CN113234694A - Application of apple MdBT2 in prevention and treatment of apple mosaic disease - Google Patents

Abstract

Experiments prove that MdBT2 interacts with ApNMV virus protein 1a and promotes 1a ubiquitination degradation through a 26S proteasome pathway. Compared with Wild Type (WT), MdBT2 overexpression (MdBT2-OE) inhibits the accumulation of ApNMV genomic RNA in apple leaves, but MdBT2 gene silencing (MdBT2-anti) enhances the accumulation of ApNMV genomic RNA in leaves. Furthermore, MdBT2 may also inhibit 1a and 2a by interacting with 1a^polThe interaction between them. Thus, the nitrate response protein MdBT2 in apple mediates ubiquitination degradation of the ApNMV 1a protein and interferes with viral replication proteins 1a and 2a^polIn betweenInteract to inhibit ApNMV viral RNA replication. Based on this, MdBT2 can be applied to gene breeding work as a potential target gene to prevent and control apple mosaic disease.

Description

Application of apple MdBT2 in prevention and treatment of apple mosaic disease

Technical Field

The invention belongs to the technical field of apple mosaic disease prevention and treatment, and relates to application of apple MdBT2 in prevention and treatment of apple mosaic disease.

Background

Apple mosaic is a viral disease that is widespread and widespread throughout the world, and seriously affects apple yield and quality. The causative agent of the disease has been conventionally considered to be apple mosaic virus (ApMV) of the genus labrocavirus (ilarovirus) of the brome mosaic virus family (bromoviliadae). However, recent studies have shown that apple necrotic mosaic virus (ApNMV) is closely related to the occurrence of apple mosaic disease in china. The A pNMV and the ApMV belong to the same genus and have similar genome structures. They all contained three plus-sense single-stranded genomic RNAs (RNA1, RNA2, and RNA3) and a subgenomic RNA4 derived from RNA 3. RNA1 encodes a 1a protein comprising an N-terminal Methyltransferase (MET) and a C-terminal NTP-binding Helicase (HEL) domain. RNA2 encodes a viral-dependent RNA polymerase. The Motor Protein (MP) is encoded by RNA3, while the Capsid Protein (CP) is encoded by subgenomic RNA 4. According to the reported model virus Brome Mosaic Virus (BMV) with similar genome structure and isogeny with ApNMV, 1a is a multifunctional protein playing a key role in virus replication, including inducing the formation of Virus Replication Complex (VRC), recruiting 2a^polAnd template RNA into the VRC and facilitates viral genomic RNA replication.

In the natural environment, plants are constantly exposed to a wide variety of biotic and abiotic stresses that compromise their growth and development. In order to cope with these adversity stresses, plants have evolved various complex and effective defense mechanisms, of which the highly conserved ubiquitin-proteasome system (UPS) in eukaryotes is one. U PS is an enzymatic process that plays a key role in a variety of biological processes within plant cells, in which a ubiquitin molecule is covalently bound to a substrate protein, causing the substrate protein to be degraded by the proteasome. The ubiquitination process is performed by the co-participation of a variety of enzymes, including ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin E3 ligase (E3). Among them, E3 is a key component that achieves specific recognition and binding of substrates through interaction with substrate proteins, and transfers ubiquitin from E2 to substrate proteins and mediates their degradation through the proteasome pathway. There are four major types of E3 ligases in plants, including the H ECT, RING, U-box and CRLs (cullin-RING ligases). CRL is the most abundant E3 ligase in plants, and exists in the form of a complex. The CRL complex comprises a Cullin (CUL) subunit as a molecular skeleton, and currently, three types of CULs (C UL1, CUL3, and CUL4) are mainly present in plants.

BTB-type E3 ligase is one of the CRL subfamilies. In the CUL3-RI NG E3 ligase (CRL3) of the model plant Arabidopsis thaliana, the BTB/POZ domain-containing protein directly interacts with CUL3 and a substrate protein, and thus the BTB protein can be used as a substrate receptor to select the substrate protein for degradation by UPS. There is now a great deal of evidence that BTB/POZ domain-containing proteins play a central role in a variety of intracellular processes. In Arabidopsis, AtBT2 contains an N-terminal BTB/POZ domain, a central TAZ domain and a C-terminal calmodulin domain. It has been reported that AtBT2 is involved in the regulation of a variety of biological responses, including responses to circadian rhythms, light, stress and nutrition. Modulating the phytohormone response by inhibiting abscisic acid (ABA) signaling while enhancing auxin signaling; TELOMERASE activity is regulated by action on TAC1(TELOMERASE ACTIVATOR 1). MdBT2 is a homologue of AtBT2, has a similar protein structure to AtBT2, and can also act as a signaling hub to regulate anthocyanin biosynthesis, leaf senescence, iron homeostasis, and malate accumulation to address a variety of hormonal and environmental signals. For example, under nitrate treatment, MdBT2 interacts with and promotes ubiquitination degradation of M dMYB1 and MdCIbHLH1, thereby inhibiting the accumulation of anthocyanin and malate, respectively. Furthermore, MdBT2 can also interact with MdbHLH93 and MdMY C2 in apples and promote their ubiquitination degradation to delay leaf senescence.

