CN114480483B - Method for silencing or knocking down gene expression in young fructus forsythiae fruits - Google Patents
Method for silencing or knocking down gene expression in young fructus forsythiae fruits Download PDFInfo
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Abstract
The invention provides a method for silencing or knocking down gene expression in young fructus forsythiae fruits, and relates to the technical field of plant gene function research; the method comprises the following steps: pretreating young fructus forsythiae fruits with hypertonic solution; constructing a target gene into pTRV2 to obtain a recombinant vector pTRV2:FsTG, transforming into agrobacterium tumefaciens to obtain recombinant agrobacterium, and mixing the recombinant agrobacterium with basic invasion solution to obtain recombinant agrobacterium invasion solution transformed with pTRV2:FsTG; mixing and culturing a vector pTRV1 and agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens, and mixing the recombinant agrobacterium tumefaciens with a basic invasion solution to obtain recombinant agrobacterium tumefaciens invasion solution transformed with pTRV 1; mixing the two infection solutions to prepare a conversion solution; and (3) putting the pretreated fructus forsythiae young fruits into a conversion solution for shake culture for 2-3 weeks. The gene silencing mode is adopted to silence the gene in the fructus forsythiae fruits in the young fructus forsythiae fruits after transcription, so that the expression quantity is reduced, and a foundation is laid for the construction of a fructus forsythiae fruit virus-induced gene silencing system.
Description
Technical Field
The invention relates to the technical field of research on plant gene functions, in particular to a method for silencing or knocking down gene expression in young fruits of fructus forsythiae.
Background
In the conventional gene function research method, construction of a stable plant mutation system for plant species is necessary for researching gene functions. In the process, firstly, plant tissue culture is needed to be carried out on the plant to be transgenic, and agrobacterium is used for transforming different transgenic vectors into plant cells in the callus period, wherein the transgenic vectors mainly comprise stable expression vectors, gene editing vectors mainly comprising CRISPR-Cas9 and the like, or a materialization treatment method is used for leading the plant to generate gene mutation and the like. Currently, only arabidopsis, as well as tobacco and some other plant species, can produce stable plant transformation systems. It is relatively difficult for perennial plants because most perennial plants have low transformation efficiency and perennial properties such that plant tissue culture time is long, cost is high, certain phenotypes have hysteresis, such as certain phenotypes of reproductive organs, which are observed after the end of a relatively long vegetative period of the plant has entered the reproductive period, and it is seen that traditional means of changing genomic information are not suitable for studying perennial plants. At present, gene function research methods for changing transcription level exist, and common methods include RNA interference (RNAi), artificial microRNA mediated gene silencing and the like, but the methods are only suitable for plants with higher transformation efficiency, stable plant transformation is required, time is wasted, the methods are not suitable for high-throughput research, and the current methods suitable for gene function research of perennial plants are few.
Disclosure of Invention
The invention aims to provide a method for silencing or knocking down gene expression in fructus forsythiae young fruits, wherein the gene in fructus forsythiae young fruits is silenced in a posttranscriptional gene silencing mode, so that the expression quantity of the gene is reduced, and a foundation is laid for construction of a fructus forsythiae fruit virus-induced gene silencing system.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for silencing or knocking down gene expression in young fructus forsythiae fruits, which comprises the following steps:
pretreating young fructus forsythiae fruits with hypertonic solution;
constructing a target gene to be detected into a second subgenomic group of the tobacco brittle fracture virus to obtain a recombinant vector pTRV2: fsTG; transforming the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens transformed with pTRV2:FstG; mixing the recombinant agrobacterium tumefaciens transformed with pTRV2: fsTG with the basic invaded dye liquor to obtain the agrobacterium tumefaciens invaded dye liquor transformed with pTRV2: fsTG;
mixing and shaking culture of a first subgenomic vector pTRV1 of tobacco brittle fracture virus and agrobacterium tumefaciens; obtaining recombinant agrobacterium transformed with pTRV 1; mixing the recombinant agrobacterium tumefaciens transformed with pTRV1 with a basic invasion solution to obtain a recombinant agrobacterium tumefaciens invasion solution transformed with pTRV 1;
mixing the recombinant agrobacterium infection solution converted with pTRV1 with the recombinant agrobacterium infection solution converted with pTRV2:FsTG to prepare a conversion solution;
and (3) putting the pretreated fructus forsythiae young fruits into a conversion solution for shake culture for 2-3 weeks.
