CN116064646A - VIGS silencing vector of jujube ZjCLA gene and application thereof - Google Patents
VIGS silencing vector of jujube ZjCLA gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of plant genetic engineering, in particular to a VIGS silencing vector of a jujube ZjCLA gene and application thereof. The invention provides application of a specific fragment in constructing a VIGS silencing vector of a jujube ZjCLA gene, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1. The VIGS silencing vector constructed by the specific fragment provided by the invention can successfully infect jujube seedlings, induce the endogenous ZjCLA of the jujube to silence, effectively reduce the expression level of the ZjCLA gene of the jujube, obtain the virus-induced gene silencing character, verify the function of the ZjCLA gene of the jujube, and has application value in the research of functional genes of the jujube. In addition, the invention also establishes a high-efficiency date VIGS system, and provides technical support for date functional genomics research.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a VIGS silencing vector of a jujube ZjCLA gene and application thereof.
Background
Virus-induced Gene silencing (VIGS) technology is to insert a target gene fragment into the genome of a Virus, inhibit the expression of an endogenous target gene homologous to the inserted fragment after infection of plant tissues and express a corresponding trait deletion, so as to rapidly verify the function of the target gene. The VIGS system has the advantages of simple operation method, short periodicity, capability of avoiding plant transformation and the like, becomes an important reverse genetics tool and is widely applied to research on plant gene function identification.
Jujube is a plant of Rhamnaleae jujube genus (Ziziphus) and is native to China, and has multiple benefits of economy, ecology, society and the like due to the drought resistance and barren resistance of the jujube. In recent years, molecular biology and genetic engineering research on important economic traits of jujube are gradually in progress, and more functional genes are excavated and identified. As perennial woody plants, reports of transferring exogenous genes into organs such as stem tips, leaves and hypocotyls of jujubes and obtaining transgenic plants by using an agrobacterium tumefaciens mediated method exist, but the exogenous genes cannot be widely applied due to long time consumption, low transformation efficiency, unstable technology and the like, so that the gene function verification of the jujubes is difficult, and a set of rapid and efficient gene function verification system is urgently needed to be established.
Disclosure of Invention
In order to solve the problems, the invention provides a VIGS silencing vector of a jujube ZjCLA gene and application thereof. The VIGS silencing vector constructed by the specific fragment provided by the invention can successfully infect jujube seedlings, establishes a jujube high-efficiency VIGS system and provides technical support for jujube functional genomics research.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a specific fragment in constructing a VIGS silencing vector of a jujube ZjCLA gene, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1.
The invention provides a VIGS silencing vector of jujube ZjCLA genes, which comprises a specific fragment and a pTRV2 vector; the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO. 1.
Preferably, the specific fragment is located between EcoR I and Xho I cleavage sites of pTRV 2.
The invention provides a primer pair for amplifying a specific fragment, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the primer pair comprises an upstream primer CLA-EcoRI-F and a downstream primer CLA-XhoI-R;
the nucleotide sequence of the upstream primer CLA-EcoRI-F is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer CLA-XhoI-R is shown in SEQ ID NO. 3.
The invention provides a VIGS silencing system of jujube ZjCLA genes, which comprises the following components: an agrobacterium solution containing a pTRV1 vector and an agrobacterium solution containing a VIGS silencing vector; the VIGS silencing vector comprises the VIGS silencing vector according to the technical scheme;
the volume ratio of the agrobacterium liquid containing the pTRV1 vector to the agrobacterium liquid containing the VIGS silencing vector is 1:1;
the OD values of the agrobacterium liquid containing the pTRV1 vector and the agrobacterium liquid containing the VIGS silencing vector are respectively 1.0-1.5.
The invention provides the application of the VIGS silencing vector or the VIGS silencing system in the technical scheme in silencing jujube ZjCLA genes, wherein the jujube ZjCLA genes comprise genes with sequence numbers of XM_ 048477782.1.
The invention provides a method for silencing jujube ZjCLA gene, which comprises the following steps:
injecting the VIGS silencing system in the technical scheme to the back of the leaf blade of the jujube seedling to obtain the infected jujube seedling.
Preferably, the jujube seedlings comprise seedlings with fully developed cotyledons.
