CN116355913A - Soybean vacuole ATPase assembled protein gene Vma12 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to soybean vacuolar ATPase assembly protein gene Vma12 and application thereof. A soybean vacuolar ATPase assembly protein gene Vma12, characterized in that the nucleotide sequence of the gene is shown in SEQ.ID.No:1. The present invention further provides the application of soybean vacuolar ATPase assembly protein gene Vma12 in cultivating soybean varieties resistant to SMV. The invention also provides a genetic engineering improvement method for transforming plant disease resistance aiming at the gene. The method has a certain effect on cultivating soybean mosaic virus-resistant plant varieties, especially soybean varieties, and can serve to improve the disease resistance of crops, especially soybean, through directional transformation of crops.

Description

A kind of soybean vacuolar ATPase assembly protein gene Vma12 and its application

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to soybean vacuolar ATPase assembly protein gene Vma12 and application thereof.

Background technique

V-ATPase is an inner membrane-localized multisubunit protein complex that is a ubiquitous proton pump, and V-ATPase-mediated acidification of intracellular compartments is important for many eukaryotic cellular processes (Huss and Wieczorek 2009). V-ATPase activity can be regulated by modifying the assembly of its cytosolic proton-hydrolyzing _V1 and membrane-localized proton-conducting _V0 subunits. Although Vma12 is not part of the V-ATPase complex, it is required for V-ATPase activity (Hirata et al., 1993). V-ATPase activity is critical for pH homeostasis and organelle acidification, and also plays an important role in membrane fusion, thus affecting viral replication by inhibiting the release of virions into the host cytoplasm (Guinea and Carrasco 1995; Hunt et al. 2011; Pavelin et al. 2017). Soybean mosaic virus (SMV) is one of the main viral diseases of soybean, which seriously affects the yield and quality of soybean. Enveloped viruses such as SMV proliferate by budding from the host cell membrane. Viral entry involves the transfer of viral RNA into the host cytoplasm and also involves membrane fusion between the viral and host membranes (Tsai 2007). The _V0 subunit can directly promote membrane fusion, independent of V-ATPase involved in acidification (Peters et al., 2001). This is related to the negative regulation of the _V0 inhibitor concanavalin A on the pathogenesis of SMV. This, together with the fact that V-ATPase inhibitors prevented receptor-mediated endocytosis and low pH-mediated entry of membrane fusion-derived viruses, suggested that SMV might follow a similar pattern of entry into soybean cells.

As an important leguminous plant, soybean has always occupied a special and important position in the nutrition of humans and animals with its high protein and high oil content. However, the soybean growth process is infected by SMV, which seriously affects the yield and quality. Therefore, in the soybean breeding process Varieties with good disease resistance are required. Therefore, it is of great theoretical and practical significance to clarify the molecular mechanism of SMV infecting soybean and provide a basis for breeding disease-resistant varieties.

Contents of the invention

The purpose of the present invention is to study the molecular mechanism of SMV infecting soybeans, and then search for genes related to the pathogenesis of SMV, and regulate the expression of the genes to achieve the purpose of inhibiting SMV and provide a basis for cultivating new disease-resistant varieties.

In order to achieve the above object, the technical means adopted by the present invention is to provide a soybean vacuolar ATPase assembly protein gene Vma12, the nucleotide sequence of which is shown in SEQ.ID.No:1.

The invention also provides the use of the soybean vacuolar ATPase assembly protein gene Vma12, which can be used to breed soybean varieties resistant to SMV.

The present invention further provides a breeding method for SMV-resistant plant varieties. The method silences the soybean vacuolar ATPase assembly protein gene Vma12 by inserting a silent fragment into the soybean vacuolar ATPase assembly protein gene Vma12 to achieve anti-SMV The function of the SMV.

Further, the amplification primers of the silent fragment are shown in SEQ.ID.No:2 and SEQ.ID.No:3.

The present invention also provides a primer for amplifying the silent fragment of the soybean vacuolar ATPase assembly protein gene Vma12, the sequences of which are shown in SEQ.ID.No:2 and SEQ.ID.No:3.