Preferably, the hypertonic solution is a 1/2Ms solution comprising 20-30% (w/v) sucrose; the pH value of the hypertonic solution is 5.5-6.5; the pretreatment time is 3-7 h.
Preferably, the target gene is a forsythia lycopene desaturase gene, and the nucleotide sequence of the forsythia lycopene desaturase gene is SEQ ID NO.1.
Preferably, the culture medium for shake culture of pTRV1 and Agrobacterium GV3101 is LB medium, and 40-60 mg/L kanamycin and 40-60 mg/L rifampicin are added.
Preferably, the temperature of the oscillation culture of the first subgenomic vector pTRV1 of the tobacco brittle virus and the agrobacterium GV3101 is 26-30 ℃, the time is 8-16 h, and the rotating speed is 200-240 rpm.
Preferably, the base counterstain is a 1/2Ms medium comprising 90-110. Mu.M acetosyringone, 65-85. Mu.M Tris-X100 and 1.5-3.5% (w/v) sucrose.
Preferably, the OD of the Agrobacterium tumefaciens invasion solution transformed with pTRV1 and the OD of the Agrobacterium tumefaciens invasion solution transformed with pTRV2:Fstg 600 Independently 0.05 to 0.15.
Preferably, the mixing volume ratio of the agrobacterium transformed with pTRV1 to the agrobacterium transformed with pTRV2:fstg is 1:1-2.
Preferably, the temperature of the pre-treated young fruit of the weeping forsythia capsule hypertonic solution in the shaking culture of the conversion solution is 19-23 ℃, the relative humidity is 45-60%, and the rotating speed is 40-80 rpm.
Preferably, the sunlight period of the young fruit pretreated by the weeping forsythia hypertonic solution and placed into the conversion solution for shake culture is 14-18 hours of illumination, 6-10 hours of darkness, and the illumination intensity is 350-450 mu mol/m 2 ·s。
The invention provides a method for silencing or knocking down gene expression in fructus forsythiae young fruits, which successfully silences lycopene desaturase genes in fructus forsythiae fruits in a posttranscriptional gene silencing mode, so that the expression quantity of the lycopene desaturase genes is reduced, a photobleaching phenotype is generated, and a foundation is laid for construction of a fructus forsythiae fruit virus-induced gene silencing system. The lycopene desaturase gene of the weeping forsythia is constructed into a vector to serve as a visual indicator gene, so that the phenotype after silencing can be observed better; the agrobacterium is used for transforming the gene into fructus forsythiae fruits, and co-culture is carried out under strong light and vibration, so that the phenotype after PDS gene silencing is more obvious; compared with the traditional method for changing genome information and transcriptome level, the method does not need to construct a stable transformation system, can keep silent through asexual propagation, can silence most plant parts, has the characteristics of high efficiency, wide range and high speed, can directly collect corresponding parts from living plants and cultivate in a asexual propagation mode for perennial plants, can carry out gene silencing in the cultivation process, can directly use seedlings for testing if researching the corresponding phenotype in the vegetative stage, and can also be applied to plant tissue culture.
Drawings
FIG. 1 is a flow chart of gene expression in silencing or knocking-down young fruits of forsythia;
FIG. 2 is a schematic diagram showing comparison of PDS gene expression levels of young fruits of Forsythia suspensa (ck shows the PDS gene expression levels in control young fruits not infected with a virus; mock shows the PDS gene expression levels in control young fruits having no target sequence in virus infected with a virus; and treated shows the PDS gene expression levels in young fruits infected with a virus having a target sequence).
Detailed Description
The invention provides a method for silencing or knocking down gene expression in young fructus forsythiae fruits, which comprises the following steps:
pretreating young fructus forsythiae fruits with hypertonic solution;
constructing a target gene to be detected into a second subgenomic group of the tobacco brittle fracture virus to obtain a recombinant vector pTRV2: fsTG; transforming the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens transformed with pTRV2:FstG; mixing the recombinant agrobacterium tumefaciens transformed with pTRV2: fsTG with the basic invaded dye liquor to obtain the agrobacterium tumefaciens invaded dye liquor transformed with pTRV2: fsTG;
mixing and shaking culture of a first subgenomic vector pTRV1 of tobacco brittle fracture virus and agrobacterium tumefaciens; obtaining recombinant agrobacterium transformed with pTRV 1; mixing the recombinant agrobacterium tumefaciens transformed with pTRV1 with a basic invasion solution to obtain a recombinant agrobacterium tumefaciens invasion solution transformed with pTRV 1;
mixing the recombinant agrobacterium infection solution converted with pTRV1 with the recombinant agrobacterium infection solution converted with pTRV2:FsTG to prepare a conversion solution;
and (3) putting the pretreated fructus forsythiae young fruits into a conversion solution for shake culture for 2-3 weeks.
