CN109402166B - Cunninghamia lanceolata virus induced gene silencing system and construction method thereof - Google Patents
Cunninghamia lanceolata virus induced gene silencing system and construction method thereof Download PDFInfo
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Abstract
The invention provides a fir virus induced gene silencing system and a construction method thereof, belonging to the field of forest genetic engineering. The method comprises the following steps: culturing fir plant materials; cloning a target gene fragment; constructing a recombinant virus plasmid vector; infecting a fir plant by the agrobacterium-mediated silencing vector; and (5) verifying the silencing effect result. The invention can be used for exploring the function mechanism of the target gene according to the change of the plant phenotype, and the establishment of the VIGS system has pioneering significance for the research of the tree gymnosperm function gene.
Description
Technical Field
The invention belongs to the field of forest genetic engineering, and particularly relates to a fir virus induced gene silencing system and a construction method thereof.
Background
Fir (China fir) (1)Cunninghamia lanceolata) Cunninghamia of Cunninghamia is a special forest species mainly growing in southern areas of China. The fir has higher economic value, straight tree body, uniform wood structure and hard material. The fir wood has use value in many aspects, is a good building material, and is widely used in shipbuilding, furniture manufacturing and the like. However, the stable genetic transformation system of the forest is difficult, and no relevant report exists at present, so that the development of a rapid and effective technical system for researching the gene function of the fir is of great significance.
Virus-Induced Gene Silencing (VIGS) is a very useful research tool for the rapid generation of Gene knockout phenotypes, which can be used to assess plant Gene function. Infection of VIGS with RNA viruses activates a conserved plant RNA-based antiviral defense response that is sequence-specific for RNA produced by the infecting virus. By inserting the target gene sequence segment into the viral vector, the transcript of the endogenous gene becomes a target for degradation, thereby leading to the significant down-regulation or knockdown of the expression level of the target gene.
Virus-induced gene silencing (VIGS) has proven to be an effective tool for studying gene function in herbaceous plant species, but is rarely tested on forest trees. The establishment of a rapid and reliable transformation system is particularly important for woody plants, and the difficulty in research is that many plants are difficult to transform. In this study, we successfully established the virus-induced gene silencing VIGS system of plants in the tree gymnosperm cedar for the first time.
Disclosure of Invention
The invention aims to provide a cunninghamia lanceolata virus-induced gene silencing system and a construction method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fir virus-induced gene silencing system comprises: obtained by infecting Chinese fir with recombinant virus plasmid carrying target gene segment mediated by agrobacterium.
A construction method of a fir virus-induced gene silencing system comprises the following steps:
(1) culturing a fir plant material: and (4) planting under a greenhouse condition, selecting seeds with plump seeds and uniform texture, performing broadcast sowing treatment and culturing for 30-60 days, and opening cotyledons for 2-3 cm.
(2) Cloning of the target gene fragment: selecting a gene to be researched of the fir, taking a nucleotide sequence in a range of 300bp to 350bp close to a 3' end UTR region of the gene to be researched as a target gene fragment, designing a forward primer and a reverse primer of the target gene fragment, taking genome DNA of the fir as a template, and carrying out high fidelity enzyme PCR amplification to obtain the target gene fragment.
(3) Construction of recombinant viral plasmid vector: the viral vector pTRV2 is connected with the obtained target gene fragment to obtain a recombinant viral plasmid which is connected with pTRV2 and the target gene fragment, and then the recombinant viral plasmid is transformed into agrobacterium.
(4) Infecting the fir plants with agrobacterium-mediated recombinant virus plasmids: and uniformly mixing the agrobacterium liquid containing pTRV1 and the agrobacterium liquid containing recombinant virus plasmids according to the volume ratio of 1:1, and then infecting the fir by using a 1mL syringe by adopting an injection method.
(5) And (3) verifying the silencing effect result: and gradually showing phenotype after 60-70 days of the infected fir plant, observing the phenotype change of the plant, performing gene expression analysis by using semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction), observing the change of the expression quantity of a target gene, and measuring the chlorophyll content change of the fir plant by using an enzyme-labeling instrument.
Further, cloning of the target gene fragment: the gene to be researched is a gene of fir Phytoene Desaturase (PDS), and the gene sequence is shown as SEQ ID NO. 1; the series of the target gene segments is shown as SEQ ID NO. 2; the forward primer sequence of the target gene segment is shown as SEQ ID NO. 3, and the reverse primer sequence is shown as SEQ ID NO. 4.
Further, the construction of the recombinant virus plasmid vector is to construct the recombinant virus plasmid vector by a Gateway method, wherein the entry vector is pDONR207, and the final vector TRV2 is a viral vector 279; the agrobacterium strain used for transforming the recombinant virus plasmid into agrobacterium is GV 3101.
Further, the step of infecting the fir plants with the agrobacterium-mediated recombinant virus plasmid comprises the following steps:
1) transferring l mL of Agrobacterium liquid containing recombinant virus plasmid to 6 mL of LB culture solution containing rifampicin with final concentration of 50mg/mL and kanamycin with final concentration of 50mg/mL, and shake-culturing at 28 ℃ and 220rpm to make OD600Is 0.8;
2) transferring l mL of the bacterial liquid obtained in the step (1) into an l.5 mL centrifuge tube for bacteria preservation, centrifuging the residual bacterial liquid at 3800 rpm for 8 min, and discarding the supernatant. With suspension (10mM MES, 10mM MgCl)2200 μ M AS) suspension precipitation, and then adjusting the inoculum concentration to OD using the suspension600 = 0.6；
3) The agrobacterium liquid containing pTRV1 and the agrobacterium liquid containing pTRV2 and recombinant virus plasmid are mixed according to the ratio of 1:1, standing at room temperature for 4h, and infecting Chinese fir plants; wherein the mixed bacteria containing pTRVI and the Agrobacterium containing pTRV2 are used as the empty vector control without recombinant virus plasmids;
4) sucking the bacterial liquid by a needle-free syringe of lmL, and slowly injecting the cedar cotyledon until the foliage is full of the infection liquid, wherein the cotyledon is damaged if the cotyledon is sprayed with air holes. Dark culture at 16 ℃ for 16h after inoculation; taking out at 25 deg.C, culturing under light for 16h, and culturing in dark for 8 h.