The present invention has the following beneficial effects: the present invention has cloned a gene Vma12 that plays an important regulatory role in soybean mosaic virus (SMV) infecting soybeans from soybeans, and the mRNA expression analysis of this gene shows that it may participate in soybean regulation of soybean mosaic virus During reproduction, SMV P3 protein interacts with soybean vacuolar ATPase (V-ATPase) assembly protein GmVma12, and the ER membrane localization of GmVma12 is related to that of SMV P3 previously confirmed. GmVma12-knockout soybean silencing material inhibited SMV accumulation and SMV-induced cell death, and exogenous spraying of VATPase inhibitor concanavalin A also inhibited SMV accumulation in soybean, indicating that V-ATPase activity and its role in soybean defense against SMV infection played an important regulatory role. The invention also provides a genetic engineering improvement method for transforming plant disease resistance aiming at the gene. The method has a certain effect on cultivating soybean mosaic virus-resistant plant varieties, especially soybean varieties, and can improve the disease resistance of crops, especially soybean, through directional transformation of crops.

Description of drawings

Fig. 1 is the amino acid sequence alignment of GmVma12-10g and GmVma12-20g in the embodiment of the present invention;

Fig. 2 is the expression situation of GmVma12-10g and GmVma12-20g in soybean root, stem, leaf, flower and cotyledon in the embodiment of the present invention;

Fig. 3 is the subcellular localization of GmVma12 protein in the embodiment of the present invention;

Figure 4 shows that GmVma12 does not interact with SMV HC-Pro;

Figure 5 shows the interaction between GmVma12 and SMV P3;

Fig. 6 is the qRT-PCR result of the transcription situation of gene GmVma12 in the mosaic symptom leaves of the silent material empty vector (V) with mosaic symptoms;

Fig. 7 is the contrast that the plant that the G5 or G7 isolate strain of SMV is inoculated with V and Sil _Vma12 appears SMV-associated symptoms;

Fig. 8 is the detection result of the plant virus coat protein (CP) of G5 or G7 isolates of SMV inoculating V and Sil _Vma12 ;

Fig. 9 is the ELISA detection result of the plant virus total protein extract of the G5 or G7 isolates of SMV inoculated with V and Sil _Vma12 ;

Figure 10 is the SMV accumulation situation of V and Sil _Vma12 plants after inoculating SMV-G5 4dpi and 7dpi;

Figure 11 is the morphological phenotype of V and Sil _Vma12 plants (cv.Rsv1) after infecting SMV-G7 (14dpi);

Figure 12 is the relative mRNA level of cell death marker GmHsr203j of V and Sil _Vma12 plants (cv.Rsv1) at 0dpi and 2dpi;

Figure 13 shows the SMV coat protein (CP) level in V and SilVma12 plants (cv.Rsv1) with SMV-G7 infecting 0-14dpi after Western blot;

Figure 14 is a Western blot display of water or concanamycin A (Con A) treatment of wild-type plants (cv Essex), two days after inoculation of SMV-G5 after 0-10dpi SMV CP protein level;

Figure 15 shows the systemic cell death phenotype after treatment with water or concanamycin A (ConA) and inoculation with SMV-G7 in the Rsv1 background.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. The following description of at least one exemplary embodiment is merely illustrative in nature and in no way taken as any limitation of the invention, its application or uses. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

1. Tissue expression analysis of gene GmVma12

The present invention clones a gene GmVma12 that plays an important regulatory role in soybean mosaic virus (SMV) infecting soybean from soybean, and its sequence is shown in SEQ.ID.No:1. There are two copies of GmVma12 in soybean, which are located on chromosome 10 and chromosome 20, and are denoted as GmVma12-10g and GmVma12-20g, respectively. The amino acid sequence alignment of GmVma12-10g and GmVma12-20g, as shown in Figure 1, the amino acid sequences of these two subtypes are 91% identical (the gray background in the figure represents that the two are the same, and the white represents that the two are different), and Contains two predicted transmembrane domains similar to the yeast Vma12 protein.

