CN116622765A - E3 ubiquitin ligase RNF170L and application of encoding gene thereof in resisting potato virus Y - Google Patents
E3 ubiquitin ligase RNF170L and application of encoding gene thereof in resisting potato virus Y Download PDFInfo
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Abstract
The application discloses an E3 ubiquitin ligase RNF170L and application of a coding gene thereof in resisting potato virus Y, belonging to the technical field of biology. The application is first studied and confirmed that E3 ubiquitin ligase RNF170L from Nicotiana benthamiana can negatively regulate PVY infection. Further researching the action mechanism, the E3 ubiquitin ligase RNF170L interacts with 6K2 protein in PVY in vivo; in addition, the E3 ubiquitin ligase RNF170L also interacts with NbRHD3 in a plant body, and the overexpression of the E3 ubiquitin ligase RNF170L can promote the degradation of the NbRHD3, thereby inhibiting the infection of PVY. Thus, the E3 ubiquitin ligase RNF170L can be a new target for plant antiviral. And is applied to the prevention and treatment of potato Y virus diseases, and has important application value.
Description
Technical Field
The application relates to the technical field of biology, in particular to an E3 ubiquitin ligase RNF170L and application of a coding gene thereof in resisting potato virus Y.
Background
Potyvirus (PVY) is a representative member of the Potyvirus family (Potyviridae) Potyvirus genus (Potyvirus), whose virions are in a spiral symmetrical curved line, non-enveloped, 730-740nm long, and 11-15nm in diameter. PVY belongs to the sense single stranded RNA virus and the genome is about 9.7kb in length. Potato virus Y is used as one of important plant diseases, and the damage degree of the potato virus Y is aggravated by factors such as continuous cropping, aphid outbreak, host variety disease and the like. Unlike diseases caused by bacteria or fungi, replication and transmission of viruses need to depend on the host and transmission medium, and general chemical agents cannot directly block the proliferation of viruses and even interfere with the normal growth of the host. Therefore, the prevention and treatment difficulty for the virus diseases caused by potato virus Y is high.
The ubiquitination system is a protein degradation system which occurs in cells, is used as an important post-translational modification effect, and is widely involved in a series of vital activities such as plant growth and development, stress response, signal transduction and the like in the life cycle of plants by mediating the degradation of specific proteins.
E3 ubiquitin ligases are key enzymes in the ubiquitination system that recognize target proteins, playing a great role in the ubiquitination process. However, the E3 ubiquitin ligases are huge in quantity, various in variety and complex in function, different types of E3 ubiquitin ligases are different in the way of transmitting ubiquitin molecules to target proteins, and different in the biological processes involved in regulation. There are still many E3 ubiquitin ligases whose function is not yet defined. RNF170L, a ubiquitin ligase, has also rarely been reported for its function.
Disclosure of Invention
Aiming at the prior art, the application aims to provide an E3 ubiquitin ligase RNF170L and application of a coding gene thereof in resisting potato virus Y.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect of the application there is provided the use of an E3 ubiquitin ligase RNF170L in (1) or (2) as follows:
(1) Inhibiting infection of potato virus Y;
(2) Improving the resistance of the plants to potato virus Y;
the amino acid sequence of the E3 ubiquitin ligase RNF170L is shown as SEQ ID NO. 1.
In the above application, the E3 ubiquitin ligase RNF170L inhibits infection of potato virus Y by:
(a) Interact with the 6K2 protein in potyvirus;
(b) Interact with NbRHD3 and promote degradation of NbRHD3.
In a second aspect of the application, there is provided the use of a gene encoding E3 ubiquitin ligase RNF170L in (1) or (2) as follows:
(1) Regulating and controlling resistance of plants to potato virus Y;
(2) Cultivating a potato Y virus resistant plant variety.
In the above application, the gene encoding E3 ubiquitin ligase RNF170L is a nucleic acid molecule as shown in the following (i) or (ii):
(i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 2;
(ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 1.
In a third aspect of the present application, there is provided the use of a recombinant expression vector or engineering bacterium comprising a gene encoding E3 ubiquitin ligase RNF170L in (1) or (2) as follows:
(1) Regulating and controlling resistance of plants to potato virus Y;
(2) Cultivating a potato Y virus resistant plant variety.
In a fourth aspect of the application, there is provided a method of increasing resistance of a plant to potyvirus comprising the steps of:
overexpression of a gene encoding E3 ubiquitin ligase RNF170L in the plant;
alternatively, the NbRHD3 gene in the plant is silenced or knocked out.
In the method, the over-expression of the coding gene of the E3 ubiquitin ligase RNF170L can be realized by a method of exogenously transferring the coding gene of the E3 ubiquitin ligase RNF 170L; or up-regulate the expression of the gene encoding E3 ubiquitin ligase RNF170L in the plant genome.
In the method, nbRHD3 gene can be silenced or knocked out by adopting the techniques of VIGS technology, T-DNA insertion, CRISPR-Cas9 gene editing technology, RNA interference and the like.
In a fifth aspect of the application, there is provided a method of growing a transgenic plant resistant to potyvirus comprising the steps of:
introducing an RNF170L gene into a wild-type plant to obtain a plant with high RNF170L gene expression; the resistance of the obtained RNF170L gene high-expression plant to potato virus Y is higher than that of the wild plant.
In the above method, the RNF170L gene is a nucleic acid molecule as shown in the following (i) or (ii):
(i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 2;
(ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 1.
The application has the beneficial effects that:
the application is first studied and confirmed that E3 ubiquitin ligase RNF170L from Nicotiana benthamiana can negatively regulate PVY infection. Further researching the action mechanism, the E3 ubiquitin ligase RNF170L interacts with 6K2 protein in PVY in vivo; in addition, the E3 ubiquitin ligase RNF170L also interacts with NbRHD3 in a plant body, and the overexpression of the E3 ubiquitin ligase RNF170L can promote the degradation of the NbRHD3, thereby inhibiting the infection of PVY.