Further, the semi-quantitative RT-PCR is used for gene expression analysis, and actin gene is used as an internal reference of the semi-quantitative analysis to detect the expression quantity condition of the full length of the PDS gene; the forward primer of the PDS gene is shown as sequence SEQ ID NO. 5, and the reverse primer is shown as sequence SEQ ID NO. 6; the forward primer of the actin reference gene is shown as a sequence SEQ ID NO. 7, and the reverse primer is shown as a sequence SEQ ID NO. 8.
Further, when measuring chlorophyll, the mixed liquor used for chlorophyll extraction measurement is: the acetone, the absolute ethyl alcohol and the distilled water are prepared according to the proportion of 4.5: 4.5: 1.
The invention has the advantages that:
the invention successfully establishes a virus-induced gene silencing (VIGS) system of plants in the gymnosperm China fir of forest trees for the first time. The system can quickly and efficiently identify the functional genes of the fir, and can explore the functional mechanism of the target genes according to the change of the phenotype of the plant. The establishment of the VIGS system has pioneering significance for the research of the tree gymnosperm functional gene.
Drawings
FIG. 1: a 60-day phenotype chart of a fir plant infected by a virus vector of a fir PDS gene sequence. Untreated: an untreated plant phenotype; no load: empty vector control pTRV1 pTRV 2; the target gene is as follows: the experimental group with the transferred target gene pTRV1 is PDS; front: the front side of the leaf; back: the back of the leaf.
FIG. 2 is a schematic diagram: and (3) detecting the expression level of the target gene by semi-quantitative RT-PCR. Mechanically: the leaves are mechanically treated by needle punching;
FIG. 3: and (4) determining the content of chlorophyll after the leaves infected with the fir turn green. The obvious reduction of the chlorophyll content in the leaf subjected to gene silencing can be definitely detected.
Detailed Description
The method for establishing the present line will be described in detail below with the PDS gene of Cunninghamia lanceolata as the target gene.
Example 1 cultivation of fir plant Material
Selecting Chinese fir seeds with full grains and uniform texture, soaking the seeds in clear water for three days, and removing the dry, shrivelled and incomplete seeds floating on the water surface. The seeds are uniformly spread on the soil and then covered with 2cm of soil with the same texture after being planted in an environment with a 2:1 ratio of nutrient soil to vermiculite. After the seeds are cultured for 30 days, the seed pods are opened, and the cotyledons are fully extended to 3cm, so that the late-stage infection treatment is carried out.
EXAMPLE 2 cloning of the Gene fragment of interest
(1) Extraction of genome DNA of fir
Cutting tender leaves at growing points of the dry ends of green robust branches of fir, wrapping with tinfoil paper, storing in liquid nitrogen, crushing with a crusher in liquid nitrogen environment, and packaging the crushed powder into centrifuge tubes.
The genomic DNA of the Cunninghamia lanceolata plant was extracted according to the instructions of the centrifugal column catalog No. DP305 of the plant genomic DNA extraction kit of TIANGEN corporation. The method comprises the following specific steps:
1) adding 700 μ L of GP1 (volume percentage concentration of mercaptoethanol is 0.1% according to the test) preheated in 65 ℃ water bath into the centrifuge tube, fully mixing the solution by using a vortex oscillation instrument, and placing the centrifuge tube in a 65 ℃ water bath to heat in water bath for 20 min. And mixing up and down in the water bath process to facilitate full dissolution.
2) Add 700. mu.L chloroform to the centrifuge tube, mix well using vortex shaking instrument, centrifuge at 12000rpm for 5 min.
3) Transferring the uppermost layer of liquid in the centrifuge tube to a brand-new clean centrifuge tube, adding 700 mu L of medicine GP2, and fully and uniformly mixing by using a vortex oscillation instrument to obtain a mixture.
4) Transferring the mixture in the step 3) to a CB3 adsorption column, centrifuging at 12000rpm for 30s, and discarding waste liquid in a collection tube.
5) To the CB3 adsorption column, 500. mu.L of GD drug (absolute ethanol was added), and the mixture was centrifuged at 12000rpm for 30 seconds, and the waste liquid in the collection tube was discarded.
6) To a CB3 adsorption column, 600. mu.L of the drug PW (absolute ethanol was confirmed to have been added), and centrifuged at 120000rpm for 30 seconds, and the waste liquid in the collection tube was discarded.
7) Repeating the previous step.
8) And continuing to centrifuge at 12000rpm for 2min, discarding the waste liquid in the collection pipe, and placing the CB3 adsorption column in a room temperature environment for 25min to completely dry the residual liquid in the adsorption column.
9) Placing the CB3 adsorption column into a brand-new clean centrifuge tube, and dropwise adding 40 mu L of elution ddH into the center of the CB3 adsorption column by using a pipette gun2O, then standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min. The obtained DNA product was collected into a centrifuge tube.