In the invention, the young fructus forsythiae fruits are treated in a co-culture mode, and the young fructus forsythiae fruits are cleaned and photographed.
In the invention, the hypertonic solution for preprocessing the young fructus forsythiae fruits is preferably a 1/2Ms solution comprising sucrose, and the concentration of the sucrose in the hypertonic solution is preferably 20-30% (w/v), more preferably 22-28% (w/v), even more preferably 24-26% (w/v); the pH of the hypertonic solution is preferably 5.5 to 6.5, more preferably 5.8 to 6.2; the pretreatment time is preferably 3 to 7 hours, more preferably 4 to 6 hours, still more preferably 4.5 to 5.5 hours. In the invention, the specific mode of pretreatment is that young fructus forsythiae fruits are soaked in hypertonic solution.
In the invention, a target gene to be detected is constructed into a second subgenomic group of tobacco brittle fracture virus to prepare a recombinant vector pTRV2:FSTG, wherein the target gene is preferably a weeping forsythiae lycopene desaturase gene, and the weeping forsythiae lycopene desaturase gene is further preferably a nucleotide sequence of SEQ ID NO.1.
In the invention, a first subgenomic vector pTRV1 of tobacco brittle fracture virus and agrobacterium tumefaciens GV3101 are mixed and cultured in an oscillating way to obtain recombinant agrobacterium transformed with pTRV1, and a culture medium for the pTRV1 and agrobacterium for the oscillating culture is preferably LB culture medium added with kanamycin and rifampicin; the concentration of kanamycin in the culture medium is preferably 40-60 mg/L, more preferably 45-55 mg/L, and still more preferably 48-52 mg/L; the concentration of rifampicin in the medium is preferably 40 to 60mg/L, more preferably 45 to 55mg/L, still more preferably 48 to 52mg/L.
In the invention, the temperature of the oscillating culture of the first subgenomic vector pTRV1 of the tobacco brittle virus and the agrobacterium GV3101 is preferably 26-30 ℃, and more preferably 27-29 ℃; the time is preferably 8 to 16 hours, more preferably 10 to 14 hours, still more preferably 11 to 13 hours; the rotation speed is preferably 200 to 240rpm, more preferably 210 to 230rpm, still more preferably 215 to 225rpm.
In the invention, the recombinant agrobacterium tumefaciens transformed with pTRV1 is mixed with a basic invader solution to obtain a recombinant agrobacterium tumefaciens invader solution transformed with pTRV 1; and mixing the recombinant agrobacterium cells transformed with pTRV2: fsTG with the basic invader solution to obtain the agrobacterium invader solution transformed with pTRV2: fsTG. In the present invention, the recombinant Agrobacterium cells transformed with pTRV 2. FstG are obtained by centrifugation at a rotation speed of preferably 4500 to 5500rpm, more preferably 4800 to 5200rpm, still more preferably 5000rpm; the time is preferably 5 to 15 minutes, more preferably 8 to 12 minutes, still more preferably 10 minutes. The basic dyeing solution is preferably a 1/2Ms culture medium comprising 90-110 mu M acetosyringone, 65-85 mu M Triton-X100 and 1.5-3.5% (w/v) sucrose; the acetosyringone concentration is preferably 90 to 110. Mu.M, more preferably 95 to 105. Mu.M, still more preferably 100. Mu.M; the concentration of Triton-X100 is preferably 65 to 85. Mu.M, more preferably 70 to 80. Mu.M, still more preferably 75. Mu.M; the sucrose concentration is preferably 1.5 to 3.5% (w/v), more preferably 2 to 3% (w/v), still more preferably 2.5% (w/v).
In the present invention, the agrobacteria invasion solution transformed with pTRV1 and the OD of the agrobacteria invasion solution transformed with pTRV2:FsTG 600 Each of the above-mentioned components is preferably 0.05 to 0.15, more preferably 0.08 to 0.12, and still more preferably 0.1.