Thus, the E3 ubiquitin ligase RNF170L can be a new target for plant antiviral. And is applied to the prevention and treatment of potato Y virus diseases, and has important application value.
Drawings
Fig. 1: e3 ubiquitin ligase NbRNF170L negatively regulates PVY infection; in the figure, a: silencing NbRNF 170L's benthamiana phenotype; b: silencing efficiency of NbRNF 170L; c: fluorescence observations of NbRNF170L silenced Nicotiana benthamiana and control friction inoculated PVY-GFP; d: counting the number of Benshi cigarettes showing virus infection symptoms after rubbing and inoculating PVY-GFP every 24 hours until all Benshi cigarettes show disease symptoms, and preparing a line graph according to the statistical data; e: western blot detects PVY CP accumulation amount; f: effect of over-expression of NbRNF170L on virus accumulation levels; g: western blot detects the effect of over-expression of NbRNF170L on the level of viral accumulation.
Fig. 2: nbRNF170L interacts with PVY 6K 2; in the figure, a: the inoculation schematic diagram in the luciferase complementation test and the leaf living imaging system after inoculation are observed; b: quantitative determination of the luciferin luminescence signal; c: co-immunoprecipitation test results; d: observing the result by a laser confocal microscope; e: the amount of 6K2-eGFP protein accumulated was measured using GFP antibody.
Fig. 3: nbRNF170L interacts with NbRHD 3; in the figure, a: the inoculation schematic diagram in the luciferase complementation test and the leaf living imaging system after inoculation are observed; b: quantitative determination of the luciferin luminescence signal; c, co-immunoprecipitation test results; d: and observing the result by a laser confocal microscope.
Fig. 4: silencing NbRHD3 inhibits PVY infestation; in the figure, a: silencing NbRHD 3's benthamiana plant phenotype; b: qPCR (quantitative polymerase chain reaction) detection of NbRHD3 silencing efficiency; c: observing the NbRHD3 silencing Benshi smoke under ultraviolet irradiation; d: counting the fluorescence occurrence time after PVY-GFP inoculation every 24 hours, and making a line graph; e: western blot detects PVY CP accumulation.
Fig. 5: the effect of over-expression of NbRNF170L on protein accumulation of NbRHD 3; in the figure, a: imaging a living body co-infiltrated by FLuc-NbRHD3 and NbRNF 170L-HA; b: quantitatively measuring a luciferin luminescence signal of the inoculated area by using an enzyme-labeled instrument; c: western blot detects normal expression of NbRNF170L-HA protein; d: the FLuc empty vector is respectively subjected to in-vivo imaging with NbRNF170L-HA and HA co-infiltration; e: the enzyme-labeled instrument quantitatively determines the luciferin luminescence signal; f: western blot detects normal expression of NbRNF170L-HA protein; g: western blot results of protein accumulation amount during co-infiltration of eGFP-NbRHD3 and NbRNF 170L; h: protein accumulation Western blot results of eGFP and NbRNF170L co-infiltration; i: qPCR detects the effect of over-expression of NbRNF170L on mRNA levels of NbRHD3.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As previously mentioned, replication and transmission of viruses requires reliance on the host and transmission mediators, and general chemical agents cannot directly block viral proliferation and even interfere with normal growth of the host. Therefore, the prevention and treatment difficulty for the virus diseases caused by potato virus Y is high.
Research shows that E3 ubiquitin ligase can regulate and control plant disease resistance, but E3 ubiquitin ligases are huge in quantity, various in variety and complex in function, different types of E3 ubiquitin ligases have different modes of transmitting ubiquitin molecules to target proteins, and biological processes involved in regulation are different. There are still many E3 ubiquitin ligases whose function is not yet defined.
RNF170L is one of E3 ubiquitin ligase family members, and the amino acid sequence of RNF170L of the present application is shown in SEQ ID NO.1, specifically as follows:
MDGPPANDVCSICHGNFHVPCQSNCSHWFCANCILQVWDHGSAVQACKCPLCRRPITLLVPSDSASRLHRDPEVPEVLRRVEHYNRHFGQHNSSLFQRMQDLPFLLRRLLRDMTDPQRSLPFVIRARVYLAVILSGIYVLSPVDIIPEGVLGIIGLLDDLIIMFICFLHVAALYRSVLLFRHGGS。
the nucleotide sequence of the coding gene of RNF170L (RNF 170L gene) is shown in SEQ ID NO.2, and is specifically as follows:
ATGGATGGACCACCAGCGAATGATGTTTGTTCAATTTGCCATGGCAATTTCCATGTCCCTTGTCAATCCAATTGTTCCCATTGGTTTTGTGCTAACTGCATTCTACAAGTTTGGGATCATGGATCTGCCGTTCAAGCATGCAAGTGTCCATTATGTCGCCGTCCGATTACGTTGTTGGTTCCTAGTGATTCTGCATCAAGGTTGCACCGAGATCCTGAAGTTCCTGAGGTTTTACGAAGAGTTGAACACTATAACCGGCATTTTGGCCAACATAACAGCAGCCTTTTCCAGAGAATGCAAGATCTTCCCTTTCTCCTCAGGAGATTGCTGCGAGATATGACAGATCCTCAAAGGTCTCTTCCATTTGTCATCAGGGCACGTGTCTATTTGGCTGTAATTTTAAGTGGAATATACGTCCTTAGCCCCGTGGACATTATCCCAGAAGGAGTTTTGGGTATAATAGGTTTACTAGATGATCTGATTATCATGTTCATCTGTTTCCTGCATGTTGCTGCTCTGTATAGATCAGTACTTCTGTTTCGCCATGGAGGTTCTTAA。
there are few studies on RNF170L at present, and in view of this, the present application has conducted intensive studies on the function of RNF170L. According to the application, RNF170L gene in Nicotiana benthamiana is silenced, and then PVY-GFP is inoculated, so that the result shows that the RNF170L gene silenced Nicotiana benthamiana GFP has stronger fluorescence and higher PVY CP accumulation, and plants silenced with the RNF170L can show symptoms earlier; co-infiltrating the RNF170L-HA over-expression vector into Nicotiana benthamiana, wherein the fluorescence intensity of the region over-expressed by the RNF170L gene is weaker, and the virus accumulation level is lower; the results indicate that RNF170L negatively regulates PVY infection.