(2) Selection of target gene fragment, PCR primer design and amplification of target gene fragment
Selecting a target gene fragment which is 330bp in size and is 300bp close to a 3' end UTR region of a fir target gene PDS;
target gene fragment
Forward primer sequence: SEQ ID NO. 3
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCACCAAAATCCATATAC-3’；
Reverse primer sequence: SEQ ID NO 4
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGATTCTGAATTTATTGAAG-3’。
The underlined parts are the Gateway recognition sites, the forward primer is the attP1 recognition site, and the reverse primer is the attP2 recognition site. The primers were synthesized by Shanghai Boshang Biotechnology, Inc.
PCR reactions were purchased from TaKaRa using PrimeSTAR GXL DNA Polymerase. The reaction system is shown in Table 1.
TABLE 1 amplification System for target Gene fragments
After the sample is added into the centrifuge tube, uniformly mixing the sample by using a vortex instrument, and setting the PCR temperature cycle parameter at 94 ℃ for 5 min; 30s at 94 ℃, 50 ℃ and 30s at 68 ℃, and 35 cycles; 68 ℃ for 5min, 1 cycle.
And (3) detecting a PCR product: and (3) carrying out 1% agarose gel electrophoresis at a voltage of 120V to obtain the correct size of the target gene fragment.
(3) Target gene fragment PCR product recovery and purification
The target gene fragment was purified and recovered according to the instructions of the DNA purification and recovery kit for use in the universal kit for DNA purification and recovery under the centrifugal column number DP214-03 of TIANGEN. The method comprises the following specific steps:
1) the adsorption column CB2 was treated to reach an equilibrium state, 500. mu.L of BL was added to CB2 and centrifuged at 12000rpm for 1 min. And (4) discarding the waste liquid.
2) The gel pieces of the correct size band indicated by UV fluorescence were cut and transferred to a fresh centrifuge tube.
3) The PC solution with the volume corresponding to the weight of the gel block (0.1 g for 100. mu.L) was added into the centrifuge tube and heated in a 50 ℃ water bath for 10 min. Shaking up and down several times during this process to facilitate complete dissolution of the gum piece.
4) The dissolved solution was transferred to a CB2 adsorption column at 12000rpm, centrifuged for 1min, and the waste liquid was discarded.
5) 600. mu.L of liquid PW (absolute ethanol was confirmed to have been added) was added to the CB2 adsorption column, and then the column was allowed to stand at room temperature for 5min at 12000rpm for 1min, and the waste liquid was discarded.
6) Repeating the operation of the previous step once.
7) The centrifugation at 12000rpm was continued for 2min to remove excess liquid, and then left to stand at room temperature for 25min to completely dry the residual liquid in the adsorption column.
8) Placing the CB2 adsorption column into a clean centrifuge tube, and dripping 40 mu L ddH into the center of the CB2 adsorption column by using a pipette gun2O (preheated by using a water bath kettle at 65 ℃), standing for 2min at room temperature, centrifuging at 12000rpm for 2min, and centrifuging to collect a DNA product.
Example 3 construction of recombinant viral plasmids
(1) BP reaction was performed using gel recovery products with correct band size, and the Gateway BP Clonase ™ II Enzyme mix was purchased from Bai Whole organisms. The reaction system is shown in Table 2.
Table 2 Gateway BP Clonase reaction system
Adding the sample into a 200 mu L centrifuge tube, uniformly mixing by using a vortex instrument, and placing into an electric heating instrument for preserving for 8 hours at 25 ℃ to obtain a BP product.
(2) Ligation products were transformed into DH5 α competent cells by thermal stimulation: transformation was performed according to the instructions of Shanghai Weidi Biotechnology Co., Ltd.model DL1001 competent cell DH 5. alpha. and positive clones were verified by amplifying the target fragment by PCR with a bacterial solution. The method comprises the following specific steps:
1) immediately placing DH5 alpha competent cells into an ice box for standing for 5min after taking out the cells from a refrigerator at minus 80 ℃, taking out 50 mu L of the cells by using a pipette gun, adding BP product, slowly pulling the bottom of a centrifuge tube by hands, placing the centrifuge tube into the ice box for standing for 25 min.
2) Placing into ice box immediately after water bath for 45s at 42 deg.C, standing for 2min, and standing while still.
3) Using a pipette gun, 600. mu.L of the antibiotic-free liquid medium LB was pipetted into a centrifuge tube and resuscitated using a shaker set at 37 ℃ with 200rpm shaking.
4) And centrifuging the recovered bacterial liquid at 5000rpm for 1 min. The upper excess liquid was removed using a pipette, 50. mu.L of the resuspended pellet was blown out using a pipette and spread onto a solid medium containing gentamicin resistance at a final concentration of 30mg/mL using a sterilized tip. The cells were then placed upside down in an incubator at 37 ℃ for 16 hours.
5) White unicellular colonies are selected and inoculated in 1mL of a liquid medium LB containing gentamicin with the final concentration of 30mg/mL, the medium is cultured for 4 hours at the temperature of 220rpm set by a shaker at 37 ℃,
(3) 200 mu.L of bacterial liquid containing the correct target gene fragment is transferred into a gentamicin liquid culture medium with the final concentration of 30mg/mL, and is subjected to shake culture at 37 ℃ and 220rpm overnight. Plasmids were extracted using a centrifugal column DP103 plasmid miniextraction kit from Tiangen corporation, according to the instructions. The method comprises the following specific steps:
1) the column was adsorbed with CP3, and the drug BL was applied thereto at 12000rpm, centrifuged for 1min, and the waste liquid was discarded to allow CP3 to reach an equilibrium state.
