CN108866096B - Method for establishing virus-induced gene silencing system on rosa multiflora plant - Google Patents
Method for establishing virus-induced gene silencing system on rosa multiflora plant Download PDFInfo
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- 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]
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- 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)
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Abstract
The invention discloses a method for establishing a virus-induced gene silencing system on a rosa plants in a field, which relates to the field of genetic engineering for improving the shape of rosa plants, and comprises five steps of determining an inoculation period, preparing an infection liquid, inoculating a virus vector, treating after inoculation and testing the gene silencing efficiency, wherein the concentration of the agrobacterium carrying the virus vector is increased to OD in the preparation of the infection liquid6001.5-2.0, which ensures that the agrobacterium carrying the virus vector in the inoculation area has enough survival base number under natural conditions, and improves the infection efficiency of the whole experiment; the low-concentration Silwet-77 component is added in the preparation of the infection liquid, so that the hydrophilicity of the surface of the plant material is increased, the infection efficiency of the infection liquid is improved, meanwhile, the low-concentration component does not produce toxic action on the plant material, the pollution rate is low, and the environment is protected; the method not only saves the cost, but also improves the gene silencing efficiency, and the gene silencing efficiency of the processed sample reaches more than 87.5 percent.
Description
Technical Field
The invention relates to the field of genetic engineering for improving the shape of rosa plants, in particular to a method for establishing a virus-induced gene silencing system on rosa plants in a field.
Background
Virus-induced gene silencing (VIGS) is a ubiquitous genetic immune mechanism in plants and belongs to post-transcriptional gene silencing (PTGS). When a host plant is infected with a viral vector carrying a fragment containing the endogenous gene, the plant's own immune system can be activated: the microRNA containing the endogenous target gene can be generated while the viral RNA is recognized and degraded, and the microRNA is combined with mRNA of a target gene and then degraded by Dicer enzyme, so that the expression level of the target gene is reduced or the function of the target gene is lost. It is this fact that the verification of the function related to the unknown gene can be accomplished. The gene silencing induced by the artificially improved Tobacco Rattle Virus (TRV) becomes a gene silencing system which is most widely applied at present due to the advantages of high silencing efficiency, long duration (about 30-40 days), light host plant virus symptoms, no covering of a silencing phenotype and the like. Even so, TRV virus-induced gene silencing is most frequently applied to the research of a series of herbaceous plants such as solanaceae and brassicaceae. At present, no report exists for establishing a virus-induced gene silencing system by taking perennial rosa plants as experimental objects under field conditions.
Some plants (such as roses) in the rosa have great scientific research value, but the existing technical operation of virus-induced gene silencing is not suitable for the rosa under the conditions, so that the establishment of an optimized and efficient virus-induced gene silencing system by taking the rosa as a target is particularly important. Under the condition that the target gene segments of the virus selection (TRV) and the embedded virus are optimal, after a recombinant virus vector with a target gene is constructed, the factors such as the activity of the mediated transformation agrobacterium, the preparation of an infection solution, the inoculation technology of the virus vector, the inoculation period, the light-temperature condition of the cultured plant material after inoculation and the like are the key factors influencing the successful establishment of the virus-induced gene silencing system.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for establishing a virus-induced gene silencing system by taking perennial rosa plants under field conditions as experimental objects, which is simple, efficient, low in cost and easy to operate outdoors.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for establishing a virus-induced gene silencing system on a rosa plants in a field comprises the following steps:
1) determining the inoculation period: during 3 to 4 middle ten days of the month, after the inoculation time is 3 pm, the weather forecast is checked in advance, and no rainfall is ensured within one week after inoculation;
2) preparing a staining solution: the infection liquid is prepared at present, and the preparation process comprises the following steps:
firstly, selecting agrobacterium positive clones with corresponding virus plasmids in 5ml LB or YEB liquid culture medium containing kan 50ug/ml and rifampicin 100ug/ml, shaking at 28 deg.C for 10-12h at 200r/min until the clones are completely white;
then, the cells were cultured in 50ml LB or YEB liquid medium containing kan 50ug/ml, rifampicin 100ug/ml, MES 10mM, AS 200uM, and shaken at 28 ℃ until the bacterial concentration reached OD600Taking out when the temperature is 1.5-2.0 ℃;