However, no description is given of the role of MdBT2 in the response of apple to apple necrotic mosaic virus (ApNMV).

Disclosure of Invention

In order to overcome the technical problems, the invention discovers that MdBT2 interacts with ApNMV virus protein 1a and promotes the degradation of the ApNMV virus protein through ubiquitin-26S proteasome pathway. Compared with Wild Type (WT), MdBT2 overexpression (MdBT2-OE) inhibits the accumulation of ApNMV genomic RNA in apple leaves, but silencing MdBT2(MdBT2-anti) enhances the accumulation of ApNMV genomic RNA in leaves. Furthermore, MdBT2 interferes with 1a and 2a by competitively interacting with 1a^polThe interaction between the two can also inhibit the genomic RNA accumulation of ApNMV to a certain extent.

In a first aspect, the invention provides application of apple MdBT2 protein or a coding gene thereof in preventing and treating apple mosaic disease, and further the apple mosaic disease is caused by apple necrotic mosaic virus (ApN MV).

Further, the MdBT2 protein or the encoding gene thereof inhibits the accumulation of apple necrotic mosaic virus (ApNMV) by interacting with 1a protein in apple necrotic mosaic virus (ApNMV).

Further, the MdBT2 protein is (a) or (b) as follows:

(a) consisting of SEQ ID NO: 1, and the protein consists of an amino acid sequence shown in the specification;

(b) converting SEQ ID NO: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and can be combined with a protein 1a in apple necrotic mosaic virus (ApNMV) and is derived from the protein (a).

Further, the encoding gene is (a) or (b) as follows:

(a) consisting of SEQ ID NO: 2;

(b) a DNA molecule which hybridizes with the DNA sequence defined in (a) under strict conditions and codes for the apple MdBT2 protein.

In a second aspect, the present invention provides a method for controlling apple mosaic disease, the method comprising the step of increasing the expression level of MdBT2 protein or mRNA transcription level of apples in plants, wherein said MdBT2 protein is as follows (a) or (b):

(a) consisting of SEQ ID NO: 1, and the protein consists of an amino acid sequence shown in the specification;

(b) converting SEQ ID NO: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and can be combined with a protein 1a in apple necrotic mosaic virus (ApNMV) and is derived from the protein (a).

Further, the method comprises the step of introducing the apple MdBT2 protein into an apple.

Furthermore, the introduction of the apple MdBT2 protein is realized by introducing a coding gene of the apple MdBT2 protein, wherein the coding gene is (a) or (b) as follows:

(a) consisting of SEQ ID NO: 2;

(b) a DNA molecule which is hybridized with the DNA sequence defined in (a) under strict conditions and codes the apple MdBT2 protein.

Further, the introduction is performed by agrobacterium tumefaciens containing a recombinant vector containing a gene encoding the apple MdBT2 protein.

Compared with the prior art, the invention has the technical effects that:

the invention identifies a nitrate response BTB protein MdBT2 in apples, which mediates ubiquitination degradation of ApNMV 1a protein and interferes with virus replication proteins 1a and 2a^polThe interaction between them serves to inhibit ApNMV viral RNA replication. Therefore, MdBT2 can be used as a target gene to be applied to gene breeding work to control apple mosaic disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 shows that nitrate treatment inhibited ApNMV viral RNA accumulation and viral protein 1a stability. Schematic representation of the construction of apnmv invasive clones. Three RNA segments are respectively constructed into plant binary expression vectors and started by CaMV 35SThe mover drives expression, followed by a ribozyme (Rz) and a Nos terminator (T). B. With different concentrations of KNO₃Levels of ApNMV genomic RNA accumulation in GL3 leaf discs were treated. Northern blot was performed with probes against DIG-labeled targeting CP coding sequences to detect the level of accumulation of viral (+) RNA3 and (+) RNA 4. The number indicates the signal intensity of the (+) RNA3 band, and the band intensity at 0mM was set to 100. rRNA was used as a loading control. C. With different concentrations of KNO₃Protein level of 1a in treated GL3 leaf. 1 a-specific antibodies were used for Western blot analysis of 1a protein levels. MdACTIN was used as loading control. The number indicates the signal intensity, and the intensity of the 0mM band is set to 1.00. D. In vitro protein degradation assays detect 1a-HIS protein degradation. E. Nitrate-mediated degradation of 1a-HIS protein was inhibited in the presence of MG 132. (D) The protein extract compound used in (A) and (E) is obtained by using KNO₃Or KCl pretreated GL3 leaf. F. Detection of KCl or KNO by in vitro protein degradation assay₃Treatment pair 2a^pol-effect of HIS protein stability.