2) Transfer 5ml of the inoculum to a clean centrifuge tube, centrifuge at 12000rpm for 1 min. After centrifugation, the upper aqueous phase was aspirated off using a pipette.
3) 250 μ L of P1 was pipetted into the centrifuge tube and then vortexed rapidly and vigorously using a vortexing instrument.
4) 250 mu L of P2 was added to the tube and mixed by gentle shaking to completely lyse the bacteria in the tube
5) mu.L of P3 was added to the tube and mixed gently by shaking to give a white precipitate, which was then centrifuged at 12000rpm for 10 min.
6) The supernatant after centrifugation was pipetted onto a CP3 adsorption column, and a pipette gun was carefully used to avoid pipetting into the pellet. 12000rpm, centrifuging for 60s, and discarding the waste liquid.
7) 600. mu.L of drug PW (absolute ethanol was confirmed to have been added) was applied to a CP3 adsorption column, and the mixture was centrifuged at 12000rpm for 1 min. And (4) discarding the waste liquid.
8) Repeating the step 7) once.
9) The CP3 adsorption column was placed in the collection tube and centrifuged at 12000rpm for 2 min. And then standing for 25min at room temperature to facilitate the complete drying of the residual liquid in the CP3 adsorption column.
10) Placing CP3 adsorption column in a new test tube, and dripping 40 μ L ddH into the central part of CP3 adsorption column2And O, standing for 2min at room temperature, 12000rpm, centrifuging for 2min, and centrifuging the plasmids into a collection tube.
(4) The LR reaction was performed using a plasmid carrying the correct target gene verified by PCR, and the Gateway LR Clonase II Enzyme mix was purchased from Xiamen Taijing Bio Inc. The reaction system is shown in Table 3.
TABLE 3 Gateway LR Clonase reaction systems
The sample is added into a 200 mu L centrifuge tube, then the sample is mixed evenly by using a vortex instrument and is placed into an electric heating instrument for preservation for 8 hours at 25 ℃.
(5) DH5 alpha competent cells were transformed by ligation products heat-activated method according to the instruction manual of DH5 alpha competent cells of DL1001, model of Shanghai Weidi Biotechnology Ltd, and positive clones were verified by PCR amplification of the target fragments with the bacterial solution. The method comprises the following specific steps:
1) immediately placing DH5 alpha competent cells into an ice box for standing for 5min after being taken out from a refrigerator at minus 80 ℃, taking out 50 mu L of the cells by using a pipette, adding LR products, slowly pulling the bottom of a centrifuge tube by hands, placing the centrifuge tube into the ice box for standing for 25 min.
2) Placing into ice box immediately after water bath for 45s at 42 deg.C, standing for 2min, and standing while still.
3) 600. mu.L of the antibiotic-free liquid medium LB was pipetted into a centrifuge tube using a pipette gun, and resuscitated using a shaker set at 37 ℃ with shaking at 200 rpm.
4) Centrifuge at 5000rpm for 1 min. The upper excess liquid was removed using a pipette, 50. mu.L of the resuspended pellet was blown out using a pipette and spread onto a solid medium containing kanamycin resistance at a final concentration of 50mg/mL using a sterilized pipette tip. The cells were then placed upside down in an incubator at 37 ℃ for 16 hours.
5) White single cell colonies were picked, inoculated into 1mL LB liquid medium containing 50mg/mL kanamycin to a final concentration, and cultured for 4 hours at 220rpm with 37 ℃ using a shaker.
(6) 200 μ L of the bacterial solution containing the correct gene fragment was transferred to a liquid medium containing kanamycin to a final concentration of 50mg/mL and cultured overnight at 37 ℃ with shaking at 220 rpm. Plasmids were extracted using a centrifugal column DP103 plasmid miniextraction kit from Tiangen corporation, according to the instructions. The method comprises the following specific steps:
1) the adsorption column of CP3 was used, and the drug BL was applied thereto at 12000rpm, centrifuged for 1min, and the waste liquid was discarded to allow the adsorption column of CP3 to reach an equilibrium state.
2) Transfer 5ml of the lysate to a clean centrifuge tube, centrifuge at 12000rpm for 1 min. After centrifugation, the upper aqueous phase was aspirated off using a pipette. mu.L of P1 was pipetted into the centrifuge tube and then vortexed rapidly and vigorously using a vortexing instrument.
3) mu.L of P2 was added to the tube and gently shaken to mix the mixture to completely lyse the bacteria in the tube.
4) mu.L of P3 was added to the tube and gently shaken and mixed to give a white precipitate, and the mixture was centrifuged at 12000rpm for 10 min.
5) The supernatant after centrifugation was pipetted into a CP3 adsorption column, carefully using a pipette to avoid aspirating the pellet, centrifuging at 12000rpm for 60s, and discarding the waste solution.
6) 600. mu.L of drug PW (absolute ethanol was confirmed to have been added) was applied to a CP3 adsorption column, and the mixture was centrifuged at 12000rpm for 1 min. The waste liquid after centrifugation is not needed to be poured.
7) And the operation of the previous step is carried out again.
8) The CP3 adsorption column was placed in the collection tube and centrifuged at 12000rpm for 2 min. And then standing for 25min at room temperature to facilitate the complete drying of the residual liquid in the CP3 adsorption column.
9) The CP3 adsorption column was placed in a new test tube, and 40. mu.L of ddH was dropped onto the center of the CP3 adsorption column2And O, standing for 2min at room temperature, 12000rpm, and centrifuging for 2min to obtain the recombinant virus plasmid pTRV2-PDS, and collecting in a tube.
