Application of turnip crinkling virus protein P38 in regulating and controlling double-chain binding protein of arabidopsis thaliana

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to application of turnip crinkling virus protein P38 to regulation and control of double-chain binding protein of arabidopsis thaliana. Research finds that turnip crinkling virus protein P38 interacts with Drb7.1 and Drb7.2, expression of Drb7.1 and Drb7.2 can be reduced, the growth capacity of yeast in an SD / -Leu / -Trp auxotroph culture medium and an SD / -Leu / -Trp / -His / -Ade auxotroph culture medium is improved, and a research result lays a foundation for clarification of a molecular mechanism of TCV P38 regulation and control of Drb7.1 and Drb7.2.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to application of Turnip Crinkle Virus protein P38 in regulating Arabidopsis thaliana double-stranded binding protein. Background Art

[0002] Post-transcriptional gene silencing (PTGS) is an important RNA regulatory mechanism in eukaryotes. Double-stranded RNA-specific endonucleases, Dicer or Dicer-like (DCL for short), cut double-stranded RNA or partially double-stranded hairpin structures with the assistance of double-stranded RNA binding proteins (DRBs) to produce siRNA (small interference RNA). siRNA guides AGO proteins to specifically recognize and degrade matching target RNAs through base complementarity. During the evolution process, plants have formed multiple antiviral PTGS signaling pathways that are participated by different proteins and have different functions but overlap with each other. PTGS not only regulates plant growth and development and responds to biotic and abiotic stresses, but also plays a very important role in plant resistance to viral invasion and is one of the important antiviral immune mechanisms. In order to resist the antiviral immune function of PTGS in the host, most plant viruses have evolved various silencing suppressors (viral suppressors of RNA silencing, VSRs). VSRs of different viruses do not have homology in sequence, and their mechanisms and modes of action are also different. The molecular mechanism of VSRs hijacking or inhibiting the antiviral defense function of host PTGS by interfering with various steps of PTGS has been a hot topic in recent years. In addition, literature reports that VSRs can also enhance viral replication and pathogenicity by interfering with signaling pathways such as host cell autophagy, RNA degradation, and RNA-mediated DNA methylation.

[0003] Turnip crinkle virus ( Turnip crinkle virus ,TCV) belongs to the genus Carnation mottle virus (Tombusviridae) Carmovirus ). TCV is a positive-sense single-stranded RNA virus (containing 4054 nt) with 5 open reading frames encoding 5 proteins including P28, P388, P38, P38 and P9. The P28 protein encoded by the 5' end of TCV gRNA and the read-through product P388 protein are responsible for replication and translation. During the transcription of TCV gRNA, subgenomes encoding movement proteins P38 and P9 and subgenomes encoding P38 protein are also produced. P38 is a structural protein of TCV - coat proteining (CP), and is also a silencing suppressor, but there is no report on its mechanism of action in regulating Arabidopsis double-stranded binding proteins 7.1 and 7.2. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of Turnip Crinkle Virus protein P38 in regulating Arabidopsis thaliana double-stranded binding protein.

[0005] The first aspect of the present invention is to provide the use of the P38 gene, or the protein encoded by the P38 gene, or a recombinant vector or host bacteria containing the P38 gene coding region in negatively regulating the expression level of Drb7.1 (Arabidopsis double-stranded binding protein 7.1) and / or Drb7.2 (Arabidopsis double-stranded binding protein 7.2).

[0006] Wherein, the nucleotide sequence of the P38 gene is shown in SEQ ID NO: 1; The nucleotide sequence of the gene corresponding to Drb7.1 is shown in SEQ ID NO: 2; The nucleotide sequence of the gene corresponding to Drb7.2 is shown in SEQ ID NO: 3; The second aspect of the present invention is to provide the use of the P38 gene, or the protein encoded by the P38 gene, or a recombinant vector containing the coding region of the P38 gene in the interaction with Drb7.1 or Drb7.2.

[0007] Among them, the interaction between P38 and Drb7.1 leads to a decrease in the expression level of Drb7.1, and the nucleotide sequence of the gene corresponding to Drb7.1 is shown in SEQ ID NO:2; the interaction between P38 and Drb7.2 leads to a decrease in the expression level of Drb7.2, and the nucleotide sequence of the gene corresponding to Drb7.2 is shown in SEQ ID NO:3.

[0008] Among them, the interaction between P38 and Drb7.1 or the interaction between P38 and Drb7.2 improves the growth ability of yeast in SD / -Leu / -Trp nutrient-deficient medium and / or SD / -Leu / -Trp / -His / -Ade nutrient-deficient medium.