Fig. 2 is the interaction of MdBT2 with ApNMV 1a in vivo and in vitro. Y2H experiments demonstrated that BACK-like and TAZ domains are collectively responsible for the interaction of MdBT2-1 a. The y2h assay showed that the full length of 1a is responsible for the 1a-MdBT2 interaction. 1a is divided into N-terminal MET and C-terminal HEL. Bifc assay demonstrated that MdBT2 interacts with 1 a. DAPI (4',6-diamidino-2-phenyli ndole) was used as a nuclear dye. Pull-down experiments showed the presence of in vitro interaction between MdBT2 and 1 a. GST and HIS antibodies were used to detect the target protein. The short lines on the right indicate the position of GST-1a, GST, MdBT 2-HIS. E. Luciferase complementation assay demonstrated that MdBT2 interacts with 1 a. The right color bar represents the captured signal strength. Empty nLuci and cluuci vectors served as controls.

FIG. 3 shows that MdBT2 promotes 1a ubiquitination degradation in vivo and in vitro. The effect of MdBT2 on the degradation of 1a-HIS protein was examined using an in vitro protein degradation assay in the presence (A) or absence (B) of MG 132. The graph on the right shows the protein degradation tendency, and the gray value of the protein band of 0h is set to 1.00. MdActin was used as loading control. C. Protein levels of 1a-HA in transgenic apple leaves. MdACTIN was used as loading control. Mdbt2 mediates ubiquitination of 1a protein in vitro. HIS and Ubi antibodies were used to detect the target protein. The 1a-HIS and Ubi (n) -1a-HIS markers are on the left. MdBT2 mediates the in vivo ubiquitination of 1a-HA proteins. HA antibodies were used for Immunoprecipitation (IP), HA and Ubi antibodies were used for Immunoblotting (IB). Input represents samples collected before IP and detected with mdactn antibody. The 1a-HA and Ubi (n) -1a-HA markers are on the left.

Fig. 4 shows that MdBT2 inhibits ApNMV genomic RNA replication by promoting ubiquitination degradation of viral protein 1 a. Protein levels of 1a in wt, MdBT2-OE and MdBT2-anti transgenic apple seedlings leaves. The 1a specific antibody is used for detecting the 1a protein by Western blot. MdACTIN was used as loading control. MdBT2 mediates ubiquitination of 1a protein in vivo. Antibody 1a was used for immunoprecipitation (I P) and antibodies 1a and Ubi were used for immunoblotting. C. Northern blot was used to detect the level of ApNMV genomic RNA in wt, MdBT2-OE and MdBT2-anti transgenic apple seedling leaves. Accumulation levels of viral (+) RNA3 and (+) RNA4 were detected with D IG labeled probes targeting the CP coding sequence. The number represents the signal intensity of (+) RNA3, and the band intensity of WT was set to 100.

FIG. 5 shows MdBT2 interference 1a and 2a^polThe interaction between them. A. Validation of 1a and 2a Using luciferase complementation assay^polThe interaction between them. B. Validation of MdBT2 pairs 1a and 2a using luciferase complementation assay^polThe effect of the interaction. Empty nLuci and cluuci vectors served as controls. C. Detection of MdBT2 pairs 1a and 2a using Pull-down technique^polThe impact of the interactions. HIS antibodies were used to detect 1a-HIS and MdBT2-HIS proteins.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are merely for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The following are materials and methods used in the present invention

In the present invention, the plant material and growth conditions used are: tissue-cultured apple seedlings (Malus domestica cv "Royal Gala") "GL 3" were used. MdBT2 overexpression (MdBT2-OE) and MdBT2 gene silencing (MdBT2-anti) transgenic apple seedlings were obtained in our previous work and stored in our laboratory. WT and transgenic "GL 3" seedlings were grown on Murashige-Skoog (MS) medium containing 0.2mg/L GA (gibberellin), 0.6 mg/L6-BA and 0.2mg/L NAA (naphthylacetic acid). Apple seedlings were cultivated at 25 ℃ under long day (16 h: 8 h/light: dark). MdBT2 overexpression (MdBT2-OE) and MdBT2 gene silencing (Md BT2-anti) transgenic apple calli were used as laboratory-stored test materials. WT and transgenic apple callus Orin were cultured at 25 ℃ in constant darkness on MS medium containing 0.4 mg/L6-BA and 1.5 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid). Burley tobacco was cured at 23 ℃ for 16 h: 8 h/illumination: culturing under dark condition.