(7) The recombinant virus plasmid pTRV2-PDS, which was used to verify the presence of the correct gene fragment of interest by PCR, was transformed into Agrobacterium GV3101 competent cells according to the Agrobacterium GV3101 competent cell manual of Bomeyer catalog number BC 304. The method comprises the following specific steps:
1) GV3101 competent cells were removed from the-80 ℃ freezer and placed in an ice-box to be thawed.
2) And (3) adding the recombinant virus plasmid pTRV2-PDS 100ng obtained in the previous step into a centrifugal tube under a super clean bench sterile environment, slightly stirring the bottom of the tube, uniformly mixing, and placing in an ice box for standing for 5 min.
3) Quickly putting the centrifugal tube into liquid nitrogen for freezing for 5min, then quickly taking out the centrifugal tube, putting the centrifugal tube into a water bath kettle at 37 ℃, keeping the liquid level, standing and heating for 5 min.
4) The centrifuge tube is put into an ice box for freezing for 5min, then 500 mu L of liquid culture medium LB without antibiotics is added in the super clean bench sterile environment, and the mixture is put into a shaking table at 28 ℃ and 220rpm for culturing for 2h to recover the thalli.
5) Recovering the bacterial liquid at 5000rpm, and centrifuging for 1 min. The upper excess liquid was removed using a pipette, 50. mu.L of the resuspended pellet was blown using a pipette and spread onto solid medium containing rifampicin at a final concentration of 50mg/mL and kanamycin resistance at a final concentration of 50mg/mL using a sterilized pipette tip. Placing the mixture upside down in a constant temperature box at 28 ℃ for about 48 hours
6) White unicellular colonies were picked, inoculated into 2mL of LB liquid medium containing rifampicin at a final concentration of 50mg/mL and kanamycin at a final concentration of 50mg/mL, and cultured for 4 hours at 220rpm with a shaker set at 28 ℃.
Example 4 Agrobacterium-mediated silencing vector infests Taxus chinensis.
1) Transferring l mL bacterial solution into 6 mL LB liquid culture solution containing rifampicin with final concentration of 50mg/mL and kanamycin with final concentration of 50mg/mL, and shake culturing at 28 ℃ and 220rpm to make OD600And was 0.8.
2) Transferring l mL of the bacterial liquid into an l.5 mL centrifuge tube for bacteria preservation, centrifuging the rest bacterial liquid at 3800 rpm for 8 min, and discarding the supernatant. With suspension (10mM MES, 10mM MgCl)2200 μ M AS) suspension precipitation, and then adjusting the inoculum concentration to OD using suspension withering600 = 0.6。
3) The agrobacterium liquid containing pTRV1 and the agrobacterium liquid containing pTRV2 and recombinant virus plasmid pTRV2-PDS are mixed according to the ratio of 1:1, standing at room temperature for 4h, and infecting Chinese fir plants; the mixed bacteria containing pTRVI and Agrobacterium containing pTRV2 were used as empty vector control without recombinant virus plasmid.
4) Sucking a proper amount of bacteria liquid by using a needleless syringe of lmL, injecting the fir, and culturing for 16h in the dark at 16 ℃ after inoculation. Taking out, standing at 25 deg.C, culturing under light for 16h, and culturing in dark for 8 h.
Example 5 results of silencing effect confirmed.
(1) And (3) phenotype verification: and observing the color change of the leaf surfaces of the photographed fir plants, wherein the whitening phenomenon of the leaf surfaces can be caused by PDS gene silencing.
The results are shown in FIG. 1.
(2) Verifying the expression quantity of the endogenous PDS gene of the fir.
The leaves showing phenotype after infection were cut and infected with no-load leaves were RNA extracted according to the instructions of the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (DP441) from Tiangen. The method comprises the following specific steps:
1) the cut leaves were pulverized in a liquid nitrogen atmosphere using a disrupter, and then 500. mu.L of drug SL (added mercaptoethanol was confirmed by examination) was added thereto, and rapidly vortexed using a vortex shaker to sufficiently lyse the drug SL. 12000rpm, 2min centrifugation.
2) Transferring the upper layer liquid after centrifugation to a CS adsorption column, centrifuging at 12000rpm for 2min, and then transferring the upper layer liquid in the bottom collecting tube to a new RNA-free centrifuge tube (which is easy to be polluted in the process of extracting RNA).
3) Measuring the volume by using a pipette gun, sucking corresponding 0.4 time of absolute ethyl alcohol into a centrifuge tube, and turning upside down and mixing uniformly. All solutions were transferred together to a CR3 adsorption column at 12000rpm, centrifuged for 15s, and the waste discarded.
4) mu.L of RW1, 12000rpm, was applied to a CR3 adsorption column using a pipette, centrifuged for 15 seconds, and the waste solution was discarded.
5) Preparing DNaseI working solution, and gently mixing the storage solution and the RDD in a volume ratio of 1: 7.
6) And transferring the prepared working solution into a CR3 adsorption column, and standing for 15min at room temperature.
7) mu.L of RW1 drug was pipetted into a CR3 adsorption column at 12000rpm for 15 s. And (4) discarding the waste liquid.
8) Using a pipette gun, 500. mu.L of drug RW was pipetted onto a CR3 adsorption column (ethanol addition was confirmed), and centrifuged at 12000rpm for 15 seconds, and the waste liquid was discarded.
9) The operation of the previous step is carried out again
10) 12000rpm, centrifugal 2min, CR3 into a new RNA-free tube, CR3 central portion of the dropwise 40L ddH2And O, standing for 2min at room temperature, 12000rpm, centrifuging for 2min, and centrifuging the RNA solution into a collection tube.