then, the cells were collected by centrifugation, and suspended in 25ml of an infection buffer containing a liquid medium of 1/2MS AS a solvent, MES 10mM, AS 100uM and MgC 1210 mM, and the OD of the concentration of the bacterial solution was adjusted600To 1.5-2.0, forming an agrobacterium infection buffer solution;
finally, respectively mixing an agrobacterium infection buffer solution containing pTRV1 with equal concentration with an agrobacterium infection buffer solution containing pTRV2 and pTRV 2-target gene fragments in equal volume, respectively adding Silwet-77 with the final concentration of 0.01% into the two mixed liquids, and standing for 4h at room temperature under a dark condition to form two agrobacterium infection solutions;
3) inoculation of viral vectors: cleaning leaflets of the whole annual whole cluster, petioles of the leaflets and twigs at the base parts of the leaflets by adopting a sharp instrument, intensively scratching the leaflets of the whole cluster and other scratched parts, completely immersing the leaflets and other scratched parts in a 5ml or 10ml centrifugal tube containing an infection solution according to the volume for infection for 10-15min for a single time, infecting the leaflets for 2-5min again if the moisture on the surfaces of the infected leaflets is evaporated quickly in dry weather or windy weather, and making a brand and noting treatment group and treatment time after infection;
4) and (3) post-inoculation treatment: after each group of the small leaves, the leafstalks and the small branches are inoculated, a large black plastic bag is adopted to completely cover the inoculation area and the whole branch where the inoculation area is located, the opening of the bag is sealed and fixed, the black plastic bag can be removed after 24 hours, so that the inoculation area is in contact with normal natural conditions, and the bag picking time is properly delayed if severe weather occurs;
5) testing the gene silencing efficiency: the groups comprise a virus empty vector group and a blank control group which is formed by combining virus and target gene composite vectors and is not processed, firstly, phenotype observation is carried out on the three groups of samples, secondly, RNA is extracted from plant materials of corresponding tissue parts processed by all the groups, first-strand cDNA is synthesized, PCR detection is carried out, and strip observation is carried out through agarose gel electrophoresis to determine existence of TRV virus and target genes, and finally, the gene silencing efficiency is determined by calculating the ratio of effective quantity of the PCR detection to total treatment quantity.
The device used in step 3) is set as a syringe needle, a fine needle or a blade.
And 3) scratching the back of the small leaf in the step 3), scratching the whole leaf surface without damaging the small leaf, increasing the scratching strength of the petiole and the small branch, deepening the wound and not breaking the small leaf.
The scratched wounds in the step 3) are alternately arranged in an inclined grid shape.
The phenotype observation of all the group samples in the step 5) is specifically operated as follows: from the first day of inoculation, before and after the date of occurrence of the trait change predicted from the function of the silencing target gene, observation and comparison between different groups were carried out on the relevant traits of the gene silencing treated region, and the corresponding amounts were counted.
The invention establishes a virus-induced gene silencing system aiming at perennial rosa plants under the field condition, and improves the thallus concentration of agrobacterium carrying a virus vector to OD (origin-destination-diameter) in the preparation of an infection solution based on the influence of natural environment under the field condition and the infection resistance of perennial plant materials600Is 1.5-2.0, and ensures the natural conditionAgrobacterium carrying virus vectors in the inoculation area has enough survival base number, so that the infection efficiency of the whole experiment is improved; the low-concentration Silwet-77 component is added in the preparation of the infection liquid, so that the hydrophilicity of the surface of the plant material is increased, the infection efficiency of the infection liquid is improved, meanwhile, the low-concentration component does not produce toxic action on the plant material, the pollution rate is low, and the environment is protected; the treated part is scratched intensively, the infiltration infection time is prolonged, the contact area of the wound and the infection liquid can be increased, the infection efficiency is improved, and meanwhile, the normal growth of the treated part is not influenced greatly under the condition of not breaking and being incomplete because the treated part grows vigorously; the bag covering after inoculation is subjected to dark treatment, so that the heat and moisture can be preserved, and severe weather can be resisted to a certain extent; the method saves cost and improves inoculation success rate, thereby improving gene silencing efficiency, the gene silencing efficiency of processed samples reaches more than 87.5 percent, greatly promoting the research of the genetic engineering field in rosa plants, promoting the conversion of research objects as experimental verification materials, and greatly shortening the time of gene function verification.
