)1C-UFGT14: 4 muL, H28 muL of O and 20 muL of Total; the connection process comprises the following steps: 20 min at 37 ℃; 10min at 37 ℃, 10min at 20 ℃ and 5 cycles; 20 min at 37 ℃; 5min at 80 ℃.
The golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing a double-gene vector is characterized in that the step 3) is implementedCnFLS+CnUFGT14The method comprises the following steps of transferring the double-gene vector into escherichia coli, extracting plasmids after correct sequencing, and transferring the plasmids into agrobacterium: by usingCnFLS+CnUFGT14Transforming the competence of colibacillus by a double-gene vector, coating a resistant LB plate for transformation, carrying out inverted culture at 37 ℃ for 12 hours, selecting a single colony for PCR identification and sequencing, extracting a plasmid with correct sequencing, and carrying out PCR amplification by a hot shock methodCnFLS+CnUFGT14The double gene vector is ligated into Agrobacterium.
The golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides 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+CnUFGT14The agrobacterium with double gene vectors is cultured in LB liquid culture medium containing 50mg/L Kan with shaking at 28 deg.c and 200rpm to OD600 of 0.4-0.5 and 8000rpm,and centrifugally collecting thalli at the temperature of 4 ℃, resuspending cells by using a liquid culture medium containing 100 mu M/L acetosyringone to prepare an infection solution, and taking the strong tobacco sterile seedling leaves with the seedling age of 4-6 weeks. Cutting into leaf discs, infecting for 5-10min, blow-drying, inoculating on a co-culture medium, culturing in the dark at 28 ℃ for 3 days, cleaning leaves with sterile water for 10 times, blow-drying, inoculating on a screening culture medium, selecting the culture medium under the conditions of illumination period of 16h/8h and 25 ℃, subculturing once every 2 weeks, cutting off and transferring into a rooting culture medium when adventitious buds grow to 1-2cm, rooting for 2-3 weeks and root length of 2-3cm, taking out the strong and strong Nicotiana benthamiana tissue cultured seedlings, and transferring to the outdoor for culturing.
The golden camellia teaCnFLS+CnUFGT14The method for promoting plant flavonol glycoside synthesis by double-gene vector construction is characterized in that the PCR identification of the positive line of the transgenic tobacco in the step 4) specifically comprises the following steps: carrying out positive identification on the transgenic tobacco by using the kit, wherein 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+CnUFGT14Relative expression amount of the double genes in the positive strain.
The golden camellia prepared by the methodCnFLS+CnUFGT14A double gene vector.
The golden camellia teaCnFLS+CnUFGT14Application of double-gene vector in promoting plant flavonol glycoside synthesis.
The invention relates to a method for preparing golden camellia through overexpressionCnFLSGenes, simultaneously over-expressedCnUFGT14Reasonable design of gene willCnFLSFlavonols synthesized by gene promotion, and thenCnUFGT14The gene glycosylation forms stable flavonol glycoside, improves the content of the flavonol glycoside in the plant, simultaneously inhibits the synthesis of polyphenol and anthocyanin to a certain extent, and further can yellow the flower color of the plant.
Drawings
FIG. 1 is a drawing ofCnFLS+CnUFGT14A double gene vector map;
FIG. 2 shows the total content of polyphenols and total content of flavonoids in transgenic positive lines;
FIG. 3 shows the contents of polyphenol and flavone components in transgenic positive lines, wherein EGC: epigallocatechin, GC: gallocatechin, CG: catechin gallate, ECG: epicatechin gallate is the 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 teaCnFLS、CnUFGT14Cloning of the genes:
(1) prepared from Camellia nitidissimaCnFLS、CnUFGT14Gene gene sequences (Genbank ID: JF343560.1, MN 276188) primers were designed (see Table 1), and ExTaq enzyme (RR 902, TaKaRa) was used for PCR amplification to obtain the CDS sequence of the full length 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).
2. CnFLS+CnUFGT14Construction of a double-gene vector:
(1) mixing Camellia ChysanthaCnFLS、CnUFGT14The genes are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-UFGT14 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-UFGT14: 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+CnUFGT14A double gene vector. By using a heat shock methodCnFLS+CnUFGT14The two-gene vector (see FIG. 1) was ligated into the GV3101 Agrobacterium.