In the present invention, plasmid construction and agrobacterium transformation: all PCR-amplified fragments were inserted into a p EASY-Blunt-simple (TransGen Biotech) cloning vector and then constructed into an expression vector. Viral proteins and the coding sequence of MdBT2 were inserted into pGAD424 and pGBT9 for yeast two-hybrid (Y2H) analysis. 1a, MdBT2 and 2a^polWas constructed into pGEX-4T-1 and pET-32 a to obtain GST-and-HIS-tagged fusion proteins for pull-down assay. For two-molecule fluorescence complementation (BiFC) analysis, the coding sequences of 1a and MdBT2 were constructed into 35S:: SPY CE-cYFP and 35S:: SPYNE-nYFP to obtain 1a-cYFP and MdBT2-nYFP, respectively. Similarly, 1a, MdBT2 and 2a^polThe coding sequence of (a) was inserted into pGreenII 62-SK-nLuc/-cLuc for luciferase complementation imaging analysis. The coding sequences of 1a and MdBT2 were inserted into pCX SN-HA to obtain pCXSN-1a-HA and pCXSN-MdBT 2-HA. The binary plant expression vector used for this work was driven by the cauliflower mosaic virus (CaMV)35S promoter. Three fragments of ApNMV were amplified from our previously isolated ApNMV-Lw and constructed into the pC AMBIA1300 vector, where the viral genes were driven by the double 35S promoter. For Agrobacterium-mediated transformation, a suitable expression vector is transformed into Agrobacterium tumefaciens LBA4404 strain and cultured in LB medium containing the corresponding antibioticCulturing in culture medium.

In the present invention, gene expression analysis: and extracting the total RNA of the apple leaves subjected to different treatments by using an OminiPlant RNA kit. First strand cDNA was synthesized using the PrimeScript first strand cDNA synthesis kit (TaKaRa) according to the manufacturer's instructions. Forward and reverse primers (0.25. mu.M each), template cDNA (50ng) and SYBR Green PCR Master Mix (10. mu.l) were mixed and assembled into a real-time quantitative PCR (qRT-PCR) reaction solution (20. mu.l). The reaction program is 95 ℃ for 5 minutes; 95 ℃, 15s, 60 ℃, 1 minute, for 40 cycles. 18S rRNA was used as a control. Relative amount of Gene expression 2^-ΔΔCtAnd (4) calculating by using the method. Three biological replicates were performed for each individual experiment.

In the present invention, a yeast two-hybrid cDNA library was constructed by Oebiotech Company using apple peel. Amplification of coding sequences of target genes, including Virus 2a^polMP and CP and truncated M dBT2 and ApNMV 1a, and inserted into pGAD424 and pGBT9 vectors. The different combinations of vectors were co-transformed into yeast and then cultured at 28 ℃ for 2-3d on medium lacking leucine (Leu) and tryptophan (Trp). The yeast colonies were then transferred to selection medium without Leu, Trp, histidine (His) and adenine (Ade) to determine the interaction between the different proteins.

In the present invention, bimolecular fluorescence complementation (BiFC): firstly, the coding sequences of ApNMV 1a and MdBT2 are respectively inserted into 35S-SPYCE-cYFP and 35S-SPYNE-nYFP vectors. The recombinant vector was transformed into Agrobacterium LBA4404 and co-injected into Nicotiana benthamiana leaves. Two days after infection, the YFP (yellow fluorescent protein) signal was observed under a confocal microscope (Zeiss, LSM 880). The different signals are obtained in "best signal" mode, in which the image is obtained by a single optical portion, with a scanning speed of 6 (scanning time of about 10 s).

In the present invention, luciferase complementation imaging assay: the coding sequences of ApNMV 1a and MdBT2 were constructed into pGreenII 62-KS-cLuc and-nLuc vectors, respectively. The recombinants were transformed into Agrobacterium LBA4404 and co-infected with Nicotiana benthamiana leaves. Two days after infection, the substrate of luciferase is sprayed on the back of the leaf and is in darknessAnd incubated for 3 minutes. Bioluminescent signals are detected under an in vivo imaging system. In the IVIS acquisition control panel, "light emission" is selected as "imaging mode", and the exposure time is set to 30 s. After the picture is taken, the tone scale (displayed as a color bar on the right side of the image) is adjusted to improve the contrast of the image. For competitive assays, ApNMV 1a and 2a were tested as described previously^polThe coding sequence of (a) was inserted into pGreenII 62-KS-nLuc and-cLuc vectors, whereas MdBT2 was constructed into p CXSN-HA vectors and all constructs were transformed into Agrobacterium LBA 4404.