To verify the expression level of the endogenous PDS gene in Cunninghamia lanceolata, cDNA was synthesized by reverse transcription using the kit for reverse transcription of quantitative Universal reverse transcriptase type number R212-01/02 from Vazyme.
The RNA template was denatured and placed in clean RNA-free centrifuge tubes and the reaction system is shown in Table 4.
TABLE 4 denaturation reaction System for RNA template
Heating with 65 deg.C water bath for 5min, immediately placing into ice box for 2min, and cooling
Genomic DNA was removed and the reaction system is shown in Table 5.
TABLE 5 reaction System for removing genomic DNA
After the sample is added into the centrifuge tube, the sample is blown and beaten by a pipette and is uniformly mixed, and the mixture is heated for 2min by a water bath kettle at 42 ℃.
First strand cDNA synthesis reaction solution was prepared in the reaction system shown in Table 6.
TABLE 6 first Strand cDNA Synthesis reaction System
And (5) lightly blowing and stirring uniformly by using a liquid transfer gun.
The first strand cDNA synthesis reaction was carried out under the following temperature conditions: 5min at 25 ℃; 45min at 50 ℃; 2min at 85 ℃. And (3) performing temperature reaction by using a PCR instrument, and performing RT-PCR semi-quantitative identification on a reaction product.
The expression level of the entire length of the PDS gene was determined using actin (reference gene) as a reference for semi-quantitative detection.
PDS full-length gene forward primer, SEQ ID NO: 5: 5'-ATGCAAGGGGTTGGCTTTTC-3'
PDS full-length gene reverse primer, SEQ ID NO: 6: 5'-TTAATACAAAGCTGCTCCAA-3'
actin forward primer sequence, SEQ ID NO: 7: 5'-GTGAGACGGTGACCATTGTG-3'
actin reverse primer sequence, SEQ ID NO: 8: 5'-TCTTGGCCCTGTACCAGTTC-3'
The actin reference gene reaction system is shown in Table 7.
TABLE 7 actin reference Gene reaction System
Adding the sample into the centrifuge tube, and mixing uniformly by using a vortex instrument, wherein the PCR temperature cycle parameter is set to 94 ℃ for 5 min; 30s at 94 ℃, 30s at 58 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃ for 1 cycle.
PDS gene reaction system is shown in table 8:
TABLE 8 PDS Gene reaction System
Adding the sample into the centrifuge tube, and mixing uniformly by using a vortex instrument, wherein the PCR temperature cycle parameter is set to 94 ℃ for 5 min; 30s at 94 ℃, 30s at 50 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃ for 1 cycle.
Adjusting the sample loading amount of cDNA of the internal reference according to the PCR system of the gel strip brightness adjusting target gene of the internal reference, and then determining the light and shade degree of the running strip of the target gene by using the same PCR system under the condition of the strip with the consistent brightness of the internal reference strip. The results are shown in fig. 2 (in the case of consistent reference brightness, the target gene infection is obviously lighter than that without any treatment, mechanical needling and no-load infection), and the expression level of the PDS gene is judged to be down-regulated according to the brightness.
Example 6 chlorophyll content determination in PDS Gene-silenced leaves
Preparing a mixed solution: acetone, absolute ethyl alcohol and distilled water are mixed according to the proportion of 4.5: 4.5: 1 to prepare a mixed solution for chlorophyll extraction.
Taking 0.1g of fir leaf, and cutting into 0.5-1cm2The fragments were then placed in a 15ml centrifuge tube, 10ml of the mixture was added, then coated with tinfoil paper and extracted directly for three days in the dark, when the leaf tissue became completely white, indicating complete extraction.
Collecting 3ml chlorophyll leaching solution, placing into 10mm cuvette, using the mixed solution as control, measuring optical density values of the leaching solution at 645nm and 663nm with Thermo Fisher Multiskan FC enzyme-linked immunosorbent assay (ELISA) reader, and calculating chlorophyll concentration according to Arnon formula
Chlorophyll a concentration (mg/g) = (12.7A 663-2.69A 645) (V/W)
Chlorophyll b concentration (mg/g) = (22.9A 645-4.68A 663) × (V/W)
Total chlorophyll concentration (mg/g) = chlorophyll a concentration + chlorophyll b concentration
V is the total volume of the leaching liquor and is expressed in L; w is the fresh weight of the leaves in g
From the results of chlorophyll content measurement, fig. 3 shows that compared with the untreated control group and the empty vector control group, the chlorophyll a and the chlorophyll b of the target gene fragment infected treated plant are both obviously reduced, and the empty vector infected and untreated phenotype is relatively unchanged.
According to the experimental verification, the expression level of phytoene desaturase gene silenced by VIGS gene is obviously reduced.