3. Leaf disc method for transforming Nicotiana benthamiana:
(1) will containCnFLS+CnUFGT14GV3101 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 overexpression aloneCnUFGT14Positive lines of the genes were compared.
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+CnUFGT14positive lines of the double gene vectors were compared.
Comparative experiment 2:
obtained by cloning in step 1CnFLSThe gene was subjected to PCR by homologous recombination method using primers designed according to the EXclone kit instructions (exv 09, Baige Gene science and technology (Jiangsu) Ltd.) (see Table 1) and the procedure was 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+CnUFGT14positive lines of the double gene vectors were compared.
Comparative experiment 3:
obtained by cloning in step 1CnUFGT14Gene, using homologous recombination method, according to EXclone kit instruction (exv 09, Baige Gene science and technology (Jiangsu) Co., Ltd.), design primer: (See table 1), PCR was performed, the procedure was 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+CnUFGT14positive lines of the double gene vectors were compared.
TABLE 1 primer sequences
The test results are shown in fig. 2 and 3. As is clear from the test results, the method of the present invention, example 1CnFLS+CnUFGT14The total flavone content and flavone component content of the positive strain constructed by the double-gene vector are obviously higher than those of comparative experiments 1, 2 and 3; the total polyphenol content and the EGC content in the example 1 and the comparative experiment 1 are obviously lower than those in the comparative experiments 2 and 3, and the content changes of three components of GC, CG and ECG are not obvious. Show thatCnFLS+CnUFGT14The double-gene vector construction promotes the synthesis of plant flavonol glycosides, and the promotion effect is larger than that of single over-expressionCnFLSGene and overexpression aloneCnUFGT14A gene. The invention relates to a method for preparing golden camellia through overexpressionCnFLSGenes, simultaneously over-expressedCnUFGT14Reasonable design of gene willCnFLSFlavonols synthesized by gene promotion, and thenCnUFGT14The gene glycosylation forms stable flavonol glycoside, improves the content of the flavonol glycoside in the plant, simultaneously inhibits the synthesis of polyphenol and anthocyanin to a certain extent, and further can yellow the flower color of the plant.
As shown in Table 2, untransformed wild type Nicotiana benthamiana control group was over-expressed as compared to tobacco aloneCnFLSGene, overexpression aloneCnUFGT14Comparison of positive strains of genes, transformationCnFLS+CnUFGT14The content of the flavonol glycosides of the double-gene vector positive strain is obviously higher than that of the control strain.
TABLE 2 content (mg/g) of polyphenol fraction and flavone fraction in comparative tests 1 to 3 with those in example 1
Sequence listing
<110> subtropical forestry research institute of China forestry science research institute
<120> method for promoting plant flavonol glycoside synthesis by constructing camellia chrysantha CnFLS + CnUFGT14 double-gene vector
<160> 4
<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> 21
<212> DNA
<213> primer pUFGT14-F (primer pUFGT14-F)
<400> 3
accaaccatg aacggtgact c 21
<210> 4
<211> 21
<212> DNA
<213> primer pUFGT14-R (primer pUFGT14-R)
<400> 4
acctctcaca tccaagaggg a 21
Claims (10)
1. Golden camellia teaCnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized by comprising the following steps of:
1) cloned golden camelliaCnFLSAndCnUFGT14a gene;
2) construction ofCnFLS+CnUFGT14A double gene vector;
3) will be provided withCnFLS+CnUFGT14Transferring 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 strains of the transgenic tobacco by adopting PCR, determining the contents of flavone components, polyphenol components and anthocyanin components in the flowers of the positive strains by using an HPLC method, and calculating the total content of the flavone, the total content of the polyphenol and the total content of the anthocyanin.
2. The Camellia chrysantha as set forth in claim 1CnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing a double-gene vector is characterized in that the step 1) specifically comprises the following steps: prepared from Camellia nitidissimaCnFLSAndCnUFGT14primers pFLS-F/pFLS-R and pUFGT 14-F/pUFGT 14-R are respectively designed for the gene sequence, ExTaq enzyme is used for PCR amplification to obtain the full-length CDS sequence of the target gene, gel electrophoresis analysis and detection are carried out, recovery and purification are carried out to obtainCnFLSAndCnUFGT14the nucleotide sequence of the primer pFLS-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer pFLS-R is shown as SEQ ID NO.2, the nucleotide sequence of the primer pUFGT14-F is shown as SEQ ID NO.3, and the nucleotide sequence of the primer pUFGT14-R is shown as SEQ ID NO. 4.