[0009] The present study found that there is an interaction between the Turnip Crinkle Virus protein P38 and Drb7.1 and Drb7.2, which can downregulate the expression of Drb7.1 and Drb7.2 and improve the growth ability of yeast in SD / -Leu / -Trp nutrient-deficient medium and SD / -Leu / -Trp / -His / -Ade nutrient-deficient medium. The research results will lay the foundation for clarifying the molecular mechanism of TCV P38 regulating Drb7.1 and Drb7.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1VIGS studies showed that Drb7.2 is involved in the DCL4-mediated, Drb4-free resistance to turnip crinkle virus and RNA silencing pathways. The PDS in Figures B and C is phytoene desaturase.

[0011] Figure 2 The AD-CP and BD-Drb7.1 and AD-CP and BD-Drb7.2 yeast interactions. AD-CP + BD-Drb7.1 and AD-CP + BD-Drb7.2 represent the AD-CP + BD-Drb7.1 and AD-CP + BD-Drb7.2 co-transformed yeast combinations, respectively. SD-LW: SD-LW / -Leu / -Trp medium; SD-LWHA: SD-LWHA / -Ade / -His / -Leu / -Trp medium.

[0012] Figure 3 Western Blot was used to detect the protein accumulation levels of pG1300-Drb7.1 and pG1300-Drb7.2 on the 5th day after infiltration of Nicotiana benthamiana leaves with pCP-Flag, where pG1300-Drb7.1: plant expression vector expressing Drb7.1-GFP; pG1300-Drb7.2: plant expression vector expressing Drb7.2-GFP; CP-Flag: plant expression vector pCP-Flag of CP-Flag; Vec: plant expression vector p1300-Flag expressing only Flag tag; α-Flag: Flag antibody detection; α-GFP: GFP antibody detection; Rubisco: Rubisco Coomassie Brilliant Blue staining. DETAILED DESCRIPTION

[0013] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0014] 1 Plant material Arabidopsis mutants dcl2drb4 (Zhang, X., Zhang, X., Wu, K., Liu, Z., Li, D., & Qu, F. (2016). Incomplete DRB4-dependence of the DCL4-mediated antiviraldefense. Scientific reports , 6 , 39244.), dcl2drb4drb7.1 (Zhang Chunwei, Zhang Xiuchun, Fu Yan, et al. CRISPR / Cas9 system edits the double-stranded binding protein 7.1 of Arabidopsis dcl2drb4[J]. Molecular Plant Breeding, 2022, 20(15): 5005-5010. DOI: 10.13271 / j.mpb.020.00500) and dcl2drb4drb7.2 (Tang Wen, Zhang Chunwei, Fu Yan, et al. CRISPR / Cas9 system knockout Arabidopsis dcl2drb4 Double-chain binding protein 7.2[J / OL]. Molecular Plant Education, 1-10) are all preserved in this laboratory.

[0015] 2 Experimental methods and results

[0016] 2.1 Vector construction (1) The construction methods of virus-induced gene silencing vectors (VIGS) CPB1B-Drb7.1A, CPB1B-Drb7.1B, CPB1B-Drb7.2A, CPB1B-Drb7.2B and the control vector CPB1B-GUSA with GUS gene fragment inserted are as follows: Shanghai Sangon Biotechnology Co., Ltd. was commissioned to artificially synthesize the target fragments Drb7.1A, Drb7.1B, Drb7.2A, Drb7.2B and GUSA (see Table 1 for specific sequences). According to the instructions of the kit, they were respectively ligated with the viral vector CPB1B (Wu, K., Wu, Y., Zhang, C., Fu, Y., Liu, Z., & Zhang, X. (2021). Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector. Plant methods, 17(1), 6.) digested with KpnI. The ligated products were transformed into Escherichia coli. After sequencing and identification, the positive clones were named CPB1B-Drb7.1A, CPB1B-Drb7.1B, CPB1B-Drb7.2A, CPB1B-Drb7.2B and CPB1B-GUSA.

[0017] (2) BD-Drb7.1 Primers Drb7.1-1F SfiI and Drb7.1-597R EcoRI (specific sequences are shown in Table 2) were used to amplify the full-length Drb7.1 (nucleotide sequence is shown in SEQ ID NO: 2) using Arabidopsis thaliana cDNA as a template. The reaction system is as follows:

[0018] The reaction procedure is as follows:

[0019] The PCR product was double-digested with SfiI and EcoRI and ligated with the large fragment of pGBKT7 (whether it was pGBKT7) recovered by the same double enzyme digestion. The positive clone was named BD-Drb7.1 after being correctly identified by PCR and sequencing.