Example 1 nitrate treatment reduces ApNMV viral RNA accumulation and 1a protein stability

To determine whether nitrate affects ApNMV infection, an invasive clone of ApNMV was first constructed by inserting RNA1, RNA2, and RNA3, respectively, of ApNMV into a binary vector driven by a dual CaMV 35S promoter (fig. 1A). And then respectively transforming the recombinant vectors into agrobacterium tumefaciens, and co-transforming the leaves of the apple seedlings treated by different nitrate concentrations by a vacuumizing method. The accumulation of viral RNA was then detected by Northern blot using DIG labeled probes targeting the CP coding sequence, and RNA levels were found to follow KNO₃The concentration increased and decreased (fig. 1B). Since replication proteins play a key role in viral genomic RNA replication, nitrate pairs 1a and 2a were tested in vivo and in vitro^pol(two replicator proteins of ApNMV) protein levels. By using 1a specific antibodies, the abundance of 1a protein was found to follow KNO₃The concentration increased and decreased (fig. 1C), which is consistent with R NA accumulation (fig. 1B). Nitrate pairs 1a and 2a were then detected using an in vitro protein degradation assay^polInfluence of protein stability. In nitrate treatment, the transcript levels of the nitrate-responsive genes mdnrt1.1 and M dBT2 increased with increasing nitrate concentration, confirming that these apple seedlings did respond to nitrate treatment. Then, the same amount of 1a-HIS or 2a obtained from the prokaryotic expression system^pol-H IS fusion protein with secondary KNO₃Or total protein extracted from KCl-treated "GL 3" leaf discs were mixed and incubated for various periods of time. Western blot analysis showed that the protein from KNO ₃1a-HIS protein of protein extracted from treated GL3 leafThe rate of degradation was faster than the protein extracted from KCl treatment (FIG. 1D), 2a^polThe protein degradation rate of HIS was similar under both treatments (FIG. 1F). This indicates that nitrate treatment reduced protein stability for 1a, but did not affect 2a^polThe protein stability of (3).

To determine whether nitrate-induced degradation of 1a-HIS protein was dependent on the proteasomal pathway, the proteasome inhibitor MG132 was added to the assay, with DMSO as a control. Will be driven from KNO₃Total protein extracted from treated "GL 3" leaves was treated with MG132 or DMSO for 30 minutes, and then 1a-HIS fusion protein was added to these samples and assayed as described above. The results show that addition of MG132 almost completely inhibited degradation of 1a-HIS protein, whereas DMSO had no effect on this (fig. 1E). These data indicate that KNO₃The induced degradation of 1a-HIS protein is mediated by the proteasomal pathway. Overall, these data indicate that nitrate treatment inhibited the accumulation of ApNMV genomic RN a and reduced the stability of viral protein 1 a.

Example 2 ApNMV 1a interaction with MdBT2

To identify host factors in apples that are involved in nitrate-promoted ApNMV 1a degradation, we screened an apple cDNA library using viral protein 1a as bait and identified MdBT2(MDP 0000643281) as a potential target protein. The protein contains an N-terminal BTB domain, an intermediate B ACK-like domain and a C-terminal TAZ domain (fig. 2A). To confirm the interaction between ApNMV 1a and MdBT2, we validated with the Y2H assay. The coding sequences for 1a and MdBT2 were first inserted into pGBT9 and pGAD424, respectively, and then co-transformed into yeast cells. The results indicated that yeast colonies grew normally in SD/-Trp/-Leu/-His/-Ade deficient medium, indicating an interaction between 1a and MdBT 2.

To determine the critical segments responsible for the interaction of the two, MdBT2 was first cut into several fragments according to the distribution of the domains (fig. 2A), which were then constructed separately into pGBD vectors with full-length MdBT2 used as a positive control. The results showed that yeast grew on S D/-Trp/-Leu/-His/-Ade deficient medium only when BACK-like and TAZ were present at the same time, indicating that these two domains are responsible for the interaction of MdBT2 with 1a (FIG. 2A). Likewise, 1a was divided into N-terminal MET domain and C-terminal HEL domain, which were found not to interact with MdBT2 when present alone (fig. 2B), suggesting that the full length of 1a is responsible for 1a-MdBT2 interaction. We then further validated the 1a-MdBT2 interaction using BiFC assay. Agrobacterium harboring MdBT2-nYFP and 1a-cYFP vectors were co-injected into Nicotiana benthamiana leaves and a strong yellow fluorescent signal was captured in the cytoplasm under a confocal microscope (FIG. 2C, top panel). However, when MdBT2-nYFP was co-injected with cYPF (FIG. 2C, middle panel), or 1a-cYFP was co-injected with nYFP (FIG. 2C, bottom panel), no signal was observed for either. These results indicate that ApNMV 1a interacts with MdBT2 in vivo. We next validated the in vitro interaction of the two using pull-down technique. The fusion proteins GST-1a or GS T were first incubated with MdBT2-HIS and magnetic beads attached with GSH, followed by elution of the target protein with a GSH solution and detection with GST and HIS antibodies. The results show that MdBT2-HIS fusion protein is recruited in the presence of GST-1a instead of GS T (FIG. 2D), indicating that GST-1a interacts with MdBT2-HIS in vitro. Finally, we validated the interaction between 1a-MdBT2 using luciferase complementation assay. Agrobacterium with MdBT2-nLuci and cLuci-1a were co-injected into B.benthamiana leaves, and the empty vector was used as a control. The results show that luminescent signal was only observed when MdBT2-nLuci and cLuci-1a were co-injected, but no signal was captured in both the MdBT2-nLuci plus cLuci combination and the nLuci plus cLuci-1a combination (FIG. 2E). These results indicate that ApNMV 1a interacts with MdBT2 in vivo.