To further investigate the mechanism of action of RNF170L, the present application confirmed that RNF170L interacted with the 6K2 protein of PVY in vivo by co-immunoprecipitation experiments.
The PVY infection can be inhibited after the cigarette RHD3 gene is silenced, and the application also verifies the interaction of RNF170L and NbRHD3 in a plant body and promotes the degradation of NbRHD3 through experiments.
Thus, RNF170L inhibits PVY infection by interacting with the 6K2 protein of PVY in vivo, or with NbRHD3 in plants and promoting degradation of NbRHD3.
This demonstrates that: e3 ubiquitin ligase RNF170L can be used as a new target for plant virus resistance, can be applied to the prevention and treatment of potato Y virus diseases, and has important application value.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present application are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers. Wherein:
plasmid extraction kits were purchased from OMEGA company; gel recovery kit was purchased from OMEGA company; the homologous recombinant enzyme required for vector construction is purchased from abm company; RNA extraction kit (TransZol) was purchased from beijing holo gold company; common restriction enzymes are purchased from Thermo company; the phusion enzyme required for amplifying the gene fragment is purchased from TaKaRa company; reverse transcriptase was purchased from TaKaRa company; GFP antibodies were purchased from TaKaRa; HA antibodies were purchased from beijing abway company; fluorescent quantitative PCR reagents were purchased from Vazyme company; protease inhibitors were purchased from MCE company; the nitrocellulose membrane used by Western Blot was manufactured by Pall Gelman company.
The Nicotiana benthamiana (Nicotiana benthamiana) used in the application was grown at a temperature of 22℃in 16h light/8 h darkness, except as specified, in a climatic chamber or incubator with a relative humidity of 65%.
The potato virus Y used in the present application is PVY N605 isolate (GenBank: X97895.1).
Example 1: investigation of the influence of E3 ubiquitin ligase NbRNF170L on PVY infection
1. The test method comprises the following steps:
(1) Effect of RNF170L gene silencing on PVY infection:
the NbRNF170L complete sequence was placed in the sequence analysis TOOL VIGS TOOL of the Sol Genomics Network website (https:// www.sgn.cornell.edu /), the Benshi smoke database was selected, the fragment length was set to 300bp, the predicted optimal fragment for RNA silencing was obtained, and the predicted 300bp NbRNF170L fragment was cloned into the TRV2 vector. The 300bp NbRNF170L fragment has a nucleotide sequence shown in SEQ ID NO.3, and is specifically as follows:
CTGAGGTTTTACGAAGAGTTGAACACTATAACCGGCATTTTGGCCAACATAACAGCAGCCTTTTCCAGAGAATGCAAGATCTTCCCTTTCTCCTCAGGAGATTGCTGCGAGATATGACAGATCCTCAAAGGTCTCTTCCATTTGTCATCAGGGCACGTGTCTATTTGGCTGTAATTTTAAGTGGAATATACGTCCTTAGCCCCGTGGACATTATCCCAGAAGGAGTTTTGGGTATAATAGGTTTACTAGATGATCTGATTATCATGTTCATCTGTTTCCTGCATGTTGCTGCTCTGTATA。(SEQ ID NO.3)
the specific operation steps are as follows:
firstly, using the complete NbRNF170L fragment amplified by cDNA as a template, designing a primer TRV2 for amplifying a 300bp silent fragment: GC (gas chromatography)TCTAGAAAGTGTCCATTATGTCGCCG(SE Q ID NO.4)；TRV2::NbRNF170L-sil-R：CGGGATCCAAACAGAAGTACTGATCTATACAG AGCAG (SEQ ID NO. 5); the first two bases in the primer are enzyme cutting site protecting bases, the underlined parts are restriction enzyme sites XbaI and BamHI respectively, the italic part is the combined area of the primer and the template, the enzyme cutting sites XbaI and BamHI are utilized to linearize the TRV2 carrier, and T4 connection is carried out on the amplified silencing fragment, so that the TRV2 is constructed to be NbRNF170L.
Selecting young Benshi tobacco with seedling age of 2-3 weeks, respectively transforming NbRNF170L and TRV1 into agrobacterium, and regulating final concentration OD of agrobacterium 600 Co-infiltrating lower lamina of burley at volume ratio 1:1 after =0.5. GUS gene is beta-glucosidase, exists in bacterial genome, and uses tobacco co-infiltrated by TRV 2:GUS and TRV1 as negative control. The PDS gene is phytoene dehydrogenase, and silencing the PDS gene can whiten plants, and the tobacco whitening degree obtained by co-infiltrating PDS and TRV1 is taken as a subsequent test indication.
About 10 days after silencing, taking the system leaf of the silencing target gene plant according to the whitening condition of the upper system leaf of Nicotiana benthamiana silencing PDS, and extracting the total RNA of the plant to detect silencing efficiency. Selecting the cigarette with higher silencing efficiency to rub and inoculate PVY virus, wherein the specific inoculation method comprises the following steps:
0.5g of PVY-GFP source (full-length PVYN605 sequence is cloned on pCB301 vector, GFP gene is inserted between NIb and CP gene) preserved in a refrigerator at-80 ℃ is weighed, placed in a sample grinding bag for full grinding, and diluted with ultrapure water according to a weight ratio of 1:5. Selecting Benshi smoke which needs to be rubbed and inoculated with virus, uniformly scattering silicon carbide on the leaves of a disease-causing system, and dripping 200 mu L of diluted PVY-GFP virus source on each leaf of the system by using a liquid transfer device.