Preferably, obtaining infected jujube seedlings further comprises dark treatment of the infected jujube seedlings in a culture room at 24 ℃ for 24 hours.
The invention provides the application of the VIGS silencing vector or the VIGS silencing system in the technical scheme in the identification of the function of the jujube ZjCLA gene, wherein the jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1.
The beneficial effects are that:
the invention provides application of a specific fragment in constructing a VIGS silencing vector of a jujube ZjCLA gene, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1. The VIGS silencing vector constructed by the specific fragment provided by the invention can successfully infect jujube seedlings, induce the endogenous ZjCLA of the jujube to silence, effectively reduce the expression level of the ZjCLA gene of the jujube, obtain the virus-induced gene silencing character, verify the function of the ZjCLA gene of the jujube, and has application value in the research of functional genes of the jujube.
In addition, the invention also establishes a high-efficiency date VIGS system, and provides technical support for date functional genomics research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a diagram of PCR amplification of CLA-specific fragments for VIGS silencing, with lanes representing amplified CLA-specific fragments at different annealing temperatures, in which the annealing temperatures are 65 ℃,64.5 ℃,63.9 ℃,62.8 ℃,61.6 ℃,60.5 ℃,59.8 ℃,58.4 ℃,57.1 ℃,55.8 ℃,55.4 ℃ in order from left to right;
FIG. 2 is a graph showing leaf albino symptoms occurring after infection of wild jujube seedlings by a silencing vector;
FIG. 3 shows the detection of ZjCLA expression using real-time fluorescent quantitative PCR (qRT-PCR).
Detailed Description
The invention provides application of a specific fragment in constructing a VIGS silencing vector of a jujube ZjCLA gene, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the specific fragment comprises the following specific sequences: GAGCAGATCTATTGGGCTTAGCTCGATTCAAGTTTAATCAGGTCAGGAAAAGGCCATGTGGGGTTTTTGCATCACTTTCAGAGATGGGGGAGTATCATTCACAGAGACCACCAACTCCTCTCTTGGATACCATAAACTATCCAATTCACATGAAAAATCTATCTGTCAAGGAGCTTAAACAGCTATCGGATGAACTAAGGTCTGATGTCAT.
The jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1.
The ZjCLA gene VIGS silencing vector is constructed by taking the ZjCLA gene specific fragment as a target fragment, so that the endogenous ZjCLA of the jujube can be induced to silence, the expression level of the ZjCLA gene of the jujube is effectively reduced, the virus-induced gene silencing character is obtained, the function of the ZjCLA gene of the jujube can be verified, and the method has application value in the study of the functional gene of the jujube.
The invention also provides a VIGS silencing vector of the jujube ZjCLA gene, which comprises a specific fragment and a pTRV2 vector; the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO. 1.
In the present invention, the specific fragment is preferably located between EcoR I and Xho I cleavage sites of pTRV 2.
The invention also provides a primer pair for amplifying the specific fragment, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the primer pair comprises an upstream primer CLA-EcoRI-F and a downstream primer CLA-XhoI-R;
the nucleotide sequence of the upstream primer CLA-EcoRI-F is shown as SEQ ID NO.2, and specifically comprises the following steps: TGTGAGTAAGGTTACCGAATTCGAGCAGATCTATTGGGCTTAGC;
the nucleotide sequence of the downstream primer CLA-XhoI-R is shown as SEQ ID NO.3, and specifically comprises the following steps: GGGACATGCCCGGGCCTCGAGATGACATCAGACCTTAGTTCATCC.
The primer pair provided by the invention can specifically amplify the specific fragment, and two ends of the amplified specific fragment contain EcoR I and Xho I cleavage sites.
The invention also provides a VIGS silencing system of the jujube ZjCLA gene, which comprises: an agrobacterium solution containing a pTRV1 vector and an agrobacterium solution containing a VIGS silencing vector; the VIGS silencing vector comprises a VIGS silencing vector as described in the above schemes;
the volume ratio of the agrobacterium liquid containing the pTRV1 vector to the agrobacterium liquid containing the VIGS silencing vector is 1:1;
the OD values of the agrobacterium liquid containing the pTRV1 vector and the agrobacterium liquid containing the VIGS silencing vector are respectively 1.0-1.5.