In the present invention, the conversion solution is prepared by mixing the recombinant Agrobacterium tumefaciens invasion solution converted into pTRV1 with the recombinant Agrobacterium tumefaciens invasion solution converted into pTRV2:FSTG, and the mixing volume ratio of the Agrobacterium tumefaciens invasion solution converted into pTRV1 to the Agrobacterium tumefaciens invasion solution converted into pTRV2:FSTG is preferably 1:1-2, more preferably 1:1.2-1.8, and still more preferably 1:1.4-1.6.
In the invention, the temperature of the conversion solution for shaking culture of the fructus forsythiae hypertonic solution pretreated young fruits is preferably 19-23 ℃, and more preferably 20-22 ℃; the relative humidity is preferably 45 to 60%, more preferably 46 to 48%; the rotation speed is preferably 40 to 80rpm, more preferably 50 to 70rpm, still more preferably 55 to 65rpm.
In the invention, the sunshine period of the fructus forsythiae hypertonic solution pretreatment young fruits in the conversion solution shaking culture is preferably 14-18 hours of illumination and 6-10 hours of darkness, and is further preferably 15-17 hours of illumination and 7-9 hours of darkness; the illumination intensity is preferably 350 to 450 mu mol/m 2 S, more preferably 380 to 420. Mu. Mol/m 2 S, still more preferably 390 to 410. Mu. Mol/m 2 ·s。
In the invention, the fructus forsythiae hypertonic solution pretreated young fruits are put into the solution of the transformation liquid shaking culture and are replaced every 24 hours.
In the invention, the shaking culture time of the pretreated fructus forsythiae young fruits in the conversion liquid is preferably 2-3 weeks, more preferably 15-20 days, still more preferably 16-18 days; after young fructus forsythiae is oscillated, a photobleaching phenomenon occurs, and fruits with suspected silencing effect are selected for RNA extraction and qRT-PCR analysis.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The young fructus forsythiae fruits are treated in a co-culture mode, the young fructus forsythiae fruits are cleaned and photographed, and then the young fructus forsythiae fruits are pretreated for 5 hours by using a hypertonic solution containing 1/2Ms solution (pH 5.8) of 25% (w/v) sucrose. Screening the fructus forsythiae PDS gene through transcriptome data, wherein the sequence is shown as SEQ ID NO. 1; after gel purification by digestion of pTRV2 with BamH1 and Xba1 enzymes, fsPDS-insert (SEQ ID NO. 1) was ligated with linearized vector using homologous recombination enzymes, and the resulting recombinant vector was transformed into E.coli DH5a cells (China full-gold) and PCR was confirmed by detecting the presence of the insert containing PDS using F-PDSBOS and R-PDSBOS primers. The nucleotide sequence of the F-PDSBOS is shown as SEQ ID NO.2, and the nucleotide sequence of the R-PDSBOS is shown as SEQ ID NO. 3. Expected size of PCR product of empty TRV2Is 0bp, while the expected size of the PCR product of TRV2-FsPDS is 309bp. Sequencing the recombinant vector to detect whether the target fragment is correctly inserted or not, so as to obtain a recombinant vector pTRV2:FsPDS; the plasmid of the recombinant vector is transformed into agrobacterium tumefaciens GV3101 by a heat shock method (42 ℃ C., 90 s) to obtain recombinant agrobacterium transformed with pTRV2:FsPDS, and the recombinant agrobacterium is cultured to OD 600 0.6, centrifuging to obtain bacterial cells, mixing with basic invader solution (1/2 Ms culture medium containing 100. Mu.M acetosyringone, 75. Mu.M Tris-X100 and 2.5% (w/v) sucrose), and adjusting OD 600 0.1. The plasmid of the first subunit vector pTRV1 of tobacco brittle virus is placed into LB culture medium containing 50mg/L kanamycin and 50mg/L rifampicin by a heat shock method (42 ℃,90 s) and the agrobacterium tumefaciens GV3101 are subjected to shaking culture for 12 hours at 28 ℃ and 220rpm to obtain recombinant agrobacterium transformed with pTRV1, and the recombinant agrobacterium is cultured to OD 600 Centrifugation at 0.6 to obtain a cell, mixing with 1/2Ms medium containing 100. Mu.M acetosyringone, 75. Mu.M Tris-X100 and 2.5% (w/v) sucrose in the basic invasive solution, and adjusting OD 600 0.1. The recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV1 was mixed with the recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV2:FsPDS 1:1 to obtain a transformed solution. The fructus forsythiae young fruits pretreated by the hypertonic solution are placed into a culture flask with a prepared conversion solution, and are cultured in a shaking table at 60rpm, and the solution is replaced every 24 hours. The temperature is 21 ℃, the relative humidity is 50 percent, and the illumination intensity is 400 mu mol/m 2 S, the sunshine period is 16h of illumination and 8h of darkness treatment. After 18 days, the young fructus forsythiae fruits are subjected to photobleaching, and the fruits with suspected silencing effect are selected for RNA extraction and qRT-PCR analysis.