The virus onset condition is observed by hand-held ultraviolet and the like every day after the virus is rubbed and inoculated, a camera is used for photographing, the number of the patients suffering from the virus onset every day and the condition that GFP fluorescence reaches the system leaves are recorded, and an onset curve is drawn. Meanwhile, different time points are selected in the observation process, the leaves are sampled, and the PVY CP antibody Western blot is used for detecting the virus accumulation amount. The inoculated TRV is used as a control, and whether the infection of viruses is affected after the tobacco is silenced is clear.
(2) Effect of RNF170L overexpression on PVY infection:
according to the coding region sequence of NbRNF170L gene in NCBI database, designing specific primer 35S of MCS-HA for amplifying NbRNF170L and then carrying out homologous recombination to transient expression vector 35S:TGGCGCGCCA CTAGTATGGATGGACCACCAGC(SEQ ID NO.6)；35S::NbRNF170L-HA-R：GACAGT ACTATCGATAGAACCTCCATGGCGAAAC(SEQ ID NO.7)。
the transient expression vector 35S is constructed based on an agrobacterium binary expression vector pCAMBIA0390, a 35S promoter is inserted before MCS, the 5' -UTR of TVMMV is inserted, and a 3 XHA coding sequence is inserted after MCS. The vector was linearized using the restriction enzyme BamHI in the multiple cloning site, with homology arms in the horizontal line and specific binding sites for primer and template in the italic), and the primer sequences were synthesized by the company Limited for biological engineering.
Extracting total RNA of Nicotiana benthamiana, removing genomic DNA by using gDNA wind, and performing reverse transcription to obtain cDNA. The target gene was amplified using the specific primers (35S:: nbRNF170L-HA-F and 35S::: nbRNF 170L-HA-R) using cDNA as a template. And (3) carrying out single enzyme digestion on the required vector by utilizing BamHI or SalI in the multiple cloning sites, and connecting the digested vector with the amplified target gene homologous recombination to construct the NbRNF170L-HA.
The two amino acid codons K at position 177 and Q at position 200 of P3N-PIPO of PVY were simultaneously mutated to stop codons to produce the mobility-deficient mutant PVYPIPOSTOP. PVY-PIPOSTOP and NbRNF170L-HA were co-infiltrated into leaf blades of Nicotiana benthamiana, and whether over-expression of NbRNF170L-HA affected PVY replication was examined by using PVY-PIPOSTOP and an empty vector of HA expressed alone (transient expression vector 35S:: MCS-HA) as controls. (PVY-PIPOSTOP requires the addition of the silencing inhibitor TBSV P19 when co-infiltrating with NbRNF170L-HA, thereby ensuring the expression level of NbRNF170L-HA in cells for a longer period of time).
The PVY-PIPOSTOP and NbRNF170L-HA and TBSV P19 are mixed according to the volume ratio of 2:4:4, mixing uniformly to obtain final concentration OD of Agrobacterium after mixing uniformly 600 =1.0, the third day after infiltration inoculationPVY-PIPOSTOP expression was observed from the initial comparison and recorded by photographing. In the observation process, different time points are selected for leaf sampling, and PVY CP antibody Western blot is used for detecting the virus accumulation amount. It was clarified whether or not the overexpression of NbRNF170L and NbRHD3 would affect PVY replication.
2. Test results:
to investigate the function of NbRNF170L and the effect of NbRNF170L on PVY infection, the silencing sequence of NbRNF170L was predicted using the VIGS TOOL of Sol Genomics Network website, and further, TRV-induced gene silencing was used to reduce NbRNF170L gene expression. Construction of NbRNF170L silencing vector TRV2:: nbRNF170L, and construction of TRV2:: GUS as a negative control. And (3) respectively co-infiltrating the constructed TRV2, nbRNF170L and TRV2, GUS and TRV1 into Benshi tobacco. Observations were made on day 10 post-inoculation, with no significant change in the benthamiana phenotype of NbRNF170L silencing compared to the control (1A). Sampling the system leaves, extracting total RNA of plants, detecting the mRNA accumulation level of NbRNF170L by real-time fluorescence quantitative PCR after reverse transcription, and significantly reducing the mRNA level of NbRNF170L in Nicotine with NbRNF170L silencing as high as 80% compared with a control (FIG. 1B). Bunge-type cigarette with NbRNF170L silencing and control were rubbed with PVY-GFP, and the symptoms of Bunge-type cigarette and fluorescence intensity of GFP were observed from the third day. Fifth day after inoculation, leaf-benthamiana leaves with NbRNF170L silencing began to curl, showing more severe symptoms of PVY infection and GFP showed more fluorescence (FIG. 1C). And taking system leaves, extracting plant total proteins, and detecting PVY CP accumulation amount through Western blot. The viral accumulation was higher in NbRNF170L N.benthamiana than in the control (FIG. 1E). The number of Benshi cigarettes showing symptoms of viral infection after rubbing and inoculating PVY-GFP was counted every 24 hours until all Benshi cigarettes appeared to have symptoms of onset. And (5) making a line graph according to the statistical data. As shown, plants silencing RNF170L were able to exhibit symptoms of viral infection faster than controls (fig. 1D).
To investigate the effect of over-expression of NbRNF170L on viral infection. With the HA empty vector as a control, the over-expression of NbRNF170L-HA was observed 24h after the agrobacterium infiltration of the over-expressed region under PVY-PIPOSTOP, and the 4 th day after the inoculation, the fluorescence intensity of GFP in the over-expressed region of NbRNF170L was weaker than that of the control, indicating that the virus accumulation level was probably lower (FIG. 1F). The inoculation area was sampled, and after total protein of plants was extracted, the level of virus accumulation was detected by Western blot, and the result showed that the level of virus accumulation in n smoke over-expressing NbRNF170L was significantly lower than that of the control (fig. 1G).