Example 3 MdBT2 promotes ubiquitination degradation of ApNMV 1a

Apple calli of wt, MdBT2-OE and MdBT2-anti were first used for in vitro proteolytic assays. Compared with WT, the gene expression level of MdBT2 was significantly increased in MdBT2-OE callus, but significantly decreased in MdBT2-anti, indicating that these transgenic calli were suitable for functional analysis of MdBT 2. Total protein was then extracted from these three types of apple callus and they were incubated with 1a-HIS fusion protein for different periods of time. After immunoblotting with HIS antibody, it was found that 1a-HIS was degraded more rapidly in total protein extracted from MdBT2-OE transgenic calli than WT, but that 1a-HIS was degraded more slowly in protein extracts of MdBT2-anti (FIG. 3A). This indicates that MdBT2 promotes in vitro degradation of 1a protein. To verify whether proteasome participates in 1a-HIS degradation, we applied the proteasome inhibitor MG132 to these experiments and found that 1a-HIS protein degradation rate was inhibited in all three types of apple callus (fig. 3B), suggesting that the protein may be degraded by the 26S proteasome pathway.

Agrobacterium carrying pCXSN-1a-HA was transformed into WT, MdBT2-OE and MdBT2-anti "GL 3" apple seedling leaves by vacuum pumping. Transgenic apple seedlings are obtained and stored in our laboratories in previous work, and we firstly use qRT-PCR to confirm that the expression level of MdBT2 gene is obviously higher than that of WT in MdBT2-OE transgenic seedlings but is obviously reduced in MdBT2-anti seedlings, which indicates that the transgenic plants are suitable for MdBT2 functional analysis. Then, total protein was extracted from apple leaves five days after transformation, and HA antibody was used for immunoblotting. The results showed that the 1a-HA protein level in MdBT2-OE leaves was lower than wild type, but the 1a-HA protein level in MdBT2-anti transgenic leaves was higher than wild type (FIG. 3C), indicating that MdBT2 promotes the in vivo degradation of 1a-HA protein. Next, we tested whether MdBT2 could promote ApNMV 1a protein ubiquitination. Active MdBT2-GFP protein was obtained by first immunoprecipitation from total protein extracts of MdBT2-OE calli using GFP antibody. The 1a-HIS protein was then incubated with human E1, E2, Ubi and active MdBT2-GFP in incubation buffer and detected by HIS and Ubi antibodies. The results show that 1a-HIS high molecular form of polyubiquitinated 1a-HIS (Ubi (n) -1a-HIS) was detected in the presence of active MdBT2 protein when immunoblotting was performed with HIS antibody (FIG. 3D, top). When immunoblotting was performed using Ubi antibodies, higher numbers of ubiquitinated proteins were detected in the presence of active MdBT2-GFP (fig. 3D, bottom). These data indicate that MdBT2 is capable of promoting the in vitro ubiquitination of 1 a-HIS. We then transformed pCXSN-1a-HA into WT, M dBT2-OE and MdBT2-anti apple seedlings leaves of 'GL3', treated with MG132, immunoprecipitated with HA antibody against total protein extracts of three types of apple leaves, and then detected the precipitate using HA (FIG. 3E, top) and Ubi (FIG. 3E, bottom) antibodies. Compared with WT, the content of Ubi (n) -1a-HA in MdBT2-OE leaf was higher, while the content of Ubi (n) -1a-HA in MdBT2-anti leaf was lower (FIG. 3E), indicating that MdBT2 promotes the ubiquitination of 1a protein in vivo. Thus, these results indicate that MdBT2 can promote ubiquitination and degradation of 1a protein through the UPS pathway.

Example 4 MdBT2 inhibits ApNMV genomic RNA accumulation by promoting 1a ubiquitination degradation

The infectious clone of ApNMV was transformed into apple leaves of WT, MdBT2-OE and Md BT2-anti by evacuation. Five days after transformation, leaves were collected and total protein and total RNA were extracted. The protein level of 1a was first tested using a1 a-specific antibody and it was found that the 1a protein level was lower in MdBT2-OE leaves but higher in MdBT2-anti leaves compared to WT (fig. 4B), indicating that MdBT2 promotes 1a protein degradation during viral infection. The amount of MdBT 2-mediated polyubiquitination 1a was found to be higher in MdBT2-OE leaves and lower in MdBT2-anti leaves compared to WT (FIG. 4C), indicating that MdBT2 enhanced the level of ubiquitination of the viral protein 1 a. The role of MdBT2 in ApNMV viral RNA replication was examined by Northern blot using DIG-labeled probes targeting the CP-encoding gene. The results indicate that the level of accumulation of viral RNA is lower in MdBT2-OE leaves compared to leaves in WT leaves, while the level of viral RNA is higher in MdBT2-an ti, indicating that MdBT2 plays a negative regulatory role in ApNMV replication in apples. These results indicate that MdBT2 may inhibit ApNMV genomic RNA replication by promoting ubiquitination degradation of the 1a protein.