In the present invention, the agrobacterium preferably includes GV3101 agrobacterium.
In the present invention, the OD value of the Agrobacterium solution containing pTRV1 vector is 1.0 to 1.5, preferably 1.3 to 1.5, more preferably 1.5; the OD value of the agrobacterium tumefaciens solution containing the VIGS silencing vector is 1.0-1.5, preferably 1.3-1.5, and more preferably 1.5.
According to the invention, the obtained VIGS silencing system can successfully infect jujube seedlings by compounding the agrobacterium tumefaciens liquid containing the pTRV1 vector and the agrobacterium tumefaciens liquid containing the VIGS silencing vector in proper concentration and volume ratio, so that the expression level of the jujube ZjCLA gene is effectively reduced, and the simple, efficient and low-cost identification of the jujube ZjCLA gene function can be realized.
In the present invention, the preparation method of the VIGS silencing system preferably comprises: and (3) regulating the agrobacterium tumefaciens liquid containing the pTRV1 vector and the agrobacterium tumefaciens liquid containing the VIGS silencing vector to OD values of 1.0-1.5 respectively, standing at room temperature for 2-3 h, and mixing the agrobacterium tumefaciens liquid containing the pTRV1 vector and the agrobacterium tumefaciens liquid containing the VIGS silencing vector in equal volumes after standing at room temperature to obtain the VIGS silencing system. In the present invention, the room temperature is preferably 20 to 25 ℃, more preferably 23 to 25 ℃, and still more preferably 24 ℃.
In the present invention, the time for the standing is preferably 2 to 3 hours, more preferably 2.5 to 3 hours, and still more preferably 3 hours. The invention can improve the activity of the bacterial liquid by standing for a proper time, thereby improving the infection effect.
The invention also provides the application of the VIGS silencing vector or the VIGS silencing system in the technical scheme in silencing jujube ZjCLA genes, wherein the jujube ZjCLA genes comprise genes with sequence numbers of XM_ 048477782.1.
The invention also provides a method for silencing the ZjCLA gene of the jujube, which comprises the following steps:
injecting the VIGS silencing system in the technical scheme to the back of the leaf blade of the jujube seedling to obtain the infected jujube seedling.
In the present invention, the jujube seedlings preferably include seedlings with fully developed cotyledons, more preferably seedlings of wild jujube with fully developed cotyledons. The seedlings with fully developed cotyledons are preferably seedlings which are continuously cultivated for 7-10 days after seeds sprout to 1 cm.
In the invention, the method for obtaining the infected jujube seedlings preferably further comprises the step of carrying out dark treatment on the infected jujube seedlings in a culture room at 24 ℃ for 24 hours, so that the infection can be completed best, and the conversion efficiency is maximized.
According to the invention, a pTRV2 virus silencing expression vector plasmid containing a ZjCLA gene specificity fragment is transformed into agrobacterium, standing is carried out for 2-3 hours at room temperature, after the activity of bacterial liquid reaches the highest, the agrobacterium bacterial liquid containing pTRV1 and the agrobacterium bacterial liquid containing pTRV2-ZjCLA are mixed in equal volume, a silencing system is used for infecting wild jujube seedling leaves in a needle injection mode, the wild jujube endogenous ZjCLA is induced to silence, the expression level of the jujube ZjCLA gene is effectively reduced, the virus-induced gene silencing character is obtained, the function of the jujube ZjCLA gene can be verified, and the method has application value in the research of the functional gene of jujube.
The invention also provides application of the VIGS silencing vector or the VIGS silencing system in identifying the functions of the ZjCLA gene of the jujube, wherein the ZjCLA gene of the jujube comprises a gene with a sequence number of XM_ 048477782.1.
For further explanation of the present invention, the VIGS silencing vector of jujube ZjCLA gene and its application provided in the present invention will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A VIGS silencing vector of jujube ZjCLA gene is constructed by the following method:
RNA of the leaf tissue of the wild jujube is extracted by adopting a quick extraction kit (DP 452) for RNA of plant tissues of Tiangen biochemical technology (Beijing) limited company, and the first strand synthesis premix kit (KR 118) for removing the genome cDNA of Tiangen biochemical technology (Beijing) limited company by a one-step method is used for carrying out reverse transcription to obtain the cDNA of the wild jujube.