The above results indicate that: e3 ubiquitin ligase NbRNF170L negatively regulates PVY infection.
Example 2: in vivo interaction study of E3 ubiquitin ligase NbRNF170L and 6K2
1. The test method comprises the following steps:
(1) And (3) constructing a carrier:
the construction strategy of NbRNF170L-eGFP, nbRNF170L-mCherry and NbRNF170L-nLuc is the same as that of NbRNF170L-HA in example 1, wherein 35S is MCS-eGFP, 35S is MCS-DsRed and 35S is MCS-mCherry is constructed by inserting eGFP, dsRed, mCherry fluorescent tags into pCAM35S basic vector after multiple cloning sites, the multiple cloning sites are the same as that of 35S, and the homologous arm sequences in the primers are the same. 35 S. MCS-nLuc vector is pCambia1300-nLuc, linearized with restriction enzymes BamHI and SalI in the multiple cloning site. Specific primers are shown in Table 1 and SEQ ID NO.8-SEQ ID NO.11 in the sequence table:
table 1: primer for constructing NbRNF170L related vector
Note that: the primers required for constructing vectors "NbRNF170L-DsRed" and "NbRNF170L-mCherry" were identical to RNF170L-eGFP F and RNF170L-eGFP R.
According to PVY 6K2 gene sequence (GenBank: X97895.1) in the database, designing a specific primer 35S for amplifying 6K2 and then carrying out homologous recombination to a transient expression vector 35S for MCS-HA, and a specific primer 35S for amplifying 6K2-HA-F:TGGCGCGCCACTAGTATGGCAACAACTTCACTCGCAAAG(SEQ ID NO.12)；35S::6K2-HA-R：GACAGTACTATCGATCTGGTGAGACACAGTTTCAACTG (SEQ ID NO. 13) the vector was linearized using the restriction enzyme BamHI in the multiple cloning site. The horizontal line part is a homologous arm, and the italic part is a specific binding site of the primer and the template). Primer sequences are sent to the engineering and bioengineering stockSynthesized by limited company.
6K2-eGFP, 6K2-DsRed and cLuc-6K2 construction strategies are the same as above. Specific primers are shown in Table 2 and SEQ ID NO.14-SEQ ID NO.17 in the sequence Listing:
table 2: primers required for constructing PVY 6K2 related vector
Note that: the primers required for the construction of the vector "6K2-DsRed" are identical to 6K2-eGFP F and 6K2-eGFP R.
PVY (PVY) N 605-GFP invasive clones were used as amplification templates and the 6K2 gene was amplified using the F, R primers described above. The vector is digested by BamHI or SalI in the multiple cloning site, and the digested vector is connected with the homologous recombination of the amplified 6K2 gene.
(2) Co-immunoprecipitation:
two proteins which are verified to interact are selected as test groups (NbRNF 170L-nLuc+cLuc-6K 2), the two proteins are respectively used as two groups of negative controls (nLuc+cLuc-6K 2 and NbRNF 170L-nLuc+cLuc) with the other side labels, and agrobacterium is co-infiltrated and inoculated into Benshi smoke, and sampling is carried out after 3 days for detection.
Cutting leaves along the agro-infiltration site, weighing 2g of fresh leaf samples, placing the fresh leaf samples in a mortar, adding liquid nitrogen, and grinding the samples to powder. The ground sample was added to a 100mL centrifuge tube at a ratio of 1:1.5 3mL of protein extract (25 mM Tris-HCl pH7.4, 150mM NaCl, 0.5wt% NP-40, 0.5wt% protease inhibitor, 10wt% glycerol) was added, and the mixture was stirred well and left on ice for 20min.12000 Xg, centrifugation at 4℃for 20min, at which time the protein was in the supernatant, and the supernatant was transferred to a new 100mL centrifuge tube and repeated centrifugation until the pellet was removed. The supernatant was filtered using a 0.22 μm aqueous filter to remove unnecessary impurities. The treated protein solution was placed in a 5mL centrifuge tube, 20. Mu. LGFP-beads was added, and the tube was incubated on ice with shaking for about 30min to bind 6K2-eGFP to GFP-beads. 2500 Xg, 4 ℃ centrifugation for 5min, careful aspiration of supernatant into waste liquid cylinder, adding 1 XPBS to the centrifuge tube to wash GFP-beads, repeating 7 times. 2500 Xg, 4 ℃ centrifugation for 5min, careful aspiration of the supernatant to a waste liquid jar, addition of 50. Mu.L of 2 XSDS loading buffer, sealing, boiling water bath for 5min, preservation at-20 ℃ in a refrigerator, and Westernblot detection of interaction.
(3) Luciferase complementation assay:
to investigate whether two proteins interact in vivo, a luciferase complementation assay may be selected for validation. Two proteins that need to be validated for interaction are used as control groups, and corresponding nLuc and cLuc are used as negative control groups, respectively. Selecting young leaves of Benshi tobacco of 4 weeks of seedling age, selecting 4 areas of agrobacteria infiltration in one leaf, respectively inoculating bacteria liquid combinations, and recording, and performing in-vivo imaging observation and enzyme-labeling instrument to determine protein interaction strength after 48 hours, wherein the method comprises the following steps of:
cutting the soaked leaves after 48h of inoculation, placing the back surfaces of the leaves on white A4 paper upwards, diluting the prepared 10mM D-Luciferin free acid mother solution by 10 times with water, spraying the 10mM D-Luciferin free acid mother solution on the back surfaces of the leaves by a small spray can, and lightly smearing the substrate uniformly by using a glove. And (5) performing light-shielding treatment for 5min, and then observing in a living body imager.