The establishment of a rapid and reliable transformation system is particularly important for woody plants, many of which are difficult to transform. The established fir virus induced gene silencing system can be used for quickly and efficiently researching the target gene of the fir with pertinence without carrying out traditional tissue culture regeneration genetic transformation on the fir.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> cunninghamia lanceolata virus induced gene silencing system and construction method thereof
<130> 8
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 3086
<212> DNA
<213> Artificial sequence
<400> 1
agccaaatcg gagatgccag ccgctcctga tacagatgcg ataagataaa acaacacaat 60
ttacacagag acgacgcatc gcttcttcga atcttacgcg ttgcccagga aatatagtta 120
acataaccaa aaacaaaatg aattgaagct tgacattttg aatgattctt cactgttagc 180
ggcttccgat tccgttaaaa ttgtttgaag attctggtgg aatacccggg ggccgaatta 240
tttagagttt aagtccatgc ttgtgctgta acaactcctc tgctccatgt tctgtgataa 300
tttctcttga aattagtagc ggtaggagga gtaggagcat gtggctttac attttttagc 360
agagtattta agcacggata tcagtgttag cttttggcag atcggaggag aaatgcaagg 420
ggttggcttt tcatcgattt caaagtcccg gagtggaaca ttgtgtgcgt cagacttcaa 480
atgcggcagt caagaggcat ttgggatcgg gaggagtgat tatttgggcc gagctgttcg 540
tcttgcttgt ttcaggaaaa gaaataacgc cggtagaaaa tttggagctc ctttgcaggt 600
tacatgcaag gactacccaa gaccagatat tgacaataca atcaattttc tggaagcttc 660
tgccttatct gcttcgttcg gaagtgcacc acgtcctaat agaccattag aggttgtgat 720
agcagggtca ggtttggctg gcttgtcaac tgcaaaatat ttggctgatg ctggtcacaa 780
acctatacta ttggaagcaa gagatgttct tggtggaaag gtagcagcat ggaaggatga 840
cgagggagac tggtacgaga caggcttaca tatcttcttc ggagcttatc ccaatgttca 900
aaacttgttt ggagagcttg ggatcaatga tagactgcag tggaaggagc attctatgat 960
ctttgctatg ccaaacaaac ctggacagta tagccgtttt gattttccgg aagttcttcc 1020
agcgcctttg aatggaattt gggcgatcct aaagaacaat gagatgttga cgtggccaga 1080
aaaaatacaa tttgcaattg gacttttacc agcaattgct ggaggtcaag cttatgttgt 1140
agcccaagat ggcttaactg tcaaagaatg gatgcgtaag cagggtgtac ctgatcgagt 1200
cagtgatgaa gtatttgtag ctatgtcgaa agctttaaac tttattaatc ctgaggagct 1260
ttcaatgcag tgtattctta tagccttgaa tcgttttctt caggaaaagc acgggtcaaa 1320
gatggccttt ctagatggaa atccgccgga gaggctatgc aagccaattg ttgatcatgt 1380
ccagtcacta ggtggtgagg ttcggataaa ttctcgatta caaaagattg agctaaatca 1440
tgatggcaca gtgaagcact ttgttctggc caatggtaac attgttgaag gagatgttta 1500
tgtatcagcc atgcctgttg acatcctcaa gctactttta cctgaagagt ggaaggaaga 1560
ttcctacttc aacaaactgg aaaagctagt aggagttcca gtaatcaaca ttcacatttg 1620
gtttgatcga aaactgaaaa acacatacga ccatttacta ttcagcagga gctctctcct 1680
tagtgtctat gcagatatgt ctgttacttg caaggaatac tatgatccaa atcgatcaat 1740
gctagagctt gtatttgcac ctgcagaaga atggattgct cgcagtgata ctgacattct 1800
tgaggctact cttaaggaac tttcaaaact gtttcctgat gaaatagcaa tagacggaag 1860
caaagcaaag gttttgaagt atcacgttgt taaaacccca cggtctgtct acaagacaat 1920
acccaattgt gagccttgtc gtcctttaca gaggtcaccc ataaaaggat tttatttggc 1980
tggtgattac accaaacaaa aatatctagc atccatggag ggtgccgtgc tatcagggaa 2040
gctttgtgca caggctattt tggaggatta tgacttgctt gtagctcgtg ctggacagaa 2100
tgaaagagtt ggagcagctt tgtattaata ccaccaaaat ccatatacag attcaagatt 2160
ggattgatag agaagcattt atatgtgatt gaaaggctac gtatcctaat gaggaacttt 2220
tcaagggcat tgtaattacc atgtagttcc aaaggaagca aggaaaactt atggttaaga 2280
ctttgagatc ctcttatttt tgtttatgta ttatatgcct tttaagcatt catgtcacaa 2340
aatagatgag aactttgtcc aggaattgaa ctagcagaag gcgcattctg ttaggaaaga 2400
ctcaagaact tcaataaatt cagaatcata aaattcggtg gctagtattt ggaaagaggt 2460
ctgaattatt ggaaagaaac attctaaggg atctaaatat ttccatggtc ttgacgagaa 2520
ctatgctggt atacgttctt gatatttgtg ttgcaccttc ttagatatga aaattggagc 2580
atttataaat cctatagtgg gtgattgaca agatactagt atttgcaggt tttaccagga 2640
tatttctcct tgataacata gtatgtaatc tcattttcat ttgactaagc tctctctcac 2700
ctattctcga acatcattat ggtccaagtt ccatacaatg gtggcaatca cattactggt 2760
aataactcta atgataatgg tggtggtagt ggttcttaat aataataaca atcatcgtca 2820
tcatcattgg taggatggtg gactttgctc aggctggatg cctgtttact cttatttgga 2880
aagtgtaaag tgttaatgcc tcttgaggag ggccatgcct ttggcaatct acaatggcca 2940
tttcccacgt gaagacatct cattgtggat ggatgtttaa aaccttgcag gtatgcatct 3000
tgatatgtct atttaatatc attacaaata caaaaaaacg gtgtatataa aatgtaaatt 3060
agaagaatgt tacattaatg ttttcc 3086
<210> 2
<211> 300
<212> DNA
<213> Artificial sequence
<400> 2
taccaccaaa atccatatac agattcaaga ttggattgat agagaagcat ttatatgtga 60
ttgaaaggct acgtatccta atgaggaact tttcaagggc attgtaatta ccatgtagtt 120
ccaaaggaag caaggaaaac ttatggttaa gactttgaga tcctcttatt tttgtttatg 180
tattatatgc cttttaagca ttcatgtcac aaaatagatg agaactttgt ccaggaattg 240
aactagcaga aggcgcattc tgttaggaaa gactcaagaa cttcaataaa ttcagaatca 300
<210> 3
<211> 51
<212> DNA
<213> Artificial sequence
<400> 3
ggggacaagt ttgtacaaaa aagcaggctt ctaccaccaa aatccatata c 51
<210> 4
<211> 50
<212> DNA
<213> Artificial sequence
<400> 4
ggggaccact ttgtacaaga aagctgggtc tgattctgaa tttattgaag 50
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
atgcaagggg ttggcttttc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
ttaatacaaa gctgctccaa 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
gtgagacggt gaccattgtg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
