CN114990155A - Method for establishing virus-induced gene silencing system at rabdosia lophanthide flower part - Google Patents
Method for establishing virus-induced gene silencing system at rabdosia lophanthide flower part Download PDFInfo
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Abstract
The invention discloses a method for establishing a virus-induced gene silencing system on the flower part of rabdosia lophanthide of iris, relating to the field of genetic engineering for improving flower color and flower type of iris, wherein the optimal inoculation part is an uncolored bud period, an infection solution needs to be prepared on site, and the thallus concentration of agrobacterium carrying a virus vector is increased to OD in the preparation of the infection solution 600 To 1.8-2.0. The agrobacterium carrying the virus vector in the inoculation area under natural conditions is ensured to have enough survival base number, the infection efficiency of the whole experiment is improved, and no light is needed after inoculation. The method is efficient and easy to operate, solves the problem of researching the gene function of the rabdosia lophanthide without a genetic transformation system, and is an effective means for analyzing the gene molecule regulation and control mechanism of the rabdosia lophanthide through rabdosia lophanthide body plants.
Description
Technical Field
The invention relates to the field of gene engineering for improving the floral character of rabdosia lophanthide, in particular to a method for establishing a virus-induced gene silencing system in the floral part of rabdosia lophanthide plants.
Background
Rabdosia Lophanthoides (Iris sanguinea) is a perennial herb of Iris genus of Iridaceae family. The strong cold resistance and high ornamental value of the plant are always considered to be an important landscaping and cut-flower plant to be developed urgently in northeast, and the plant is widely applied as an ornamental plant in many places. Although a series of achievements are obtained in the breeding of rabdosia lophanthide, because the establishment of a monocotyledon genetic transformation system such as rabdosia lophanthide is very difficult, and the verification of the flower color gene function can only be realized by heterologous plants such as tobacco and petunia, the research on the flower color regulation and control mechanism is relatively slow, and the breeding of special flower colors is difficult to break through.
Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing phenomenon discovered in recent years and can cause sequence-specific degradation of endogenous mRNA. VIGS mediated by Tobacco rattle virus (Tobacco rate virus) has become the virus vector with the widest application range at present due to wide host range, convenient construction and obvious effect, has been used as a novel high-efficiency reverse genetics technology for researching flower color gene functions of a plurality of plants, including butterfly orchid, petunia, China rose, tomato and the like, is very suitable for carrying out gene function analysis on species lacking a stable genetic transformation system, and has been successfully used in key gene function research in the growth and development process of a plurality of plants. At present, no research on establishing a virus-induced gene silencing system by taking perennial field iris as an experimental object has been reported, and a technology system for establishing rablodes gene function research by using the VIGS technology is probably an effective means for analyzing a gene molecule regulation and control mechanism by rablodes bulk plants.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for establishing a flower virus induced gene silencing system by taking rabdosia lophanthide plants as experimental subjects under field conditions, and the method is simple, efficient and easy to operate outdoors.
The technical scheme adopted by the invention for solving the technical problems is as follows:
(1) determining the inoculation period: the rabdosia lophanthide is in the bud uncolored period, and the inoculation time is 3 pm in the clear and windless environment;
(2) preparing a staining solution: the infection liquid is prepared at present, and the preparation method comprises the following steps:
firstly, selecting agrobacterium positive clones with corresponding virus plasmids in 1mL YEB liquid culture medium containing 50 mug/mL kanamycin and 20 mug/mL rifampicin, and shaking for 6-8h at 200r/min in a glass test tube with a rubber plug at 28 ℃ until the agrobacterium positive clones are completely white;
secondly, the turbid bacteria liquid is cultured by 50mL YEB liquid culture medium containing 50 ug/mL kanamycin and 20 ug/mL rifampicin, and the culture medium is shaken under 200r/min conditions, and the concentration of the bacteria liquid reaches OD 600 Taking out when the temperature reaches 1.8-2.0;
then, the cells were collected by centrifugation, and suspended in 25mL of an infection buffer containing 10mM MES and 200. mu. M, MgCl mM AS 200 in sterile water AS a solvent 2 10mM, adjusting the concentration of the bacterial liquid of the agrobacterium liquid to OD 600 To 1.8-2.0, preparing Agrobacterium infection buffer solution, buffer solution OD 600 Preferably 0.8-1.0;
finally, respectively mixing the agrobacterium infection buffer solution containing the pTRV1 with equal concentration with the agrobacterium infection buffer solution containing pTRV2 and pTRV 2-target gene fragments in equal volume, and standing at room temperature under dark conditions to form two kinds of agrobacterium infection solutions;
(3) selection of vaccination site: selecting buds of rabdosia lophanthide in an uncolored period as an inoculation part;
(4) inoculation of viral vectors: a 1mL injector is adopted, a wound is pricked from the top of a bud, then 1mL infection liquid is injected into the bud of the rabdosia lophanthide by using a needle, the bud is ensured to be upright, the bacterial liquid is not left, the light is not needed to be shielded after inoculation, and the treatment group and the treatment time are marked by a label;
(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.