3. The camellia nitidissima of claim 2CnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized in that the PCR 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.
4. The Camellia chrysantha as set forth in claim 1CnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized in that the step 2) specifically comprises the following steps: will be provided withCnFLSAndCnUFGT14the genes are respectively constructed into pBWA (V) and pBWD (LA) vectors with enzyme cutting sites to obtain pBWA (V) KS-FLS and pBWD (LA)1C-UFGT14 intermediate vectors, and the intermediate vectors are obtained by enzyme cutting connection by adopting an OSCR cloning technology and then recoveredCnFLS+CnUFGT14A double gene vector.
5. The Camellia chrysantha as set forth in claim 4CnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing the double-gene vector is characterized in that the enzyme cutting system is as follows: 2 muL Buffer, BsmBI/Esp3I: 1 muL T4_ ligase: 1 muL, 4 muL pBWA (V) KS-FLS, 4 muL pBWD (LA)1C-UFGT14: 4 muL, H28 muL of O and 20 muL of Total; the connection process comprises the following steps: 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+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides by constructing a double-gene vector is characterized in that the step 3) is implementedCnFLS+CnUFGT14The method comprises the following steps of transferring the double-gene vector into escherichia coli, extracting plasmids after correct sequencing, and transferring the plasmids into agrobacterium: by usingCnFLS+CnUFGT14Transforming the competence of colibacillus by a double-gene vector, coating a resistant LB plate for transformation, carrying out inverted culture at 37 ℃ for 12 hours, selecting a single colony for PCR identification and sequencing, extracting a plasmid with correct sequencing, and carrying out PCR amplification by a hot shock methodCnFLS+CnUFGT14The double gene vector is ligated into Agrobacterium.
7. The Camellia chrysantha as set forth in claim 1CnFLS+CnUFGT14The method for promoting the synthesis of plant flavonol glycosides 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+CnUFGT14The agrobacterium of the double-gene carrier is put in LB liquid culture medium containing 50mg/L Kan, the temperature is 28 ℃ to rotatePerforming shaking culture at 200rpm until OD600 is 0.4-0.5, centrifuging at 8000rpm and 4 deg.C, collecting thallus, resuspending cells in a liquid culture medium containing 100 μ M/L acetosyringone, making into an infection solution, taking strong tobacco sterile seedling leaves with seedling age of 4-6 weeks, cutting into leaf discs, infecting for 5-10min, blow-drying and inoculating on a co-culture medium, performing dark culture at 28 deg.C for 3 days, cleaning the leaves with sterile water for 10 times, blow-drying and inoculating on a screening culture medium, selecting the culture medium under the conditions of illumination period of 16h/8h and 25 deg.C, performing subculture once every 2 weeks, cutting and transferring into a rooting culture medium when the adventitious bud grows to 1-2cm, rooting for 2-3 weeks, and taking out the strong tobacco group seedling, and transferring to outdoor culture.
8. The Camellia chrysantha as set forth in claim 1CnFLS+CnUFGT14The method for promoting plant flavonol glycoside synthesis by double-gene vector construction is characterized in that the PCR identification of the positive line of the transgenic tobacco in the step 4) specifically comprises the following steps: carrying out positive identification on the transgenic tobacco by using the kit, wherein 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+CnUFGT14Relative expression amount of the double genes in the positive strain.
9. Camellia chrysantha prepared by the method of claims 1-8CnFLS+CnUFGT14A double gene vector.
10. The camellia nitidissima of claim 9CnFLS+CnUFGT14Application of double-gene vector in promoting plant flavonol glycoside synthesis.
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| EP1254960A1 (en) * | 2001-05-02 | 2002-11-06 | Unilever Plc | A process for increasing the flavonoid content of a plant and plants obtainable thereby |
| CN107190015A (en) * | 2017-07-11 | 2017-09-22 | 山东大学 | Corn glycosyltransferase gene UFGT2 is improving the application in plant in flavones content |
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| EP1254960A1 (en) * | 2001-05-02 | 2002-11-06 | Unilever Plc | A process for increasing the flavonoid content of a plant and plants obtainable thereby |
| CN107190015A (en) * | 2017-07-11 | 2017-09-22 | 山东大学 | Corn glycosyltransferase gene UFGT2 is improving the application in plant in flavones content |
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