Detailed Description
The present invention is described in detail below with reference to specific examples:
a method for establishing a virus-induced gene silencing system on a rosa plants in a field comprises the following steps:
1) determining the inoculation period: and during 3 to 4 middle of the month, after the inoculation time is 3 pm, checking the weather forecast in advance, and ensuring no rainfall within one week after inoculation.
2) Preparing a staining solution: the infection liquid is prepared at present, and the preparation process comprises the following steps:
firstly, selecting agrobacterium positive clones with corresponding virus plasmids in 5ml LB or YEB liquid culture medium containing kan 50ug/ml and rifampicin 100ug/ml, shaking at 28 deg.C for 10-12h at 200r/min until the clones are completely white;
then, the cells were cultured in 50ml LB or YEB liquid medium containing kan 50ug/ml, rifampicin 100ug/ml, MES 10mM, AS 200uM, shaking at 28 ℃ and concentrated in bacterial solutionDegree reaches OD600Taking out when the temperature is 1.5-2.0 ℃;
then, the cells were collected by centrifugation, and suspended in 25ml of an infection buffer containing a liquid medium of 1/2MS AS a solvent, MES 10mM, AS 100uM and MgC 1210 mM, and the OD of the concentration of the bacterial solution was adjusted600To 1.5-2.0, forming an agrobacterium infection buffer solution;
and finally, mixing the agrobacterium infection buffer solution containing the pTRV1 with the agrobacterium infection buffer solution containing pTRV2 and pTRV 2-target gene fragments in equal volume, respectively adding Silwet-77 with the final concentration of 0.01% into the two mixed liquid, and standing for 4h at room temperature under a dark condition to form two kinds of agrobacterium infection solutions.
3) Inoculation of viral vectors: cleaning leaflets of the whole annual whole cluster, petioles of the leaflets and twigs at the base parts of the leaflets by adopting a sharp instrument, intensively scratching the leaflets of the whole cluster and other scratched parts, completely immersing the leaflets and other scratched parts in a 5ml or 10ml centrifugal tube containing an infection solution according to the volume for infection for 10-15min for a single time, infecting the leaflets for 2-5min again if the moisture on the surfaces of the infected leaflets is evaporated quickly in dry weather or windy weather, and making a brand and noting treatment group and treatment time after infection; the adopted apparatus is preferably a syringe needle or a fine needle, the back surface of each lobule is preferably scratched in a scratching mode, the number of wounds can be increased as required, the whole leaf surface is scratched, the leaves are not damaged as much as possible, the scratching strength of the petioles, the small branches at the base parts of the leaves and the like can be properly increased, the wounds can be deepened but cannot be broken, and the scratched wounds are preferably arranged in an inclined grid-shaped alternating mode.
4) And (3) post-inoculation treatment: according to the set inoculation flow, the inoculation is started after 3 pm, and the temperature gradually drops after the inoculation is finished, generally in the evening. After each group of lobules, petioles and small branches is inoculated, a large black plastic bag is adopted to completely cover the inoculation area and the whole branch where the inoculation area is located, and the opening of the bag is sealed and fixed. The method can meet the requirement of dark treatment after inoculation, and can preserve heat and moisture for the inoculated area at night so as to ensure that the agrobacterium carrying the virus can be infected and permeated as much as possible within 24 hours of infection, and can shield rainwater to a certain extent in rainy days. The black plastic bag can be removed after 24h, so that the inoculation area is contacted with normal natural conditions, and the bag picking time is properly delayed if severe weather occurs;
5) testing the gene silencing efficiency: the groups comprise a virus empty vector group and a virus and target gene composite vector combined untreated blank control group, and phenotype observation is firstly carried out on the three groups of samples, preferably, from the first day of inoculation, before and after the date of occurrence of character change predicted according to the function of a silent target gene, observation comparison among different groups is carried out on related characters of a gene silencing treated area, and corresponding quantity is counted. Secondly, extracting RNA from plant materials of corresponding tissue parts processed by all groups, synthesizing first strand cDNA, carrying out PCR detection, carrying out band observation through agarose gel electrophoresis to determine existence of TRV virus and target genes, and finally determining the gene silencing efficiency by calculating the ratio of the effective amount of the PCR detection to the total processing amount.