PCR amplification is carried out by taking the cDNA as a template and adopting an upstream primer CLA-EcoRI-F and a downstream primer CLA-XhoI-R, and a radix angelicae Biochemical technology (Beijing) limited company 2X TaqPCR premix reagent II (KT 211) is used for purification to obtain a jujube ZjCLA specific fragment gene, which is shown in figure 1; the nucleotide sequence of the upstream primer CLA-EcoRI-F is shown as SEQ ID NO. 2; the nucleotide sequence of the downstream primer CLA-XhoI-R is shown in SEQ ID NO. 3;
the PCR amplification reaction system comprises: cDNA 1. Mu. L, CLA-EcoRI-F1. Mu. L, CLA-XhoI-R1. Mu.L, 2 XTaqPCRMasterMixII 12.5. Mu.L and ddH 2 O 9.5μL；
The reaction process of the PCR amplification is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for 30s, 40 cycles total; reacting for 10min at 72 ℃; the annealing temperature is 65 ℃,64.5 ℃,63.9 ℃,62.8 ℃,61.6 ℃,60.5 ℃,59.8 ℃,58.4 ℃,57.1 ℃,55.8 ℃,55.4 ℃ and 55 ℃ respectively.
As can be seen from FIG. 1, zjCLA-specific bands can be amplified in the above temperature range.
Simultaneously carrying out double enzyme digestion on pTRV2 by using restriction enzymes EcoR I and Xho I produced by Thermo company, wherein the enzyme digestion system is 50 mu L, 20 mu L of water, 20 mu L of plasmid, 2 mu L of restriction enzyme EcoR I, 2 mu L of restriction enzyme Xho I and 6 mu L of 10X FastDigest Green Buffer are adopted, the enzyme digestion temperature is 37 ℃ and the time is 30min, and after enzyme digestion, agarose gel treatment is carried out on the reaction product, and the agarose gel is digested and recovered by using a common agarose gel DNA recovery kit produced by TIANGEN company, and the method is described in the specification of the kit;
connecting the ZjCLA specific fragment to the digested pTRV2 by using a pEASY seamless cloning kit produced by the full gold company, wherein the reaction system is 10 mu L, the connection temperature is 50 ℃, the connection time is 15min, and the method refers to the specification of the kit to obtain a connection product;
transferring 5 mu L of the ligation product into TIANGEN company to produce escherichia coli DH5 alpha, culturing in a solid LB culture medium added with kanamycin (Kan) for 24 hours, selecting a positive single colony, adding glycerol to preserve bacterial liquid after bacterial liquid PCR identification of the positive clone, and carrying out sequencing verification by Tianjin Optimaceae biotechnology Co., ltd, wherein the ligation product obtained after verification is the successful ligation of the VIGS silencing vector pTRV2-ZjCLA.
Example 2
A VIGS silencing system of the jujube ZjCLA gene, the VIGS silencing system consisting of: an agrobacterium solution 1 containing pTRV1 vector and an agrobacterium solution 2 containing VIGS silencing vector prepared in example 1;
the volume ratio of the agrobacterium liquid 1 to the agrobacterium liquid 2 is 1:1;
OD values of the agrobacterium liquid 1 and the agrobacterium liquid 2 are 1.5.
The VIGS silencing system is prepared by the following method:
respectively transferring pTRV1 plasmid and pTRV2-ZjCLA prepared in example 1 into GV3101 competent cells by adopting a freeze thawing method, respectively picking transformed positive agrobacterium colonies, performing PCR verification, and obtaining agrobacterium tumefaciens pTRV1 and agrobacterium tumefaciens pTRV2-ZjCLA after verification;
before carrying out VIGS infection for 3d, respectively streaking and activating agrobacterium tumefaciens pTRV1 and pTRV2-ZjCLA on LB solid media (Kan and Rif) containing 50mg/L kanamycin (Kan, sigma) and 50mg/L rifampicin (Rif, sigma), culturing for 2d at 28 ℃, respectively picking monoclonal colonies pTRV1 and pTRV2-ZjCLA to 2mL LB liquid media, and carrying out shaking culture at 28 ℃ for overnight;
small amounts of the shaken Agrobacterium solutions pTRV1 and pTRV2-ZjCLA were each inoculated into 100mL of LB liquid medium containing 100. Mu.L of rifampicin (50 mg/mL), 50. Mu.L of kanamycin (100 mg/mL), 5mL of MES (pH 5.7, 0.2M) and 20. Mu.L of acetosyringone (0.1M) and cultured overnight at 28 ℃.