[0020] (3) BD-Drb7.2 build Primers Drb7.2-1F EcoRI and Drb7.2-570R BamHI (specific sequences are shown in Table 2) were used to amplify the full-length Drb7.2 (nucleotide sequence is shown in SEQ ID NO:3) using Arabidopsis cDNA as a template. The reaction system and reaction procedure used were the same as those in 2.1 (2). The PCR product was double-digested with EcoRI and BamHI and ligated with the large fragment of pGBKT7 recovered by the same double enzyme digestion. The positive clone was named BD-Drb7.2 after being confirmed by PCR and sequencing.

[0021] (4) pG1300-Drb7.1 The primers Drb7.1-1F SalI and Drb7.1-597R BamHI (specific sequences are shown in Table 2) were used to amplify the full-length Drb7.1 (nucleotide sequence is shown in SEQ ID NO:2) using the successfully constructed BD-Drb7.1 as a template. The reaction system and reaction procedure used were the same as those in 2.1 (2). The PCR product was double-digested with SalI and BamHI and ligated with the large pG1300 fragment recovered by the same double enzyme digestion. The positive clone was named pG1300-Drb7.1 after being correctly identified by PCR and sequencing.

[0022] (5) Construction of pG1300-Drb7.2 The primers Drb7.2-1F KpnI and Drb7.2-570R BamHI (specific sequences are shown in Table 2) were used to amplify the full-length Drb7.2 (nucleotide sequence is shown in SEQ ID NO:3) using the successfully constructed BD-Drb7.2 as a template. The reaction system and reaction procedure used were the same as those in 2.1 (2). The PCR product was double-digested with KpnI and BamHI and ligated with the large fragment of pG1300 recovered by the same double enzyme digestion. The positive clone was named pG1300-Drb7.2 after being correctly identified by PCR and sequencing.

[0023] Table 1 Synthetic fragments for constructing VIGS vector

[0024] Table 2 Primers used

[0025] 2.2 Drb7.1 and Drb7.2 participate in a new pathway of DCL4-mediated resistance to turnip crinkle virus and RNA silencing that does not require Drb4 like Figure 1 As shown, the VIGS vectors CPB1B-Drb7.1A, CPB1B-Drb7.1B, CPB1B-Drb7.2A, CPB1B-Drb7.2B, and CPB1B-GUSA were digested with XbaI and used to infect and transform Arabidopsis thaliana after digestion and in vitro transcription. dcl2drb4 Double mutants, the results are as Figure 1 As shown in A in Figure 1. Except for the Mock control plant, the virus-infected plants all showed typical virus symptoms. On the 14th day after virus inoculation, total RNA was extracted from the uninoculated leaves on the upper part of the plant and subjected to qRT-PCR. The results are shown in Figure 1 B and Figure 1 As shown in C, CPB1B-Drb7.1A and CPB1B-Drb7.1B and CPB1B-Drb7.2A and CPB1B-Drb7.2B can be effectively silenced respectively. Drb7. 1 and Drb7.2 , Drb7. 1 and Drb7.2 The accumulation of TCV virus increased significantly after being silenced. The above results indicate that Drb7.1 and Drb7.2 are involved in the DCL4-mediated, Drb4-free, anti-Turnip Crinkle Virus and RNA silencing pathway.

[0026] 2.3 Yeast two-hybrid experiments demonstrated that TCV P38 interacts with both Drb7.1 and DRB7.2 For ease of description, during the experiment, the "pGADT7" vector is abbreviated as "AD", the "pGBKT7" vector is abbreviated as "BD", and "P38" is abbreviated as "CP". The yeast expression vectors pGADT7 (AD), pGBKT7 (BD), AD-T, BD-53 and AD-CP (Liu, L., Wang, H., Fu, Y., Tang, W., Zhao, P., Ren, Y., Liu, Z., Wu, K., & Zhang, X. (2023). Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection. Molecular plantpathology, 24(2), 154–166.) are all preserved in this laboratory.

[0027] Yeast vectors were co-transformed into competent yeast cells with different yeast expression vectors according to Table 3. The monoclonal colonies positively identified by PCR were picked and put into 25 μL sterile water, resuspended to make a suspended bacterial solution, and gradiently diluted with sterile water to 10 times, 100 times, and 1000 times. 2 μL of each was inoculated into SD / -Leu / -Trp (SD-LW) nutrient-deficient medium and SD / -Leu / -Trp / -His / -Ade (SD-LWHA) nutrient-deficient medium. Yeast co-transformed with AD-T and BD-Lam and AD-T and BD-53 were used as negative and positive controls, respectively. They were inoculated into solid culture medium together with the yeast solution transformed with the target plasmid, and cultured inverted in a constant temperature incubator at 28°C for 3 days to observe and record the growth conditions to determine the interaction between proteins.