Example 1:
a method for establishing virus-induced flower color related gene silencing by taking perennial purple branch roses under field conditions as objects comprises the following steps:
(1) determining the inoculation period: according to the growth conditions of the past annual flowering period and the current stage branch sprouting of the purple branch rose and the weather forecast of the last week, the determined inoculation date is 3 and 18 months to 20 days in 2018 for three days. The specific time is as follows: half at 3 pm to half at 5 pm on the inoculation date.
(2) Preparing a staining solution: according to the use principle of the existing preparation, the preparation time is the morning of the day of inoculation. First, Agrobacterium positive clones with the corresponding viral plasmid were picked up in 5ml LB or YEB liquid medium (with kan 50ug/ml, rifampicin 100ug/ml) at 28 ℃; shaking for 10h at 200r/min until the white color is completely removed; then, the cells were cultured in 50ml of LB or YEB liquid medium (kan 50ug/ml; rifampicin 100 ug/ml; MES 10 mM; AS 200uM), respectively, and shaken at 28 ℃ until the bacterial liquid concentration reached OD600Taking out when the concentration is not less than 1.8, centrifuging to collect thallus, and soaking with 25mlStaining buffer (MES 10 mM; AS 100uM; MgC 1)210 mM; sterile water as solvent) suspension of the cells, adjustment of OD600To between 1.5 and 2.0; finally, an agrobacterium infection buffer containing an equal concentration of pTRV1 is mixed with an agrobacterium infection buffer containing pTRV2 and pTRV 2-target gene fragments in equal volume (wherein an empty vector mixed infection solution without any target gene fragment is used as a control group infection solution), Silwet-77 with the final concentration of 0.01 percent is added into the two mixed infection buffers respectively, and the mixture is kept standing for 4 hours at room temperature and 24 ℃ under dark conditions.
(3) Inoculation of viral vectors: the clean whole-year-old leaflet clusters, petioles of the leaflets and the small branches at the bases of the leaflets are wiped and intensively scratched by a syringe needle, and the method comprises the following specific operations: the back of each lobule is scratched, the wounds are alternately arranged in an inclined grid shape, the number of the wounds can be increased according to the needs, the whole leaf surface is scratched completely, but the wounds are not damaged as much as possible. The strength of the leaves can be properly increased at the petioles where the leaves are planted, the small branches at the base parts of the leaves and the like, so that the wounds can be deepened but cannot be broken. And then completely immersing the whole cluster of small leaves and other scratched parts in a 10ml centrifugal tube filled with staining solution according to the volume for infection, wherein the infection is carried out for 10min in a single time. And (5) after infection, the treatment groups and the treatment time are marked by the tag.
(4) And (3) post-inoculation treatment: according to the set inoculation flow, the inoculation completion time is about 5 o' clock and half afternoon, and the air temperature gradually drops. And (3) completely sheathing the inoculation area and the whole branch where the inoculation area is located by using a large black plastic bag after each group of inoculation is finished, and sealing and fixing the bag opening. The cap bag is removed 24h later, i.e. the same time the day the next, to expose the inoculation area to normal natural conditions.
(5) Testing the gene silencing efficiency: the total number of treatments of all the groups of samples at this time is 40, wherein the total number of treatments of the virus empty vectors is 20, and the total number of treatments of the virus and the target gene complex vectors is 20. The gene-silenced region flowers sequentially 35 days after the first day of inoculation. Based on the function of the silenced gene, we predicted that the trait changed to lighter flower color. Counting, compared with a blank control group, the sample treated by the virus empty vector has no flowers with obvious lighter colors; in the samples treated with the virus-target gene complex vector, 17 flowers in total produced a flower color with reduced color compared to the blank control group and the virus empty vector group. Extracting RNA from all groups after petal treatment, synthesizing first strand cDNA, performing statistics after PCR and agarose gel electrophoresis detection, wherein the blank control group cannot detect the existence of TRV and the expression quantity of the target gene is normal; the virus empty vector treated sample has 19 flowers which can detect the existence of TRV virus and the expression level of the target gene is normal, while the virus and target gene composite vector treated sample has 18 flowers which can detect the existence of TRV virus and detect that the abundance of endogenous transcripts of the silenced target gene is obviously reduced, wherein the flowers comprise 17 flowers with lighter flower colors. Therefore, the overall silencing efficiency of this virus-induced gene silencing reached 92.5%.