Taking Benshi cigarettes 48h after inoculation, cutting out the infiltrated leaf blades, punching and sampling different infiltrated areas in the leaf blades by using a 6mm puncher, carefully placing tobacco leaf discs into a white 96-hole ELISA plate by using tweezers, and selecting at least 8 leaf discs in the same infiltrated area. 100. Mu.L of D-Luciferin free acid substrate at a concentration of 1mM was added to the wells containing the leaf discs with a pipette and the wells were treated in the dark for 5min. And (3) performing fluorescence scanning by using an enzyme-labeled instrument, and drawing a histogram according to the data to judge the interaction strength.
(4) And (3) observing laser confocal:
and (3) co-infiltrating two agrobacterium proteins with different fluorescent labels into the leaf of the Nicotiana benthamiana, sampling after 48 hours, and carrying out confocal observation.
Taking the leaf pieces of Benshi tobacco at 48h after inoculation, and cutting square small pieces with the size of 2mm in a leaf inoculation area by a blade. A drop of clear water is dropped on a clean glass slide, the back surface of the cut blade is upwards placed on the drop of the glass slide by using tweezers, a cover glass is slowly covered on one side, and redundant bubbles are removed. The prepared sample was taken under a confocal microscope to see if co-localization was present.
2. Test results:
to verify whether NbRNF170L interacted with PVY 6K2 in plants, a luciferase complementation assay was performed. The enzyme cutting sites in pCAMBIA1300-nLuc and pCAMBIA1300-cLuc are utilized to construct pCAMBIA1300-NbRNF170L-nLuc and pCAMBIA1300-cLuc-6K2. The leaf discs were inoculated according to the combinations shown in the figures. 48h after inoculation, taking inoculated leaves, and using a seed containing 1 mmol.L –1 The reaction solution of the luciferin substrate D-luciferin is uniformly sprayed on the back of the blade, uniformly smeared, subjected to light-shielding treatment for 5 minutes and then placed in a living body imaging system for observation. Only the combination of NbRNF170L-nLuc and cLuc-6K2 was able to acquire luminescence images, none of the combination of NbRNF170L-nLuc and cLuc and nLuc-6K 2 acquired luminescence images, indicating that NbRNF170L and 6K2 interacted in vivo (fig. 2A). 8 leaf discs with a diameter of 6mm were randomly taken from each combined inoculation area, the luciferin luminescence signals were quantitatively determined by using a microplate reader, and the obtained data were processed and analyzed, and the luciferin luminescence signals of the co-infiltrated areas of NbRNF170L-nLuc and cLuc-6K2 were significantly higher than those of the other three groups of negative controls (FIG. 2B).
To verify the interaction of NbRNF170L with PVY 6K2 in plants, an immunoprecipitation assay was performed. NbRNF170L-HA and 6K2-eGFP were constructed and inoculated into Nicotiana benthamiana leaves using the combination of HA and 6K2-eGFP and the combination of eGFP and NbRNF170L-HA as negative controls, respectively, by co-infiltration. NbRNF170L-HA was successfully detected using the HA antibody after capture of 6K2-eGFP, whereas the corresponding band was not detected by capture of eGFP, further demonstrating the interaction of NbRNF170L with PVY 6K2 in plants (FIG. 2C).
To investigate the localization of NbRNF170L and PVY 6K2 in plants, nbRNF170L-eGFP and 6K2-DsRed were constructed. The NbRNF170L-eGFP and 6K2-DsRed were co-infiltrated into the leaf of Benshi smoke, and after 48 hours of inoculation, the NbRNF170L-eGFP was observed with a laser confocal microscope to co-localize with the 6K2-DsRed (FIG. 2D).
To investigate whether PVY 6K2 was ubiquitously modified as a substrate for NbRNF170L and degraded by the 26S protease system. We co-infiltrated NbRNF170L-3HA and HA with 6K2-eGFP, respectively, into Nicotiana benthamiana leaves. After 60h, total plant protein was extracted and the amount of 6K2-eGFP protein accumulated was measured using GFP antibody. There was no significant difference in protein accumulation of 6K2-eGFP when the NbRNF170L and 6K2-eGFP co-infiltrated as compared to the HA empty vector (FIG. 2E).
Example 3: research on action relation of E3 ubiquitin ligase NbRNF170L and NbRHD3
1. The test method comprises the following steps:
(1) Construction of NbRHD 3-related vectors:
based on the NbRHD3 gene coding sequence (XM_016635561, NCBI) in NCBI database, a specific primer 35S for amplifying NbRHD3 and then carrying out homologous recombination to a transient expression vector 35S for HA-MCS is designed for HA-RHD3-F:TGGCGCGCCACTAG TATGCTTTCAGATAATAAGGATGGG(SEQ ID NO.18)；35S::HA-RHD3-R：GACAGTACTATCGATCTGCATTTTGTCATGTAACGAG (SEQ ID NO. 19). The vector was linearized using the restriction enzyme BamHI in the multiple cloning site, with homology arms in the horizontal line and specific binding sites for primer and template in the italic), and the primer sequences were synthesized by the biological engineering Co.
The construction strategies of eGFP-NbRHD3, mCherry-NbRHD3, fluc-NbRHD3 and cLuc-NbRHD3 are the same as above, wherein 35S is eGFP-MCS, 35S is mCherry-MCS and 35S is formed by inserting fluorescent tags eGFP, mCherry and luciferase Fluc into a pCAM35S basic vector in front of a multiple cloning site, the multiple cloning site is the same as 35S is HA-MCS, and the homologous arm part sequences in the primers are the same. The vector was linearized using the restriction enzyme BamHI and SalI in the multiple cloning site. Specific primers are shown in Table 3 and SEQ ID NO.20-SEQ ID NO.23 in the sequence Listing:
table 3: primer for constructing NbRHD3 related vector
Note that: primers required for constructing the vectors "mCherry-NbRHD3" and "Fluc-NbRHD3" are identical to eGFP-RHD 3F and eGFP-RHD 3R.