Example 5 MdBT2 interference 1a and 2a^polInteraction between them

The relationship between these three proteins was first explored using the luciferase complementation imaging assay. We first identified 1a and 2a^polThe interaction between the two shows that only 1a-nLuci and cLuci-2a exist at the same time^polIn this case, a strong luminescence signal was observed (FIG. 5A). After addition of pCXSN-Md BT2-HA, the signal intensity gradually decreased with increasing ratio of MdBT2-HA (FIG. 5B), indicating MdBT2-HA is reacted with 2a in vivo^polCompete and interfere with its interaction with 1 a. To further validate the MdBT2 pair 1a-2a^polInterference of the interaction, we used GST-2a obtained from prokaryotic expression systems^polMdBT2-HIS and 1a-HIS fusion proteins were subjected to competitive pull-down analysis. The recruitment of 1a-HIS protein gradually decreased with the increase of MdBT2-HIS protein, indicating that MdBT2-HIS is associated with GST-2a^polCompetitive in vitro interaction with 1a-HIS, thereby inhibiting 1a-2a^polThe interaction between (fig. 5C). Thus, in addition to promoting 1a ubiquitination degradation, MdBT2 may also replicate proteins 1a and 2a by interfering with viruses^polInhibit ApNMV viral genomic RNA replication.

In summary, the present invention identifies a nitrate-responsive BTB protein MdBT2 in apple by mediating ubiquitination degradation of the ApNMV 1a protein and interfering with viral replication proteins 1a and 2a^polThe interaction between them serves to inhibit ApNMV viral RNA replication. Therefore, MdBT2 can be used as a potential target gene in gene breeding work to prevent and control apple mosaic disease.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

SEQUENCE LISTING

<110> Shandong university of agriculture

Application of <120> apple MdBT2 in prevention and treatment of apple mosaic disease