Example 2
The young fructus forsythiae fruits are treated in a co-culture mode, the young fructus forsythiae fruits are cleaned and photographed, and then the young fructus forsythiae fruits are pretreated for 3 hours by using a hypertonic solution containing 1/2Ms solution (pH 5.5) of 20% (w/v) sucrose. Screening the fructus forsythiae PDS gene through transcriptome data, wherein the sequence is shown as SEQ ID NO. 1; digestion of pTRV2 with BamH1 and Xba1 enzymes, gel purification, ligation of FsPDS-insert (SEQ ID NO. 1) with a linearized vector using a homologous recombinase, transformation of the resulting recombinant vector into E.coli DH5a cells (China Total gold) PCR, confirmation of the presence or absence of PDS-containing inserts by detection of the corresponding pTRV2 using F-PDSBOS and R-PDSBOS primersAt the point. The nucleotide sequence of the F-PDSBOS is shown as SEQ ID NO.2, and the nucleotide sequence of the R-PDSBOS is shown as SEQ ID NO. 3. The expected size of the PCR product of empty TRV2 is 0bp, while the expected size of the PCR product of TRV2-FsPDS is 309bp. Sequencing the recombinant vector to detect whether the target fragment is correctly inserted or not, so as to obtain a recombinant vector pTRV2:FsPDS; the plasmid of the recombinant vector is transformed into agrobacterium tumefaciens GV3101 by a heat shock method (42 ℃ C., 90 s) to obtain recombinant agrobacterium transformed with pTRV2:FsPDS, and the recombinant agrobacterium is cultured to OD 600 Centrifugation at 0.6 to obtain a bacterial cell, mixing the bacterial cell with 1/2Ms medium containing 90. Mu.M acetosyringone, 65. Mu.M Tris-X100 and 1.5% (w/v) sucrose in a basal invasion solution, and adjusting OD 600 0.05. The plasmid of the first subunit vector pTRV1 of tobacco brittle virus is placed into LB culture medium containing 40mg/L kanamycin and 40mg/L rifampicin by a heat shock method (42 ℃,90 s) with agrobacterium tumefaciens GV3101, and is subjected to shaking culture at 26 ℃ and 200rpm for 8 hours to obtain recombinant agrobacterium transformed with pTRV1, and the recombinant agrobacterium is cultured to OD 600 0.6, centrifuging to obtain bacterial cells, mixing with basic invader solution (1/2 Ms culture medium containing 90 μm acetosyringone, 65 μm MTriton-X100 and 1.5% (w/v) sucrose), and adjusting OD 600 0.05. The recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV1 was mixed with the recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV2:FsPDS 1:2 to obtain a transformed solution. The fructus forsythiae young fruits pretreated by the hypertonic solution are placed into a culture flask with a prepared conversion solution, and are cultured in a shaking table at 40rpm, and the solution is replaced every 24 hours. The temperature is 19 ℃, the relative humidity is 45 percent, and the illumination intensity is 350 mu mol/m 2 S, the sunshine period is 14h of illumination and 10h of darkness treatment. After 14 days, the young fructus forsythiae fruits are subjected to photobleaching, and the fruits with suspected silencing effect are selected for RNA extraction and qRT-PCR analysis.