Example 2:
a method for establishing virus-induced flower color related gene silencing by taking perennial 'Zilonghou' roses under field conditions as objects comprises the following steps:
(1) determining the inoculation period: according to the growth conditions of the former-year flowering period and the current-stage branching and sprouting of the 'Zilong crouchi' rose and the weather forecast of the last week, the determined inoculation date is 4 months and 5 days to 7 days in 2018 for three days. The specific time is as follows: half at 3 pm to half at 5 pm on the inoculation date.
(2) Preparing a staining solution: according to the use principle of the existing preparation, the preparation time is the morning of the day of inoculation. Firstly, selecting agrobacterium positive clones with corresponding virus plasmids in 5ml LB or YEB liquid culture medium (containing kan 50ug/ml, rifampicin 100ug/ml), shaking at 28 deg.C and 200r/min for 12h until the clones are completely white; then, the cells were cultured in 50ml of LB or YEB liquid medium (kan 50ug/ml; rifampicin 100 ug/ml; MES 10 mM; AS 200uM), respectively, and shaken at 28 ℃ until the bacterial liquid concentration reached OD600Taking out when the value is not less than 2.0; then, the cells were collected by centrifugation and each was infected with 25ml of infection buffer (MES 10 mM; AS 100uM; MgC 1)210 mM; sterile water as solvent) suspension of the cells, adjustment of OD600To between 1.5 and 2.0; finally, the straw containing pTRV1 at equal concentration was usedThe bacterial infection buffer solution is respectively mixed with the agrobacterium infection buffer solution containing pTRV2 and pTRV 2-target gene fragments in equal volume (wherein the empty vector mixed infection solution without any target gene fragment is used as a control group infection solution), Silwet-77 with the final concentration of 0.01 percent is respectively added into the two mixed infection solutions, and the mixture is kept stand for 4 hours at the room temperature of 23 ℃ under the dark condition.
(3) Inoculation of viral vectors: the clean whole annual lobules, petioles of the lobules and small branches at the base of the leaves are wiped and intensively scratched by a fine needle, and the method comprises the following specific operations: the back of each lobule is scratched, the wounds are alternately arranged in an inclined grid shape, the number of the wounds can be increased according to the needs, the whole leaf surface is scratched completely, but the wounds are not damaged as much as possible. The strength of the leaves can be properly increased at the petioles where the leaves are planted, the small branches at the base parts of the leaves and the like, so that the wounds can be deepened but cannot be broken. And then completely immersing the whole cluster of small leaves and other scratched parts in a 5ml centrifugal tube containing an infection solution according to the volume for infection, wherein the infection is carried out for 12min in a single time. And (5) after infection, the treatment groups and the treatment time are marked by the tag.
(4) And (3) post-inoculation treatment: according to the set inoculation flow, the inoculation completion time is about 5 o' clock and half afternoon, and the air temperature gradually drops. The measure adopted by the inventor is that after each group of inoculation is finished, the inoculation area and the whole branch where the inoculation area is located are completely sleeved by a large-size black plastic bag, and the bag opening is sealed and fixed. The cap bag is removed 24h later, i.e. the same time the day the next, to expose the inoculation area to normal natural conditions.
(5) Testing the gene silencing efficiency: the total number of treatments of all the groups of samples at this time is 40, wherein the total number of treatments of the virus empty vectors is 20, and the total number of treatments of the virus and the target gene complex vectors is 20. The gene-silenced region flowers successively 33 days after the first day of inoculation. According to the function of the silenced gene, the character is predicted to change to lighter flower color. Counting, compared with a blank control group, the sample treated by the virus empty vector has no flowers with obvious lighter colors; in the samples treated with the virus-target gene complex vector, 15 flowers in total produced a flower color with reduced color compared to the blank control group and the virus empty vector group. Extracting RNA from all groups after petal treatment, synthesizing first strand cDNA, performing statistics after PCR and agarose gel electrophoresis detection, wherein the blank control group cannot detect the existence of TRV and the expression quantity of the target gene is normal; the virus empty vector treated sample has 18 flowers which can detect the existence of TRV virus and the expression level of the target gene is normal, while the virus and target gene composite vector treated sample has 17 flowers which can detect the existence of TRV virus and detect that the abundance of endogenous transcripts of the silenced target gene is obviously reduced, wherein the flowers comprise 15 flowers with lighter flower colors. Therefore, the overall silencing efficiency of this virus-induced gene silencing reached 87.5%.