In the infection liquid preparation process in the step (2), the agrobacterium is placed in a 250mL conical flask by 50mL of YEB liquid culture medium, and is vibrated at 200r/min, and when the volume of the liquid bacteria is smaller than the bacteria shaking container 1/5, the bacteria shaking and infection effects are optimal.
And (4) selecting a period in the step (3) as a flower bud period of the rabdosia lophanthide, wherein the rabdosia lophanthide flower bud is not colored. The length is 2.5-3cm, the buds are complete, the flower pavilions are upright, and the flower pavilions can bear bacteria liquid. If the injected buds are laid down, the support device can be used for keeping the upright state of the pavilion and the buds and keeping the invasion liquid and the buds in a fully contact state.
And (4) during virus inoculation in the step (4), the amount of the infection liquid is kept at 1mL, and the buds are pricked from top to bottom by using a sterile syringe with a needle head, so that the buds are guaranteed to have wounds and can be fully contacted with the infection liquid.
When the virus is inoculated in the step (4), the same amount of no-load and target gene treatment is carried out in the same plant.
The phenotype observation of all group samples in the step (5) is specifically performed by observing and comparing the related characters of the gene silencing treated region between different groups before and after the date of occurrence of character change predicted according to the function of the silencing target gene from the first day of inoculation, and counting the corresponding number.
Because the environmental influence factors are more under field conditions, the rabdosia lophanthide has stronger resistance, and the bacteria liquid with low concentration does not reach the corresponding infection effect, the concentration of the agrobacterium is increased to OD 600 To 1.8-2.0, the amount of Agrobacterium carrying the viral vector was sufficient and the resuspension concentration was adjusted to OD 600 0.8-1.0, not only increasing the quantity of bacteria liquid carriers, but also ensuring the infection success rate, observing the plant state after the infection experiment is finished, and needing no bagging and coveringThe light can be properly covered in rainy days.
Drawings
FIG. 1 is a schematic representation of the operation of virus-induced gene silencing;
FIG. 2 is an electrophoretogram of recombinant plasmid fragments detected by PCR in IsANS gene silencing Raynaldia dictyophora obtained by VIGS technology;
FIG. 3 is a table diagram of IsANS gene silencing rabdosia lophanthide petals obtained by rabdosia lophanthide via VIGS technology;
FIG. 4 is a graph showing the results of the detection of the variation of the expression level of IsANS gene in the blank control, the empty load control and the IsANS gene silencing rabdosia pallidiflora petals.
Detailed Description
The invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
A method for establishing a virus-induced gene silencing system on a plant of Iris of the field adopts Anthocyanin synthetase (ANS) as a reporter gene, constructs a VIGS system of the flower part of rabdosia lophanthide based on TRV, and is realized by the following steps:
determining the inoculation period: the inoculation time is 3 pm later from 5 to 6 pm.