Extracting total RNA of Nicotiana benthamiana, removing genomic DNA by using gDNA wind, and performing reverse transcription to obtain cDNA. The target gene was amplified using the F, R primer described above using cDNA as a template. And (3) carrying out enzyme digestion on the vector by utilizing BamHI or SalI in the multiple cloning sites, and connecting the digested vector with homologous recombination of the amplified target gene.
In constructing the silencing vector, nbRHD3 was predicted as the optimal fragment for RNA silencing in the same manner as in example 1, and the predicted 300bp NbRHD3 fragment was cloned into the TRV2 vector: the complete NbRHD3 fragment amplified by cDNA is used as a template, and a primer TRV2 with the amplification length of 300bp silencing fragment is designed, wherein the primer is NbRHD3-sil-F: GC (gas chromatography)TCTAGAGATAATAAGGATGGGTGTTGTTC(SEQ ID NO.24)；TRV2::NbRHD3-sil-R：CGGGATCCAACAGTCTTCAAAAGAGGCTTA (SEQ ID NO. 25); the first two bases are enzyme cutting site protecting bases, the underlined parts are restriction enzyme sites XbaI and BamHI respectively, and the italic parts are primer and template binding regions. And (3) linearizing the TRV2 vector by using enzyme cutting sites XbaI and BamHI, and carrying out T4 connection with the amplified silencing fragment to construct the TRV2 of NbRHD3.
(2) NbRNF170L interacts in vivo with NbRHD 3:
the interaction of NbRNF170L with NbRHD3 in plants was verified by the luciferase complementation assay according to the method of example 2. The combination shown in fig. 3A was inoculated into lamina of benthamiana, and 48 hours after inoculation, placed in a living body imaging system for observation.
To study the in vivo localization of NbRNF170L and NbRHD3, nbRNF170L-mCherry and eGFP-NbRHD3 were co-infiltrated into Benshi smoke and observed with a laser confocal microscope 48h after inoculation.
(3) Silencing effects of the Nicotiana benthamiana NbRHD3 gene on PVY infection:
the NbRHD3 gene in Nicotiana benthamiana was silenced with TRV 2:NbRHD 3, as described in example 1. About 10 days after silencing, taking systematic leaves of a plant with a silencing target gene according to the phenotype of the Nicotiana benthamiana, and extracting total RNA of the plant to detect silencing efficiency. And selecting the cigarette with higher silencing efficiency to rub and inoculate PVY virus.
The virus onset condition is observed by hand-held ultraviolet and the like every day after the virus is rubbed and inoculated, a camera is used for photographing, the number of the patients suffering from the virus onset every day and the condition that GFP fluorescence reaches the system leaves are recorded, and an onset curve is drawn. Meanwhile, different time points are selected in the observation process, the leaves are sampled, and the PVY CP antibody Western blot is used for detecting the virus accumulation amount. The inoculated TRV is used as a control, and whether the infection of viruses is affected after the tobacco is silenced is clear.
(4) NbRNF170L degradation NbRHD3 study:
to investigate whether NbRNF170L can degrade NbRHD3, we constructed FLuc-NbRHD3. With HA as a control, FLuc-NbRHD3 was co-infiltrated with NbRNF170L-HA. After 60 hours, observation was performed under a living body imaging system.
2. Test results:
(1) In vivo interaction investigation result of NbRNF170L and NbRHD3
To verify the interaction of NbRNF170L with NbRHD3 in vivo. We first constructed pCAMBIA1300-cLuc-NbRHD3, and performed luciferase complementation assay using this vector, using pCAMBIA1300-nLuc and pCAMBIA1300-cLuc as negative controls. The leaf discs were inoculated according to the combinations shown in the figures. 48h after inoculation, taking inoculated leaves, and using a seed containing 1 mmol.L –1 The reaction solution of luciferin substrate D-luciferin is used for uniformly smearing the back of the leaf blade, and the leaf blade is placed in a living body imaging system for observation after being processed for 5 minutes in a dark place. As shown, only the co-infiltrated region of NbRNF170L-nLuc and cLuc-NbRHD3 acquired fluorescence, and no fluorescence signal was observed in the other three inoculated regions, indicating that NbRNF170L-nLuc interacted with cLuc-NbRHD3 in the plant (FIG. 3A). 8 leaf discs of 6mm diameter were randomly taken for each combined inoculation area. The luciferin luminescence signal was quantitatively determined by using a microplate reader, and the obtained data was processed and analyzed, so that the luciferin luminescence signal of the co-infiltrated region of NbRNF170L-nLuc and cLuc-NbRHD3 was significantly higher than that of the other three negative controls (FIG. 3B).
To further verify the interaction of NbRNF170L with NbRHD3 in vivo, we performed an immunoprecipitation assay. eGFP-NbRHD3 was constructed, and NbRNF170L-HA and eGFP-NbRHD3 were used as test groups, and HA and eGFP were used as negative controls, respectively. NbRNF170L-HA was successfully detected using the HA antibody after capturing eGFP-NbRHD3, whereas the corresponding band was not detected by the control capturing eGFP, again demonstrating the in vivo interaction of NbRNF170L with NbRHD3 (FIG. 3C). In addition, it is apparent from the figure that the protein accumulation amount of eGFP-NbRHD3 is lower when eGFP-NbRHD3 is co-infiltrated with HA than when it is co-infiltrated with NbRNF170L-HA.
To study the localization of NbRNF170L and NbRHD3 in vivo, nbRNF170L-mCherry was constructed. NbRNF170L-mCherry was co-infiltrated with eGFP-NbRHD3 into Benshi smoke, and after 48h of inoculation, the presence of NbRNF170L-mCherry was co-localized with eGFP-NbRHD3 (FIG. 3D).