The next morning the shaken pTRV1 and pTRV2-ZjCLA bacterial solutions were poured into 50mL centrifuge tubes, centrifuged at 6000rpm at room temperature for 10min, the supernatant discarded and the buffer suspension (preparation method: 1mL MgCl) 2 (1M)，5mL MES(pH5.7，0.2M) 200 mu L of acetosyringone (0.1M) are respectively suspended in the bacterial cells to 100mL, and OD is regulated 600 1.5, after vortex mixing, standing for 3 hours at room temperature to obtain pTRV1 agrobacterium tumefaciens heavy suspension and pTRV2-ZjCLA agrobacterium tumefaciens heavy suspension;
and (3) fully mixing the pTRV1 agrobacterium heavy suspension and the pTRV2-ZjCLA agrobacterium heavy suspension in equal volume to obtain the VIGS silencing system (namely the infectious microbe liquid).
Comparative example 1
An infectious microbe liquid is prepared by the following method:
transferring pTRV1 plasmid and pTRV2 plasmid into GV3101 competent cells respectively by adopting a freeze thawing method, respectively picking out positive agrobacterium colonies after transformation, performing PCR verification, and obtaining agrobacterium tumefaciens pTRV1 and agrobacterium tumefaciens pTRV2 after verification;
before carrying out VIGS infection for 3d, respectively streaking and activating agrobacterium strains pTRV1 and pTRV2 on LB solid media (Kan and Rif) containing 50mg/L kanamycin (Kan, sigma) and 50mg/L rifampicin (Rif, sigma), culturing for 2d at 28 ℃, and then respectively picking monoclonal colonies pTRV1 and pTRV2 to 2mL LB liquid media for small shaking, and shake culturing at 28 ℃ overnight;
small amounts of the shaken Agrobacterium solutions pTRV1 and pTRV2 were each inoculated into 100mL of LB liquid medium containing 100. Mu.L of rifampicin (50 mg/mL), 50. Mu.L of kanamycin (100 mg/mL), 5mL of MES (pH 5.7, 0.2M) and 20. Mu.L of acetolyrinone (0.1M) and cultured overnight at 28 ℃.
The next morning the shaken pTRV1 and pTRV2 bacterial solutions were poured into 50mL centrifuge tubes, centrifuged at 6000rpm at room temperature for 10min, the supernatant discarded and the buffer suspension (preparation method: 1mL MgCl) 2 (1M), 5mLMES (pH 5.7, 0.2M), 200. Mu.L of acetosyringone (0.1M), and volume to 100 mL) were suspended respectively to adjust OD 600 1.5, after vortex mixing, standing for 3 hours at room temperature to obtain pTRV1 agrobacterium tumefaciens heavy suspension and pTRV2 agrobacterium tumefaciens heavy suspension;
and fully mixing the pTRV1 agrobacterium tumefaciens heavy suspension and the pTRV2 agrobacterium tumefaciens heavy suspension in equal volumes to obtain an infectious bacteria liquid.
Application example 1
A method for identifying ZjCLA gene functions of dates comprises the following steps:
(1) Culturing of jujube seedling material
Selecting undamaged wild jujube seeds, sterilizing with 0.5wt.% sodium hypochlorite solution for 10min, washing with distilled water for 3 times, soaking overnight, absorbing water on the surface layer of the seeds, wrapping with two layers of gauze, and placing in a culture dish. Placing 30 wild jujube seeds in each culture dish, placing in a constant-temperature illumination incubator, setting the temperature to 25 ℃ and 16h illumination/8 h darkness, and culturing for 3d. Transplanting the wild jujube seeds into a culture pot after the wild jujube seeds sprout to 1cm, and carrying out the culture until the cotyledons are completely unfolded for infection.