Connecting the amplified gene specificity fragment of the dictyophora rivieri anthocyanin synthetase (IsANS) to a TRV virus induced gene silencing vector p TRV2 by using a genetic engineering method to obtain p TRV-IsANS;
the method comprises the following specific steps:
extracting total RNA of rabdosia lophanthide petals according to the RNA extraction kit operation instruction of Kangji century, synthesizing a first strand of cDNA (complementary deoxyribonucleic acid) by taking 1 mu g of total RNA as a template according to the TaKaRa cDNA synthesis kit operation instruction, and then diluting by 10 times for later use;
amplification of IsANS gene: an upstream primer 5'-ATGGCAAGCAGCAAGGTCATGCC-3' and a downstream primer 5'-CTAGTTCTGGATGGCTTCGG-3', the first chain of cDNA of the rabdosia lophanthide petals is taken as a template, and a 50 mu L system of KOD PCR Kits is used for amplification; the amplification procedure was pre-denaturation at 95 ℃ for 5 min; 35 cycles of denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 45s, and extension at 72 ℃ for 1min for 30 s; extending for 10min at 72 deg.C, and storing at 4 deg.C; detecting the PCR product as a single band by electrophoresis, and purifying for later use; the PCR amplified product of IsANS is a gene fragment of 1221 bp. And (3) obtaining a PCR product by agarose gel electrophoresis and gel cutting recovery, connecting a 15min Blunt-Zero (all-type gold) vector, transferring the connected product into escherichia coli DH5 alpha, screening and picking single clone on a resistant culture medium, carrying out PCR identification on positive clone by using the vector with a universal primer M13, and sequencing to confirm the correctness of the PCR product.
Obtaining IsANS gene conserved region sequence by sequence comparison, wherein the method comprises the steps of designing conserved region sequence primers by using NCBI website and SGN VIGS Tool website, the product length is 200-300bp optimally, adding homologous arm base sequences of pTRV2 vector at the 5 'ends of forward primer and reverse primer respectively, finally selecting the sequence of 1-242bp as the conserved region sequence of IsANS, and using upstream primer 5' -TGAGTAAGGTTACCGAATTCTCTAGAATGGCAAGCAGCAAGGTCATGCC-3' and downstream primer 5-GCCTCGAG ACGCGTGAGCTCGGTACCGCCTCCTTCACTTCCTCCAC-3’。
Construction of pTRV-IsANS plasmid: the purified PCR gene fragment was ligated with XbaI-Knpi double-digested linearized pTRV2 plasmid using Novonopathy homologous recombination Kit Clonexpress Ultra One Step Cloning Kit (C115-01) ligase.
And transforming the ligation product into escherichia coli DH5 alpha competence, screening and selecting a single clone on a resistance culture medium, designing detection primers pTRV2-F and pTRV2-R according to sequences on two sides of the restriction enzyme site of the pTRV2 vector, carrying out bacteria liquid PCR, identifying positive clone and sequencing.
The positive clone with correct sequence is selected to extract the plasmid, the agrobacterium GV3101 is transformed by a freeze-thaw method, the monoclonal is selected after two days of dark culture at 28 ℃, and the PCR detection of bacterial liquid is carried out by using primers pTRV2-F and p TRV 2-R.
Separately adding 50. mu.L of Agrobacterium solution containing pTRV1, pTRV2 and pTRV2-IsANS into 1mL of glass test tube with rubber plug containing 50mg/L kanamycin and 25mg/L rifampicin YEB culture solution, shaking at 28 deg.C and 200r/min for 6-8h, shaking to turbid state without whitening; then transferring the bacterial liquid into a 250mL conical flask containing 50mL YEB liquid culture medium containing 50mg/L kanamycin, 25mg/L rifampicin, 10mM MES and 20 mu M AS, shaking at 28 ℃ and 200 r/min;
OD of bacterial liquid 600 When the concentration was about 1.8 to 2.0, the cells were collected by centrifugation at 6000rpm at 25 ℃ for 3 minutes. With a solution containing 10mM MgCl 2 10mM MES, 200. mu.M AS 200mL of the infection solution resuspended in the bacterial cell, and the resuspended OD 600 About 0.8 to about 1.0.