Example 3:
a method for establishing virus-induced flower color related gene silencing by taking perennial rosa davurica pall under field conditions as a subject comprises the following steps:
(1) determining the inoculation period: according to the growth conditions of the past-year flowering period and the current-stage branch sprouting of the rosa davurica and the weather forecast of the last week, the determined inoculation date is 3 and 3 months and 25 to 27 days in 2018 for three days. The specific time is as follows: half at 3 pm to half at 5 pm on the inoculation date.
(2) Preparing a staining solution: according to the use principle of the existing preparation, the preparation time is the morning of the day of inoculation. Firstly, selecting an agrobacterium positive clone with a corresponding virus plasmid in 5ml LB or YEB liquid culture medium (containing kan 50ug/ml and rifampicin 100ug/ml), shaking at 28 ℃ and 200r/min for 11h until the agrobacterium positive clone is completely white; then, the cells were cultured in 50ml of LB or YEB liquid medium (kan 50ug/ml; rifampicin 100 ug/ml; MES 10 mM; AS 200uM), respectively, and shaken at 28 ℃ until the bacterial liquid concentration reached OD600Taking out when the value is 1.5; the cells were then collected by centrifugation and the cells were infected with 25ml of infection buffer (MES 10 mM; AS 100uM; MgC 1)210 mM; sterile water as solvent) suspension of the cells, adjustment of OD600To between 1.5 and 2.0; finally, the agrobacterium infection buffer containing pTRV1 with equal concentration is mixed with the agrobacterium infection buffer containing pTRV2 and pTRV 2-target gene fragments in equal volume (wherein empty vector mixed infection solution without any target gene fragment is used as control group infection solution),and respectively adding Silwet-77 with the final concentration of 0.01 percent into the two mixed staining solutions, and standing for 4 hours at room temperature of 28 ℃ under the dark condition.
(3) Inoculation of viral vectors: the clean whole annual lobules, petioles of the lobules and small branches at the base of the leaves are wiped and intensively scratched by a blade, and the method comprises the following specific operations: the back of each lobule is scratched, the wounds are alternately arranged in an inclined grid shape, the number of the wounds can be increased according to the needs, the whole leaf surface is scratched completely, but the wounds are not damaged as much as possible. The strength of the leaves can be properly increased at the petioles where the leaves are planted, the small branches at the base parts of the leaves and the like, so that the wounds can be deepened but cannot be broken. And then completely immersing the whole cluster of small leaves and other scratched parts in a 10ml centrifugal tube containing an infection solution according to the volume for infection, wherein the infection is carried out for 15min once. The infection is repeated for 5min due to dry weather. And (5) after infection, the treatment groups and the treatment time are marked by the tag.
(4) And (3) culturing the plant material after inoculation under the light-temperature condition that the temperature gradually drops when the inoculation is about 5 o' clock in the afternoon according to the set inoculation flow. The measure adopted by the inventor is that after each group of inoculation is finished, the inoculation area and the whole branch where the inoculation area is located are completely sleeved by a large-size black plastic bag, and the bag opening is sealed and fixed. The cap bag is removed 24h later, i.e. the same time the day the next, to expose the inoculation area to normal natural conditions.
(5) Testing the gene silencing efficiency: the total number of treatments of all the groups of samples at this time is 40, wherein the total number of treatments of the virus empty vectors is 20, and the total number of treatments of the virus and the target gene complex vectors is 20. The gene-silenced region flowers successively 30 days after the first day of inoculation. Based on the function of the silenced gene, we predicted that the trait changed to lighter flower color. Counting, compared with a blank control group, the sample treated by the virus empty vector has no flowers with obvious lighter colors; in the samples treated with the virus-target gene complex vector, 18 flowers in total produced a lighter flower color than the blank control group and the virus empty vector group. Extracting RNA from all groups after petal treatment, synthesizing first strand cDNA, performing statistics after PCR and agarose gel electrophoresis detection, wherein the blank control group cannot detect the existence of TRV and the expression quantity of the target gene is normal; the virus empty vector treated sample has 19 flowers which can detect the existence of TRV virus and the expression level of the target gene is normal, while the virus and target gene composite vector treated sample has 19 flowers which can detect the existence of TRV virus and can detect that the abundance of endogenous transcripts of the silenced target gene is obviously reduced, wherein the flowers comprise 18 flowers with lighter flower colors. Therefore, the overall silencing efficiency of the virus-induced gene silencing can reach 95%.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art may make modifications, alterations, additions or substitutions within the spirit and scope of the present invention.