The RNA of soybean root, stem, leaf, flower and cotyledon were extracted respectively, and after reverse transcription, fluorescent quantitative PCR was carried out with specific primers of GmVma12-10g and GmVma12-20g.

The fluorescent quantitative PCR specific primer of table 1GmVma12-10g and GmVma12-20g

As shown in Figure 2, fluorescent quantitative PCR analysis showed that the transcript of GmVma12-10g was most abundant in leaf tissue, and some expression was also detected in cotyledon, flower and root tissue, but the amount was less, similarly, GmVma12-20g transcript It is also most abundant in leaf tissue.

2. Analysis of subcellular localization of GmVma12

The present invention uses laser confocal analysis to analyze the subcellular localization of GmVma12 protein, and GFP-labeled GmVma12 is localized in endoplasmic reticulum when transiently expressed in N. benthamiana, as shown in FIG. 3 . This correlates with a previous report by the present research team demonstrating SMV P3ER localization (Luan et al., 2016).

3. Interaction between GmVma12 protein and P3 protein

The coding genes of P3 and GmVma12 were recombined from the entry vector to the BiFc terminal vector pSITE-nEYFP vector and pSITE-cEYFP vector respectively, and the plant expression vector containing the target gene was transformed into Agrobacterium tumefaciens LBA4404, and added in a medium containing 50 mg/ml spectinomycin and 50mg/ml streptomycin LB liquid medium, 28°C, 220rpm shaking culture, to the logarithmic growth phase (OD ₆₀₀ =0.6 or so), 3000rpm centrifugation for 10min, remove the supernatant to collect bacteria. Resuspend the bacteria in the injection culture solution, which contains a final concentration of 10mmol/L MgCl ₂ , 100mmol/L MES (pH=5.6), and 100μmol/L acetosyringone, and adjust the concentration of the bacteria solution so that the OD ₆₀₀ is about 1.0 , placed at room temperature for 3h.

Take the cell suspension expressing the fusion protein of GmVma12-nEYFP and P3-cEYFP, GmVma12-cEYFP and P3-nEYFP in equal amounts, and mix well; use plant protein GST (glutathione-S-transferase) and SMV protein Hc- Pro is a negative control, and the combination is as follows: bacterial suspension of GmVma12-nEYFP and GST-cEYFP, GmVma12-nEYFP and Hc-Pro-cEYFP, Hc-Pro-nEYFP and GmVma12-cEYFP, P3-nEYFP and GST-cEYFP fusion protein solution, mixed evenly, ready to inject cyan fluorescent protein nuclear localized transgenic Nicotiana benthamiana. Scissor the injected leaf tissue with a clean scalpel, place the leaf back on a glass slide, add sterile ddH ₂ O to cover it dropwise, add a cover slip, and gently tap with tweezers to expel air bubbles. The prepared slides were placed under a laser confocal microscope, the excitation wavelength of YFP was selected at 488nm, the focal length was adjusted until the image was clear, observations were made and photographs were taken, and the positions where the fluorescence appeared were recorded. At the same time, identify the expression of the negative protein to ensure that the negative control has no fluorescence because there is no interaction.

Through LR recombination reaction, GmVma12 was recombined into the plant expression vector pGWB11 containing the FLAG tag, P3 was recombined into the pGWB20 vector containing the MYC tag, heat-shock transformed Agrobacterium LBA4404, and GmVma12-FLAG and P3-MYC alone were transiently expressed, and Mixture of GmVma12-FLAG and P3-MYC in wild-type Nicotiana benthamiana. After 2 days, collect the tobacco leaves injected with the bacterial solution, extract the total protein with GTNE buffer, take part of the total protein as a control, add the remaining total protein to the centrifuge tube containing FLAGbeads, put it in a rotary mixer at 4°C and mix overnight . The next day, centrifuge at 2000rpm for 2min, remove the supernatant, add 1000ml of elution buffer, put it in a refrigerator at 4°C for 10min, and repeat washing 3 times. Centrifuge at 2000rpm for 2min, remove all elution buffer and leave only the beads, add loading buffer and boil for 10min, and detect the expression of FLAG and MYC by Western blot.