Respectively mixing the following bacterial liquid components in each group according to the volume ratio of 1: 1, standing and activating for 3 hours at 25 ℃ in the dark, and injecting the agrobacterium GV3101 bacterial liquid containing p TRV1 and p TRV2 and the agrobacterium GV3101 bacterial liquid containing p TRV1 and p TRV2-IsANS into a stream fungus bud period with uniform and uncolored growth, wherein the length of the bud is 2.5-3cm, the bud is complete and the flower pavilion is upright, and can bear the bacterial liquid by using a 1mL injector. If the injected buds are laid down, the support device can be used for keeping the straight state of the buds and the buds, so that the invasion liquid and the buds are kept in full contact, and the amount of the injected bacteria liquid is 1 mL. (attached FIG. 1)
The buds subjected to gene silencing treatment bloom in succession 10 to 14 days after the first day of inoculation, and the character is predicted to change to lighter flower color according to the function of the silenced gene IsANS. Compared with the blank control group, the samples treated by the virus empty vector have no flowers with obviously lightened flower color, and compared with the blank control group and the virus empty vector group, the samples treated by the virus and the target gene composite vector have lightened flower color. (FIG. 2)
Taking 100mg of each of the control and infected plant leaves to extract total RNA, carrying out reverse transcription to obtain cDNA, and using TRV1 and TRV2 vector detection primers to determine whether the recombinant plasmid enters the plant. The detection result is shown in figure 3, the TRV2 vector sequence is not amplified in the control sample, the TRV2 vector sequence is only in the unloaded sample, the length is about 281bp, and the size of the amplified band in the sample of the silent IsANS plant is about 500bp, so that the recombinant plasmid successfully enters the plant body.
Taking blank control, no-load control and 100mg of rabbitia venosa IsANS silent plant petals after 14 days to extract total RNA, carrying out reverse transcription to obtain cDNA, and carrying out expression quantity detection by using a fluorescent quantitative PCR instrument, wherein a reaction system is detailed in the specification of the Kangji Ultra SYBR Mixture (CW 0958M). Specific primers used for amplification of IsANS were IsANS-qpcr-F: GGTGGTGGTGACGAAGATGCTATC, IsANS-qpcr-R is: CGCCGTTGTGGAGTATGAAGGAG, the internal reference gene is: and (3) CGGTATGGAGGCTGCTGGTA is the Actin-7-qpcr-F, GTGCTCAATGAAGCGAGGATAG is the Actin-7-qpcr-R.
By use of 2 -ΔΔCT The relative expression level of the gene is calculated.
The results are shown in figure 4, and the expression level of the IsANS gene in petals of a silent plant is found to be significantly lower than that of a blank control and no load. Indicating successful silencing of IsANS.
The above description is only a preferred embodiment of the present invention, and any person skilled in the art can modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent replacements made according to the technical solution of the present invention belong to the protection scope of the present invention.
Claims (6)
1. A method for establishing virus-induced gene silencing system on flower part of Dictyophora rivularis of Iris comprises infecting part of unpigmented Dictyophora rivularis bud period of 2.5-3cm in length with complete bud and erect scape.
2. The target gene is connected with pTRV2 virus vector, and is respectively introduced into agrobacterium with pTRV1 plasmid, and then the two kinds of agrobacterium are mixed according to the volume ratio of 1: 1 for infection.
3. The method of claim 2, wherein the invader is prepared AS is, and sterile water is used AS a solvent, wherein the solvent comprises MES 10mM and AS 200. mu. M, MgCl 2 10mM, adjusting the concentration of the agrobacterium liquid to OD 600 When the concentration reaches 1.8-2.0, preparing the agrobacterium infection buffer solution.
4. The method of claim 3, wherein the OD of the infection buffer is adjusted 600 Is 0.8-1.0.
5. A wound is pricked into the top of the bud from top to bottom by using a sterile syringe with a needle, and 1mL of staining solution is injected into the bud of the rabdosia lophanthide, so that the bud is ensured to be upright, the bacterial solution cannot be left, and the light is not required to be cut after inoculation.
6. The method according to claim 2, wherein the Agrobacterium is GV 3101.
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