tcttggccct gtaccagttc 20
Claims (6)
1. A fir virus-induced gene silencing system is characterized in that: infecting Chinese fir by agrobacterium-mediated recombinant virus plasmid carrying target gene segment;
the method comprises the following steps:
(1) culturing a fir plant material: planting under a greenhouse condition, selecting seeds with plump seeds and uniform texture, performing broadcast sowing treatment and culturing for 30-60 days, opening seed pods, and completely extending seed leaves for 2-3 cm;
(2) cloning of the target gene fragment: selecting a target gene of the fir, taking a nucleotide sequence of 300bp to 350bp of the target gene close to a 3' end UTR region as a target gene fragment, designing forward and reverse primers of the target gene fragment, taking genome DNA of the fir as a template, and carrying out high-fidelity enzyme PCR amplification to obtain the target gene fragment;
(3) construction of recombinant viral plasmids: connecting the virus vector pTRV2 with the obtained target gene fragment to obtain a recombinant virus plasmid connected with pTRV2 and the target gene fragment, and then transforming the recombinant virus plasmid into agrobacterium;
(4) infecting the fir plants by agrobacterium-mediated recombinant virus plasmids: uniformly mixing agrobacterium liquid containing pTRV1 and agrobacterium liquid containing recombinant virus plasmids according to the volume ratio of 1:1, and then infecting the fir by using a 1mL syringe by adopting an injection method;
(5) and (3) verifying the silencing effect result: gradually expressing phenotypes of infected Chinese fir plants in 60-70 days, observing the phenotype changes of the plants, performing gene expression analysis by using semi-quantitative RT-PCR, observing the change condition of the expression quantity of target genes, and measuring the chlorophyll content changes of the Chinese fir plants by using an enzyme-labeling instrument;
cloning of the target gene fragment: the target gene is a fir PDS gene, and the gene sequence is shown as SEQ ID NO 1; the sequence of the target gene fragment is shown as SEQ ID NO. 2; the forward primer sequence of the target gene segment is shown as SEQ ID NO. 3, and the reverse primer sequence is shown as SEQ ID NO. 4.
2. The system of claim 1, wherein the recombinant viral plasmid vector is constructed by a Gateway method, wherein the entry vector is pDONR207, and the final vector is pTRV2 viral vector 279; the agrobacterium strain used for transforming the recombinant virus plasmid into agrobacterium is GV 3101.
3. The system of claim 1, wherein the agrobacterium-mediated recombinant viral plasmid infects a fir plant, comprising the steps of:
1) transferring l mL of Agrobacterium strain solution containing recombinant virus plasmid into 6 mL of LB culture solution containing rifampicin with final concentration of 50mg/mL and kanamycin with final concentration of 50mg/m L, and shake-culturing at 28 ℃ and 220rpm to make OD600Is 0.8;
2) transferring l mL of the bacterial liquid obtained in the step (1) into l.5 mL of a centrifugal tube for bacteria preservation, centrifuging the residual bacterial liquid at 3800 rpm for 8 min, and discarding the supernatant; by suspension of suspensionsFloating and precipitating, and then adjusting the concentration of the bacterial liquid to OD by suspension600 = 0.6；
3) The agrobacterium liquid containing pTRV1 and the agrobacterium liquid containing pTRV2 and recombinant virus plasmid are mixed according to the ratio of 1:1, standing at room temperature for 4h, and infecting a fir plant, wherein a mixed bacterium containing pTRVI and pTRV2 agrobacterium is used as an empty vector control without recombinant virus plasmids;
4) sucking the bacterial liquid by using a syringe needle-free syringe of lmL, slowly injecting the cedar cotyledon, and carrying out dark culture at 16 ℃ for 16h after inoculation on the premise that the leaf surface is filled with the infection liquid; taking out, standing at 25 deg.C, culturing under light for 16h, and culturing in dark for 8 h.
4. The system of claim 3, wherein the suspension comprises: 10mM MES, 10mM MgCl2，200 μM AS。
5. The fir virus-induced gene silencing system according to claim 1, wherein the semi-quantitative RT-PCR is used for gene expression analysis, and actin gene is used as an internal reference for the semi-quantitative analysis to detect the expression level of the full length of the PDS gene; the PDS gene full-length forward primer is shown as sequence SEQ ID NO. 5, and the reverse primer is shown as sequence SEQ ID NO. 6; the forward primer of the actin reference gene is shown as a sequence SEQ ID NO. 7, and the reverse primer is shown as a sequence SEQ ID NO. 8.
6. The system of claim 1, wherein the mixture for chlorophyll extraction is: the acetone, the absolute ethyl alcohol and the distilled water are prepared according to the proportion of 4.5: 4.5: 1.
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| CN108624617A (en) * | 2018-03-29 | 2018-10-09 | 江苏省农业科学院 | A method of improving eggplant Gene Silencing efficiency |
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