Example 3
The young fructus forsythiae fruits are treated in a co-culture mode, the young fructus forsythiae fruits are cleaned and photographed, and then the young fructus forsythiae fruits are pretreated for 7 hours by using a hypertonic solution containing 30% (w/v) of 1/2Ms (pH 6.5) of sucrose. Screening the fructus forsythiae PDS gene through transcriptome data, wherein the sequence is shown as SEQ ID NO. 1; pTRV2 was digested with BamH1 and Xba1 enzymes, after gel purification, fsPDS-insert (SEQ ID NO. 1) was ligated with a linearized vector using a homologous recombinase, and the resulting recombinationTransformation of the vector into E.coli DH5a cells (China full gold) PCR was confirmed by detecting the presence of inserts containing PDS using F-PDSBOS and R-PDSBOS primers by detecting the corresponding pTRV 2. The nucleotide sequence of the F-PDSBOS is shown as SEQ ID NO.2, and the nucleotide sequence of the R-PDSBOS is shown as SEQ ID NO. 3. The expected size of the PCR product of empty TRV2 is 0bp, while the expected size of the PCR product of TRV2-FsPDS is 309bp. Sequencing the recombinant vector to detect whether the target fragment is correctly inserted or not, so as to obtain a recombinant vector pTRV2:FsPDS; the plasmid of the recombinant vector is transformed into agrobacterium tumefaciens GV3101 by a heat shock method (42 ℃ C., 90 s) to obtain recombinant agrobacterium transformed with pTRV2:FsPDS, and the recombinant agrobacterium is cultured to OD 600 Centrifugation at 0.6 to obtain a cell, mixing the cell with 1/2Ms medium containing 110. Mu.M acetosyringone, 85. Mu.M Tris-X100 and 3.5% (w/v) sucrose in a basal invasion solution, and adjusting OD 600 0.15. The plasmid of the first subunit vector pTRV1 of tobacco brittle virus is placed into LB culture medium containing 60mg/L kanamycin and 60mg/L rifampicin by a heat shock method (42 ℃,90 s) with agrobacterium tumefaciens GV3101, and is subjected to shaking culture at 30 ℃ and 240rpm for 16 hours to obtain recombinant agrobacterium transformed with pTRV1, and the recombinant agrobacterium is cultured to OD 600 Centrifugation at 0.6, the resulting cells were mixed with basal invasion solution (1/2 Ms medium containing 110. Mu.M acetosyringone, 85. Mu.M Tris-X100 and 3.5% (w/v) sucrose) to adjust OD 600 0.15. The recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV1 was mixed with the recombinant Agrobacterium tumefaciens invasion solution transformed with pTRV2:FsPDS at a ratio of 1:1.5 to obtain a transformed solution. The fructus forsythiae young fruits pretreated by the hypertonic solution are placed into a culture flask with a prepared conversion solution, and are cultured in a shaking table at 80rpm, and the solution is replaced every 24 hours. The temperature is 23 ℃, the relative humidity is 60 percent, and the illumination intensity is 450 mu mol/m 2 S, the sunshine period is 18h of illumination and the darkness is 6h. After 21 days, the young fructus forsythiae fruits are subjected to photobleaching, the fruits with suspected silencing effect are selected for RNA extraction and qRT-PCR analysis (pTRV 1 and pTRV2 vectors are taught by the university of mountain and western agriculture student, academy of life sciences Wang Defu, china).
As can be seen from fig. 2, after the fructus forsythiae fruits are infected by the method for silencing or knocking down the gene expression in the young fructus forsythiae fruits, the PDS gene expression level in the fruits is obviously reduced, which indicates that the vigs gene silencing system in the fructus forsythiae fruits is successfully constructed; the traditional gene knockout technology needs to construct a transgenic system, and flowers can be opened after 4 years even if transgenic plants are obtained, so that the method is very unfavorable for researching the gene functions related to fruit traits, and has the characteristics of rapidness, convenience and strong tissue specificity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Shanxi university of agriculture
<120> a method for silencing or knocking down gene expression in young fruits of Forsythia suspensa
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 309
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggaaagat gatgatggag actggtacga aactggatta cacatatttt ttggggctta 60
cccaaatgtg cagaacctat ttggagagct gggcattaat gatcggctac agtggaagga 120
gcattctatg atatttgcaa tgccaagcaa gccaggggag tttagccggt tcgattttcc 180
tgaagtctta cctgcaccat taaatggaat atgggcaatc ttgaagaaca gtgaaatgct 240
tacttggcca gagaaagtca agttcgctat tggactcttg ccagcaataa ttggtggaca 300
gtcgtatgt 309
<210> 2
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aaggttaccg aattctctag aatggaaaga tgatgatgga gactgg 46
<210> 3