The fusion proteins of the full-length proteins of GmVma12 and SMV P3 and the N-terminal or C-termini of enhanced yellow fluorescent protein (nEYFP and cEYFP) were transiently expressed in tobacco, and the co-expression of GmVma12 and SMV P3 (G5) fused nEYFP and cEYFP together , leading to the reconstruction of EYFP, fluorescence can be observed under the laser confocal microscope, indicating that there is an interaction between GmVma12 and SMV P3. In contrast, SMV P3 does not interact with GST, and GmVma12 does not interact with SMV HC-Pro, as shown in the figure 4. Co-immunoprecipitation (IP) detection of MYC-tagged SMV P3 and FLAG-tagged GmVma12, respectively, further confirmed the interaction between GmVma12 and SMVP3, as shown in Figure 5.

4. Knockout study of GmVma12 gene in soybean

Design the silencing primer of 192bp (G9-G71) for the silencing fragment of the GmVma12 gene:

F: CGTGGATCCGGTTAGTGATATCCAGA, SEQ.ID.No: 2;

R: CGGTGGCCAAGTCGGGTCAAGTCGGGTCT, SEQ.ID.No:3.

Inserts were amplified with these primers and linearized. Perform in vitro transcription on the linearized product, and the transcription system is shown in Table 2:

Table 2 In vitro transcription system

Take 1 μl of the above in vitro transcription product, and use 1.0% agarose gel electrophoresis to detect the RNA yield of in vitro transcription. The remaining in vitro transcription product BPMV-RNA1 was mixed with the recombinant BPMV-RNA2 in equal amounts for in vitro inoculation experiments.

Healthy soybeans with just flattened opposite true leaves were used as materials for recombinant virus inoculation. Inoculate 5-6 plants in each pot, spray a small amount of carborundum on the leaves, drop 20 μl of the mixture of in vitro transcription product BPMV-RNA1 and recombinant BPMV-RNA2 on each true leaf with a pipette, and rub the leaves to inoculate. The mixture of soybean plants transfected with BPMV-RNA1 virus vector and BPMV-RNA2 empty vector in vitro transcription product was used as control (V) to compare the phenotypic changes of soybean caused by silencing material and virus itself. Soybean plants transfected with BPMV-RNA1 viral vector and recombinant BPMV-RNA2 vector in vitro transcription mixture were used for preliminary functional analysis of target gene silencing.

Take the silencing materials with mosaic symptoms, and detect the transcription of the target gene GmVma12. GmVma12 contains two copies. The qRT-PCR results are shown in Figure 6. The silencing efficiency of the target gene GmVma12-10g is 78%, while GmVma12-20g up-regulates the expression of 24 %.

5. Detection of disease resistance of silent materials

Place the preserved freeze-dried silencing material (soybean leaves of silencing GmVma12-10g) in a mortar, add appropriate amount of corundum and 0.05mol/L potassium phosphate buffer solution (pH 7.2) to fully grind, and inoculate the grinding solution in the tested soybean Essex ( rsv1) and Rsv1 on the opposite true leaves, inoculated with V as a control. When the first pair of three compound leaves unfolded and there were obvious mosaic symptoms, the grinding liquid of SMV G5 and G7 strains and soybean yellow mosaic virus (BYMV) were rubbed inoculated respectively.

Take the leaves before virus inoculation as 0dpi, as a control, collect the inoculated leaves of 4dpi and 7dpi, and the upper leaves of 10dpi and 14dpi respectively, collect 0.1-0.3g of each sample into a 1.5ml centrifuge tube, and store in a -80°C refrigerator. The collected samples were fully ground in liquid nitrogen, added 400 μl GTEN buffer, mixed well, and placed on ice. 12,000g, 4°C, centrifuge for 10min to remove impurities, transfer the supernatant to a new centrifuge tube and place on ice. The protein concentration was measured by the Bradford method, and 1-2 μl of the protein extract was added to 1ml of the Bio-rad chromogenic reagent, and the absorbance was measured with a spectrophotometer OD ₅₉₅ . Use BSA to develop a standard curve, calculate the concentration of the protein extract based on the absorbance value of the sample, take an equivalent amount of total plant protein 20-50 μg for each sample, add an appropriate amount of protein loading buffer, and cook in boiling water for 10 minutes. Protein denaturation for SDS-PAGE protein electrophoresis.