(2) The infectious microbe liquid prepared in example 2 was sucked by a disposable 1ml syringe (without needle), wound was gently scraped by needle, and then the microbe liquid was directly injected to the back of the cotyledon of the wild jujube seedling by gentle pressure, so that the microbe liquid was spread in the cotyledon. Culturing the wild jujube seedlings injected with the infectious microbe liquid in the dark at 24 ℃ for 24 hours, then culturing the wild jujube seedlings in 16 hours of light and 8 hours of dark culture conditions, and marking the wild jujube seedlings as an experimental group. A blank control group and a negative control group were additionally provided, wherein the blank control group was set as a heavy suspension injected with no bacterial liquid, and the negative control group was set as a plant injected with the infectious microbe liquid prepared in comparative example 1.
(4) Phenotypic observation
Obvious circular marks can be observed on the leaves after the injection of the infection, and the leaf infiltration is full of liquid. After 10d of infestation, as can be seen from FIG. 2, the experimental group inoculated with pTRV2-ZjCLA started to develop leaf albinism, indicating that the silencing vector started to function and that the leaf of the control plant did not develop albinism.
(5) Virus detection
The leaf RNA was extracted using TIANGEN RNA extraction kit and reverse transcribed into cDNA using the kit instructions for each of the blank control group (untreated), the empty control group (empty control), and the experimental group leaf (TRV-ZjCLA), three replicates were performed.
In order to detect the silencing effect of the virus vector, zjCLA real-time quantitative primers are designed, real-time fluorescence quantitative PCR is adopted to detect the expression quantity in the leaves of the wild jujube seedlings, and the internal reference uses action, wherein the sequences of the primers are as follows:
ZjCLA-F:ATCAATTGGAGGCTTTGGAT，SEQ ID NO.4；
ZjCLA-R:ACTGTTGCTGCAATATGAGA，SEQ ID NO.5；
ZjACT-F:AGCCTTCCTGCCAACGAGT，SEQ ID NO.6；
ZjACT-R:TTGCTTCTCACCCTTGATGC，SEQ ID NO.7；
the reaction system for real-time fluorescence quantitative PCR amplification is as follows: cDNA 1 mu L, zjCLA-F or ZjACT-F0.4 mu L, zjCLA-R or ZjACT-R0.4 mu L, 2 XSuperRealPreMixPlus 10 mu L and ddH 2 O 8.2μL；
The reaction process of the real-time fluorescence quantitative PCR amplification is as follows: pre-denaturation at 95℃for 15min; denaturation at 95℃for 10s, annealing at 50℃for 30s, extension at 72℃for 30s for 40 cycles; the reaction was carried out at 72℃for 10min.
The results of the detection are shown in FIG. 3 and Table 1.
TABLE 1 relative expression levels of ZjCLA Gene in different leaves
| Group of | Untreated process | No-load control | TRV-ZjCLA |
| Relative expression level | 1 | 1.07 | 0.17 |
As can be seen from FIG. 3 and Table 1, the VIGS silencing vector constructed by the specific fragment provided by the invention can successfully infect jujube seedlings, induce the silencing of jujube endogenous ZjCLA, and effectively reduce the expression level of jujube ZjCLA genes.
Comparative application example 1
The shaken pTRV1 and pTRV2 bacterial solutions were poured into 50mL centrifuge tubes, centrifuged at 6000rpm at room temperature for 10min, the supernatant was discarded, and the buffer suspension (preparation method: 1mL MgCl) was used 2 (1M), 5mL MES (pH 5.7, 0.2M), 200. Mu.L acetosyringone (0.1M), and volume to 100 mL) were suspended respectively to adjust OD 600 After vortex mixing to 0.8, standing for 3 hours at room temperature to obtain pTRV1 agrobacterium tumefaciens heavy suspension and pTRV2 agrobacterium tumefaciens heavy suspension; and fully mixing the pTRV1 agrobacterium tumefaciens heavy suspension and the pTRV2 agrobacterium tumefaciens heavy suspension in equal volumes to obtain an infectious bacteria liquid.