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cgtgagctcg gtaccggatc cacatacgac tgtccaccaa ttattg 46
Claims (7)
1. A method for silencing or knocking down gene expression in young fruits of forsythia, comprising the steps of:
pretreating young fructus forsythiae fruits with hypertonic solution;
constructing a target gene to be detected into a second subgenomic group of the tobacco brittle fracture virus to obtain a recombinant vector pTRV2: fsTG; transforming the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens transformed with pTRV2:FstG; mixing the recombinant agrobacterium tumefaciens transformed with pTRV2: fsTG with the basic invaded dye liquor to obtain the agrobacterium tumefaciens invaded dye liquor transformed with pTRV2: fsTG;
mixing and shaking culture of a first subgenomic vector pTRV1 of tobacco brittle fracture virus and agrobacterium tumefaciens; obtaining recombinant agrobacterium transformed with pTRV 1; mixing the recombinant agrobacterium tumefaciens transformed with pTRV1 with a basic invasion solution to obtain a recombinant agrobacterium tumefaciens invasion solution transformed with pTRV 1;
mixing the recombinant agrobacterium infection solution converted with pTRV1 with the recombinant agrobacterium infection solution converted with pTRV2:FsTG to prepare a conversion solution;
placing the pretreated fructus forsythiae young fruits into a conversion solution for shake culture for 2-3 weeks;
the hypertonic solution is a 1/2Ms solution comprising 20-30% (w/v) sucrose; the pH value of the hypertonic solution is 5.5-6.5; the pretreatment time is 3-7 hours;
the basic dyeing solution is a 1/2Ms culture medium comprising 90-110 mu M acetosyringone, 65-85 mu M Triton-X100 and 1.5-3.5% (w/v) sucrose;
the sunlight period of the pretreatment young fruits of the weeping forsythia hypertonic solution in the shaking culture of the conversion solution is 14-18 hours of illumination, 6-10 hours of darkness, and the illumination intensity is 350-450 mu mol/m 2 ·s。
2. The method for silencing or knocking down gene expression in young fructus forsythiae according to claim 1, wherein the target gene is a lycopene desaturase gene of fructus forsythiae, and the nucleotide sequence of the lycopene desaturase gene of fructus forsythiae is SEQ ID No.1.
3. The method for silencing or knocking down gene expression in young fructus forsythiae according to claim 1, wherein the culture medium of the shake culture of pTRV1 and agrobacterium GV3101 is LB medium, and 40-60 mg/L kanamycin and 40-60 mg/L rifampicin are added.
4. The method for silencing or knocking down gene expression in young fructus forsythiae as claimed in claim 3, wherein the temperature of the shaking culture of the first subgenomic vector pTRV1 of tobacco embrittlement virus and the agrobacterium GV3101 is 26-30 ℃, the time is 8-16 h, and the rotating speed is 200-240 rpm.
5. The method for silencing or knocking down gene expression in young fruits of forsythia suspensa according to claim 1, wherein the OD of the agrobacteria invasion solution transformed with pTRV1 and the OD of the agrobacteria invasion solution transformed with pTRV2:fstg 600 Independently 0.05 to 0.15.
6. The method for silencing or knocking down gene expression in young fructus forsythiae according to claim 1, wherein the mixing volume ratio of the agrobacteria invasion solution transformed with pTRV1 to the agrobacteria invasion solution transformed with pTRV2:fstg is 1:1-2.
7. The method for silencing or knocking down gene expression in fructus forsythiae young fruits according to claim 1, wherein the temperature of the fructus forsythiae hypertonic solution pretreatment young fruits in the transformation liquid shaking culture is 19-23 ℃, the relative humidity is 45-60%, and the rotating speed is 40-80 rpm.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949809A (en) * | 2018-07-05 | 2018-12-07 | 北京林业大学 | A kind of method of the carrier mediated Gene Silencing of Fructus Forsythiae blade TRV |
| CN109810988A (en) * | 2019-02-18 | 2019-05-28 | 广东省农业科学院蔬菜研究所 | A kind of eggplant fruit gene silencing system and its construction method |
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2021
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949809A (en) * | 2018-07-05 | 2018-12-07 | 北京林业大学 | A kind of method of the carrier mediated Gene Silencing of Fructus Forsythiae blade TRV |
| CN109810988A (en) * | 2019-02-18 | 2019-05-28 | 广东省农业科学院蔬菜研究所 | A kind of eggplant fruit gene silencing system and its construction method |
Non-Patent Citations (1)
| Title |
|---|
| Establishment and Verification of An Efficient Virus-Induced Gene Silencing System in Forsythia;Jianshuang Shen等;Horticultural Plant Journal(第1期);1-10 * |
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