V and Sil _Vma12 plants (soybean plants silenced for Vma12) were inoculated with G5 or G7 isolates of SMV (cv. Essex). Notably, Sil _Vma12 plants developed significantly fewer SMV-associated symptoms compared with V plants treated with both G5 or G7 isolates, as shown in Figure 7. In order to compare the degree of virus accumulation in V and Sil _Vma12 plants, the present invention used Western blot analysis of total protein extracts from inoculated and _upper leaves. In leaves and upper leaves, SMV content was significantly reduced, as shown in Figure 8.

This was not the case for another potato virus, BYMV. Unlike SMV, Sil _Vma12 plants showed a similar level of BYMV accumulation as V plants, as shown at the bottom of Figure 8. ELISA detection of total protein extracts in V and Sil _Vma12 also showed significantly more SMV content in V plants compared to Sil _Vma12 , as shown in FIG. 9 . This correlates with lower SMV positive strand RNA levels in Sil _Vma12 plants than in V plants at both 4dpi and 7dpi, as shown in FIG. 10 .

Plants harboring the Rsv1 locus exhibit a systemic cell death phenotype known as lethal systemic hypersensitivity response to SMV G7. Notably, SMV G7-infected Rsv1 Sil _Vma12 plants did not exhibit a systemic cell death phenotype, as shown in Figure 11. This correlated with a marked reduction in the expression of the cell death marker Hsr203j in SMV G7-infected V and Sil _Vma12 plants (Rsv1 ), as shown in FIG. 12 . Virus accumulation analysis showed that the accumulation level of SMV G7 was significantly reduced in Rsv1 Sil _Vma12 plants compared with Rsv1 V plants, as shown in Figure 13.

The fact that Vma12 is essential for V-ATPase activity prompted the present inventors to test whether inhibition of V-ATPase function would alter soybean response to SMV. In order to test this point, the present invention detected soybean plants (cv Essex) pretreated with water or the V-ATPase inhibitor concanavalin A, and then detected the accumulation of SMV in the plants, and Western blot analysis showed that in concanavalin A-treated SMVs not detected at early time points in plants are shown in Figure 14. Furthermore, concanamycin A-treated Rsv1 plants exhibited a reduced systemic necrotic phenotype in response to SMV G7, as shown in FIG. 15 . Taken together, this suggests that V-ATPase function is important for SMV virulence in both rsv1(Essex) and Rsv1 contexts, and supports the hypothesis that the role of GmVma12 in soybean resistance to SMV may be related to its function in V-ATPase assembly.

Claims (5)

1. A soybean vacuolar ATPase assembly protein gene Vma12, characterized in that: the nucleotide sequence of the gene is shown in SEQ.ID.No:1. 2. The use of soybean vacuolar ATPase assembly protein gene Vma12 as claimed in claim 1, characterized in that: the gene is used to breed soybean varieties resistant to SMV. 3. a kind of cultivation method of anti-SMV plant variety, it is characterized in that: the method is by inserting silencing fragment in soybean vacuolar ATPase assembly protein gene Vma12 as claimed in claim 1, the described soybean vacuolar ATPase assembly protein gene of silencing Vma12, realizing the function of anti-SMV. 4 . The method for cultivating SMV-resistant plant varieties according to claim 3 , characterized in that: the primers for amplifying the silent fragment are as shown in SEQ.ID.No: 2 and SEQ.ID.No: 3. 5. A primer for amplifying the silent fragment of soybean vacuolar ATPase assembly protein gene Vma12 as claimed in claim 1, characterized in that: the sequence of the primer is as SEQ.ID.No: 2 and SEQ.ID.No: 3 shown.

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