(2) Silencing effects of the Nicotiana benthamiana NbRHD3 gene on PVY infection:
to investigate the effect of silencing NbRHD3 on PVY infection, TRV2:: nbRHD3, TRV2:: GUS was constructed as a negative control. On day 10 after silencing, compared with the control, the NbRHD 3-silenced Nicotiana benthamiana plants showed obvious dwarf and leaf curl and the like symptoms, and seriously affected the growth and development of plants (figure 4A). qPCR detected NbRHD3 silencing efficiency, significantly reduced mRNA accumulation levels of NbRHD3 as compared to control, up to 80% (fig. 4B). Fifth day after rubbing PVY-GFP, no GFP fluorescence or lower fluorescence intensity was observed in NbRHD 3-silenced benshisha under uv irradiation (fig. 4C). Western blot examined PVY CP accumulation, significantly reduced virus accumulation in the silenced NbRHD3 plants compared to the control (FIG. 4E). The time of fluorescence occurrence after PVY-GFP inoculation was counted every 24 hours, and it was evident that silencing NbRHD3 Benshisha inhibited PVY infection to a large extent after line drawing (FIG. 4D).
These results indicate that silencing NbRHD3 inhibits PVY GFP infection.
(3) NbRNF170L degradation NbRHD3 study results:
to investigate whether NbRNF170L can degrade NbRHD3, we constructed FLuc-NbRHD3, and co-infiltrated FLuc-NbRHD3 with NbRNF170L-HA as a control. After 60 hours, the observation was carried out under an in vivo imaging system, and it was found from the figure that the luciferase protein accumulation level in the region co-infiltrated with NbRNF170L-HA and FLuc-NbRHD3 was extremely low, and the luminescence signal could hardly be detected. Whereas the level of FLuc-NbRHD3 protein accumulation co-infiltrated with HA was higher (FIG. 5A). The luciferin luminescence signal in the inoculated area was quantitatively measured by using a microplate reader, and it can be seen from the figure that the FLuc-NbRHD3 luciferin luminescence signal co-infiltrated with NbRNF170L-HA was significantly reduced (FIG. 5B). Western blot detection of normal expression of NbRNF170L-HA protein (FIG. 5C). To exclude the effect of FLuc, it was investigated whether NbRNF170L specifically degraded NbRHD3. As is clear from the observation of in vivo imaging after 60 hours by co-infiltrating NbRNF170L-HA and HA with FLuc empty vector, there was no significant difference in FLuc fluorescence intensity when co-infiltrating NbRNF170L-HA (FIG. 5D). The enzyme-labeled instrument quantitatively determines the luciferin luminescence signal. As can be seen from the bar graph, the luciferin luminescence signal when FLuc co-infiltrated with NbRNF170L-HA was slightly lower but not significantly different from that of the control (FIG. 5E), and Western blot detected that NbRNF170L-HA protein was normally expressed (FIG. 5F). It was demonstrated that degradation of NbRHD3 by NbRNF170L may be specific.
To further verify that NbRNF170L was able to specifically degrade NbRHD3, eGFP-NbRHD3 or eGFP was co-infiltrated with HA and NbRNF170L-HA, and after 60h, samples were taken to extract total plant protein, and GFP antibodies were used to detect the accumulation of eGFP-NbRHD3 and eGFP protein. Western blot results showed that the protein accumulation was significantly reduced at the time of co-infiltration of eGFP-NbRHD3 with NbRNF170L, approximately 1/10 of that of the control (FIG. 5G). In addition, in the figure, we can see that eGFP-NbRHD3 has more obvious degradation trace. Whereas there was no significant difference in the co-infiltration of eGFP with both separately (fig. 5H). qPCR examined the effect of over-expression of NbRNF170L on mRNA levels of NbRHD3, showing no significant differences (fig. 5I).
Thus, the above results indicate that NbRNF170L is capable of specifically degrading NbRHD3.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (8)
- Use of the e3 ubiquitin ligase RNF170L in (1) or (2) as follows:(1) Inhibiting infection of potato virus Y;(2) Improving the resistance of the plants to potato virus Y;the amino acid sequence of the E3 ubiquitin ligase RNF170L is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein the E3 ubiquitin ligase RNF170L inhibits infection by potyvirus by:(a) Interact with the 6K2 protein in potyvirus;(b) Interact with NbRHD3 and promote degradation of NbRHD3.
- Use of a gene encoding the e3 ubiquitin ligase RNF170L in (1) or (2) as follows:(1) Regulating and controlling resistance of plants to potato virus Y;(2) Cultivating a potato Y virus resistant plant variety.
- 4. The use according to claim 3, wherein the gene encoding E3 ubiquitin ligase RNF170L is a nucleic acid molecule as shown in (i) or (ii):(i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 2;(ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 1.
- 5. The application of a recombinant expression vector or engineering bacteria containing the coding gene of E3 ubiquitin ligase RNF170L in the following (1) or (2):(1) Regulating and controlling resistance of plants to potato virus Y;(2) Cultivating a potato Y virus resistant plant variety.
- 6. A method of increasing resistance of a plant to potyvirus comprising the steps of:overexpression of a gene encoding E3 ubiquitin ligase RNF170L in the plant;alternatively, the NbRHD3 gene in the plant is silenced or knocked out.
- 7. A method of growing a transgenic plant resistant to potyvirus comprising the steps of:introducing an RNF170L gene into a wild-type plant to obtain a plant with high RNF170L gene expression; the resistance of the obtained RNF170L gene high-expression plant to potato virus Y is higher than that of the wild plant.
- 8. The method of claim 7, wherein the RNF170L gene is a nucleic acid molecule as set forth in (i) or (ii) below:(i) A nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 2;(ii) A nucleic acid molecule other than (i) encoding the amino acid sequence shown in SEQ ID NO. 1.
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