Claims (3)
1. A method for establishing a virus-induced gene silencing system on perennial 'purple branch' roses, 'purple dragon crouchi' roses and rosa davurica pall plants in a field is characterized by comprising the following steps:
1) determining the inoculation period: during 3 to 4 middle ten days of the month, after the inoculation time is 3 pm, the weather forecast is checked in advance, and no rainfall is ensured within one week after inoculation;
2) preparing a staining solution: the infection liquid is prepared at present, and the preparation process comprises the following steps:
firstly, selecting agrobacterium positive clones with corresponding virus plasmids in 5ml LB or YEB liquid culture medium containing kan 50ug/ml and rifampicin 100ug/ml, shaking at 28 deg.C for 10-12h at 200r/min until turbid without whitening;
then, the cells were cultured in 50ml LB or YEB liquid medium containing kan 50ug/ml, rifampicin 100ug/ml, MES 10mM, AS 200uM, and shaken at 28 ℃ until the bacterial concentration reached OD600Taking out when the temperature is 1.5-2.0 ℃;
then, the cells were collected by centrifugation, and suspended in 25ml of an infection buffer containing 1/2MS liquid medium containing MES 10mM and AS 100 AS a solventuM、MgC1210mM, adjusting the concentration OD of the bacterial liquid600To 1.5-2.0, forming an agrobacterium infection buffer solution;
finally, respectively mixing an agrobacterium infection buffer solution containing pTRV1 with equal concentration with an agrobacterium infection buffer solution containing pTRV2 and pTRV 2-target gene fragments in equal volume, respectively adding Silwet-77 with the final concentration of 0.01% into the two mixed liquids, and standing for 4h at room temperature under a dark condition to form two agrobacterium infection solutions;
3) inoculation of viral vectors: scraping clean whole-year-old leaflets, leaflet stems and small branches at the base parts of the leaflets densely by using a sharp instrument, scraping the leaflets on the back surface, and can scrape the whole leaf surface without making the leaflets incomplete, wherein the scraping force of the leaflet stems and the small branches is increased, the wounds are deepened but cannot be broken, the scratched wounds are in inclined grid-shaped alternate arrangement, then completely immersing the whole-year-old leaflets and other scratched parts in a 5ml or 10ml centrifugal tube filled with an infection solution according to the size to infect for 10-15min for a single time, if the moisture on the surface of the infected leaves is evaporated quickly in dry weather or windy weather, infecting the leaves can be infected for 2-5min again, and after infection, branding, identifying the treatment group and the treatment time;
4) and (3) post-inoculation treatment: after each group of the small leaves, the leafstalks and the small branches are inoculated, a large black plastic bag is adopted to completely cover the inoculation area and the whole branch where the inoculation area is located, the opening of the bag is sealed and fixed, the black plastic bag can be removed after 24 hours, so that the inoculation area is in contact with normal natural conditions, and the bag picking time is properly delayed if severe weather occurs;
5) testing the gene silencing efficiency: the groups comprise a virus empty vector group, a virus and target gene composite vector group and an untreated blank control group, the phenotype observation is firstly carried out on the three groups of samples, RNA is extracted from plant materials of corresponding tissue parts of all the groups after treatment, first-strand cDNA is synthesized, PCR detection is carried out, and the strip observation is carried out through agarose gel electrophoresis to determine the existence of TRV virus and target genes, and finally the gene silencing efficiency is determined by calculating the ratio of the effective number of the PCR detection to the total number of the treatment.
2. The method of claim 1, wherein the device used in step 3) is configured as a syringe needle, a fine needle or a blade.
3. The method for establishing a virus-induced gene silencing system on perennial 'purple branch' rose, 'purple dragon crouchi' rose and rosa davurica plants in field according to claim 1, wherein the phenotype observation of all the group samples in step 5) is specifically: from the first day of inoculation, before and after the date of occurrence of the trait change predicted from the function of the silencing target gene, observation and comparison between different groups were carried out on the relevant traits of the gene silencing treated region, and the corresponding amounts were counted.
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