The infectious microbe liquid prepared in example 2 was sucked by a disposable 1mL syringe (without needle), wound was gently scraped by needle, and then the microbe liquid was directly injected to the back of the cotyledon of the wild jujube seedling by gentle pressure, so that the microbe liquid was spread in the cotyledon. Culturing the wild jujube seedlings injected with the infectious microbe liquid in the dark at 24 ℃ for 24 hours, then culturing the wild jujube seedlings in 16 hours of light and 8 hours of dark culture conditions, and marking the wild jujube seedlings as an experimental group. A blank control group and a negative control group were additionally provided, wherein the blank control group was set as a heavy suspension injected with no bacterial liquid, and the negative control group was set as a plant injected with the infectious microbe liquid prepared in comparative example 1.
The leaf RNA was extracted using TIANGEN RNA extraction kit and reverse transcribed into cDNA using the kit instructions for each of the blank control group (untreated), the empty control group (empty control), and the experimental group leaf (TRV-ZjCLA), three replicates were performed.
In order to detect the silencing effect of the virus vector, zjCLA real-time quantitative primers are designed, real-time fluorescence quantitative PCR is adopted to detect the expression quantity in the leaves of the wild jujube seedlings, and reference is made to application example 1 by using action, primer sequences, reaction systems and reaction processes.
The detection results are shown in Table 2.
TABLE 2 relative expression levels of ZjCLA Gene in different leaves
As shown in Table 2, in comparative application example 1, when the OD value of the bacterial liquid is 0.8, the effect of the VIGS on infecting the jujube seedlings is poor, and the reduction of the expression level is not remarkable.
In conclusion, the VIGS silencing vector constructed by the specific fragment provided by the invention can successfully infect jujube seedlings, effectively reduces the expression level of jujube ZjCLA genes, and can realize simple, efficient and low-cost identification of the jujube ZjCLA gene functions.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The application of a specific fragment in constructing a VIGS silencing vector of a jujube ZjCLA gene comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, wherein the jujube ZjCLA gene comprises a gene with a sequence number of XM_ 048477782.1.
2. A VIGS silencing vector of jujube ZjCLA gene, characterized in that the VIGS silencing vector comprises a specific fragment and pTRV2 vector; the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO. 1.
3. The VIGS silencing vector of claim 2, wherein the specific fragment is located between the two cleavage sites of EcoR I and Xho I of pTRV 2.
4. A primer pair for amplifying a specific fragment, wherein the specific fragment comprises a sequence with a nucleotide sequence shown as SEQ ID NO.1, and the primer pair comprises an upstream primer CLA-EcoRI-F and a downstream primer CLA-XhoI-R;
the nucleotide sequence of the upstream primer CLA-EcoRI-F is shown as SEQ ID NO. 2;
the nucleotide sequence of the downstream primer CLA-XhoI-R is shown in SEQ ID NO. 3.
5. A VIGS silencing system of jujube ZjCLA gene, comprising: an agrobacterium solution containing a pTRV1 vector and an agrobacterium solution containing a VIGS silencing vector; the VIGS silencing vector comprising the VIGS silencing vector of claim 2 or 3;
the volume ratio of the agrobacterium liquid containing the pTRV1 vector to the agrobacterium liquid containing the VIGS silencing vector is 1:1;
the OD values of the agrobacterium liquid containing the pTRV1 vector and the agrobacterium liquid containing the VIGS silencing vector are respectively 1.0-1.5.
6. Use of the VIGS silencing vector of claim 2 or 3 or the VIGS silencing system of claim 5 to silence the jujube ZjCLA gene comprising the gene sequence No. XM 048477782.1.
7. A method for silencing jujube ZjCLA gene, comprising the steps of:
injecting the VIGS silencing system of claim 5 into the back of leaf blades of jujube seedlings to obtain infected jujube seedlings.
8. The method of claim 7, wherein the jujube seedlings comprise seedlings with fully expanded cotyledons.
9. The method of claim 7, wherein obtaining the infected jujube seedlings further comprises darkly treating the infected jujube seedlings in a culture chamber at 24 ℃ for 24 hours.
10. Use of the VIGS silencing vector of claim 2 or 3 or the VIGS silencing system of claim 5 to identify the function of the jujube ZjCLA gene comprising the gene sequence No. XM 048477782.1.
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