<130> 2021

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 361

<212> PRT

<213> Malus domestica

<400> 1

Met Glu Ala Asn Pro Thr Ala Thr Ser Gly Thr Ser Val Asp Leu Tyr

1 5 10 15

Gly Leu Ser Gly Thr Lys Tyr Leu Pro Glu Pro Asp Val Asp Ile Leu

20 25 30

Thr Cys Asp Ala Ile Arg Ile Pro Val His Ser Cys Ile Leu Ala Ser

35 40 45

Val Ser Pro Val Leu Glu Asn Ile Ile Asp Arg Pro Arg Lys His Arg

50 55 60

Ser Ser Glu Arg Val Ile Pro Ile Leu Gly Val Pro Tyr Asp Ala Val

65 70 75 80

Leu Ala Phe Val Arg Phe Leu Tyr Ser Ser Arg Cys Thr Glu Glu Asn

85 90 95

Met Glu Lys Tyr Gly Ile His Leu Leu Ala Leu Ser His Val Tyr Leu

100 105 110

Val Pro Gln Leu Lys Asn Arg Cys Thr Lys Glu Leu Gly Gln Arg Leu

115 120 125

Thr Ile Glu Asn Val Val Asp Val Leu Gln Leu Ala Lys Met Cys Asp

130 135 140

Ala Ala Asp Leu Tyr Leu Lys Cys Met Lys Leu Val Ala Asn His Phe

145 150 155 160

Lys Val Val Glu Thr Thr Glu Gly Trp Lys Phe Leu Gln Ala His Asp

165 170 175

Pro Trp Leu Glu Leu His Ile Met Gln Phe Ile Asp Glu Ile Glu Ser

180 185 190

Arg Lys Lys Arg Thr Arg Arg His Arg Glu Glu Gln Arg Leu Tyr Leu

195 200 205

Gln Leu Ser Glu Ala Met Glu Cys Leu Glu His Ile Cys Lys Glu Gly

210 215 220

Cys Thr Ser Val Gly Pro Tyr Asp Met Glu Pro Gly Tyr Lys Lys Gly

225 230 235 240

Pro Cys Ser Lys Phe Ser Thr Cys Gln Gly Leu Gln Met Leu Ile Gln

245 250 255

His Phe Ala Thr Cys Lys Arg Arg Val Asn Gly Gly Cys Leu Arg Cys

260 265 270

Lys Arg Met Trp Gln Leu Leu Lys Leu His Ser Ser Met Cys Glu Glu

275 280 285

Pro Asp Ser Cys Arg Val Pro Leu Cys Arg Gln Phe Lys Leu Lys Met

290 295 300

Gln Gln Glu Lys Lys Thr Asp Asp Ala Arg Trp Lys Leu Leu Val Lys

305 310 315 320

Lys Val Met Ser Ala Lys Thr Leu Ser Ser Leu Ser Leu Pro Lys Arg

325 330 335

Lys Arg Glu Glu Glu Leu Gly Glu Gly Arg Ser Thr Ser Thr Ile Thr

340 345 350

Ala His Gly Ile Arg Ser Phe Arg Leu

355 360

<210> 2

<211> 1086

<212> DNA

<213> Malus domestica

<400> 2

atggaagcta atccgaccgc aacctccggc acctccgtcg acctgtacgg tctctccggc 60

accaaatatc tccccgaacc cgacgtcgac atcctcacct gcgacgccat ccgcatcccg 120

gtgcactctt gcatcctggc ctcagtgtcg ccggtgctgg agaacataat cgaccggcct 180

cgtaagcacc ggagctcgga gcgggtcatt ccgattctcg gcgttccgta cgacgccgta 240

ttggccttcg tccgctttct ctactcctcc aggtgcacag aagagaacat ggagaagtac 300

gggatccatc tgctggcgct gtcccacgtg tacttggtcc cgcagctgaa gaacagatgc 360

acgaaggagc ttggtcaacg tttgaccatc gaaaacgtgg tggacgtgct gcaactggcg 420

aagatgtgcg atgcagcgga tctctacctc aaatgcatga agttggtcgc taatcacttc 480

aaggttgttg agacaactga aggatggaaa ttcttgcaag ctcacgaccc ttggctcgaa 540

ctccacatca tgcaattcat cgacgaaatt gaatcgagga agaagaggac aaggaggcat 600

agagaggagc agaggttgta tctccagctg agtgaggcaa tggagtgctt ggagcacata 660

tgcaaggaag gttgcactag tgttgggccc tacgacatgg agccaggtta caagaagggc 720

ccatgcagca agttctccac gtgtcaaggc ctccagatgt tgatccagca ctttgccacg 780

tgtaagagga gggtgaatgg agggtgctta cgttgcaagc gcatgtggca gcttcttaag 840

cttcactctt caatgtgcga agaacctgat tcttgcagag tccctctttg caggcaattc 900

aagttgaaaa tgcagcaaga gaagaagacg gacgatgcca ggtggaaact gcttgtgaag 960

aaggtgatgt cggccaaaac cttatcttcc ctgtctctgc caaagaggaa gagggaggaa 1020

gaattaggag aaggaagaag cacttctact attactgctc atggaattag aagcttcaga 1080

ttgtga 1086

Claims (9)

1. Application of apple MdBT2 protein or its coding gene in preventing and treating apple mosaic disease.

2. The use according to claim 1, wherein the apple mosaic disease is caused by apple necrotic mosaic virus (ApNMV).

3. The use according to claim 2, wherein said MdBT2 protein or its encoding gene inhibits the accumulation of apple necrotic mosaic virus (ApNMV) by interacting with the 1a protein in apple necrotic mosaic virus (ApNMV).

4. The use according to claim 1, wherein the MdBT2 protein is as follows (a) or (b):

(a) consisting of SEQ ID NO: 1, and the protein consists of an amino acid sequence shown in the specification;

(b) converting SEQ ID NO: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and can be combined with 1a protein in apple necrotic mosaic virus (ApNMV) and derived from (a).

5. The use according to claim 1 or 2, wherein the coding gene is (a) or (b) as follows:

(a) consisting of SEQ ID NO: 2;

(b) a DNA molecule which is hybridized with the DNA sequence defined in (a) under strict conditions and codes the apple MdBT2 protein.

6. A method for preventing and treating apple mosaic disease, which is characterized by comprising the step of increasing the expression level or mRNA transcription level of MdBT2 protein of apples in plants, wherein the MdBT2 protein is as follows (a) or (b):

(a) consisting of SEQ ID NO: 1, and the protein consists of an amino acid sequence shown in the specification;

(b) converting SEQ ID NO: 1 through substitution and/or deletion and/or addition of one or more amino acid residues, and can be combined with 1a protein in apple necrotic mosaic virus (ApNMV) and derived from (a).

7. The method of claim 6, comprising the step of introducing the apple MdBT2 protein into an apple.

8. The method of claim 7, wherein the introduction of the apple MdBT2 protein is achieved by introducing a gene encoding the apple MdBT2 protein, wherein the gene is (a) or (b):

(a) consisting of SEQ ID NO: 2;

(b) a DNA molecule which is hybridized with the DNA sequence defined in (a) under strict conditions and codes the apple MdBT2 protein.

9. The method according to claim 8, wherein said introduction is carried out by Agrobacterium tumefaciens containing a recombinant vector containing the gene coding for the apple MdBT2 protein.

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