[0028] Table 3 Pairing combinations of yeast bait plasmids and prey plasmids combination Bait carrier Prey carrier 1 (positive control) AD-T BD-53 2 (Negative control) AD-T BD-lam 3 AD-CP BD-Drb7.1 4 AD-CP BD-Drb7.2 The results of yeast two-hybrid studies are as follows Figure 2 As shown, all co-transformed yeasts can grow normally in SD-LW defective medium, but in SD-LWHA defective medium, only yeast positive control, yeast co-transformed with AD-CP and BD-Drb7.1, and yeast co-transformed with AD-CP and BD-Drb7.2 can grow normally, while yeast negative control cannot grow normally. The results indicate that there is an interaction between AD-CP and BD-Drb7.1 and between AD-CP and BD-Drb7.2.

[0029] 2.4 P38 negatively regulates the expression of Drb7.1 and Drb7.2 (1) Plant expression vector transformation into Agrobacterium GV3101 The plant expression vectors pG1300Drb7.1, pG1300Drb7.2, pCP-Flag (Liu, L., Wang, H., Fu, Y., Tang, W., Zhao, P., Ren, Y., Liu, Z., Wu, K., & Zhang, X. (2023). Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection. Molecular plant pathology, 24(2), 154–166.), and the silencing suppressor expression vector pZP-P19 (Qu, F., Ren, T., & Morris, TJ(2003). The coat protein of turnip crinkle virus suppressesposttranscriptional gene silencing at an early initiation step. Journal ofvirology, 77(1), 511–522.) were used to transform Agrobacterium GV3101 competent cells, and the transformed Agrobacterium was evenly spread on LB solid culture medium containing corresponding resistance (pG1300Drb7.1, pG1300Drb7.2, and pCP-Flag were spread on LB plates containing Kan and Rif resistance, and pZP-P19 was spread on LB plates containing Spec and Rif resistance, with a working concentration of 50 μg / mL) and cultured inverted at 28°C for 72-90 h.

[0030] (2) Agrobacterium infiltration of Nicotiana benthamiana Use a pipette tip to pick up a single colony that is positive by colony PCR and place it in 5 mL of LB liquid culture medium containing the corresponding antibiotics, and place it in a constant temperature shaker at 28°C and 200 r / min for overnight culture. After centrifugation of the bacterial solution at 7000 rmp / min for 15 min, discard the supernatant, and use 5 mL of injection buffer to resuspend the precipitate. After sufficient vortexing and even shaking, dilute the bacterial solution to an OD600 of 0.5. After the diluted bacterial solution is placed at room temperature for 2-3 hours, the resuspensions of different recombinant vectors are mixed in a ratio of 1:1:1 (see Table 4 for specific combinations). The mixture is fully mixed and used for injection into Nicotiana benthamiana leaves that are growing vigorously and in the 5-7 leaf stage. Each combination is injected with at least 6 replicates. The injected Nicotiana benthamiana is kept away from light overnight and then cultured under normal conditions. Proteins were extracted on the 5th day after injection, and Western Blot detection was performed using GFP antibody and Flag antibody, respectively. The results are shown in Figure 3 As shown, the expression levels of Drb7.1 and Drb7.2 were significantly decreased after co-expression with P38, indicating that P38 negatively regulates the expression of Drb7.1 and Drb7.2.

[0031] Table 4 Combinations of different recombinant plasmids Agrobacterium combination Recombinant plasmid name 1 pG1300Drb7.1 + pZP-P19 + Vec 2 pG1300Drb7.1 + pZP-P19 + pCP-Flag 3 pG1300Drb7.2 + pZP-P19 + Vec 4 pG1300Drb7.2 + pZP-P19 + pCP-Flag The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Application of P38 gene, or protein encoded by P38 gene, or recombinant vector or host bacteria containing the coding region of P38 gene in negatively regulating the expression level of Drb7.1 and / or Drb7.2; in, The nucleotide sequence of the P38 gene is shown in SEQ ID NO: 1; The nucleotide sequence of the gene corresponding to Drb7.1 is shown in SEQ ID NO: 2; Among them, the nucleotide sequence of the gene corresponding to Drb7.2 is shown in SEQ ID NO:

3.

2. Use of the P38 gene, or the protein encoded by the P38 gene, or a recombinant vector containing the coding region of the P38 gene in the interaction with Drb7.1 or Drb7.2; in, The nucleotide sequence of the P38 gene is shown in SEQ ID NO: 1; The nucleotide sequence of the gene corresponding to Drb7.1 is shown in SEQ ID NO: 2; Among them, the nucleotide sequence of the gene corresponding to Drb7.2 is shown in SEQ ID NO:

3.

3. The use according to claim 2, characterized in that The interaction between P38 and Drb7.1 leads to a decrease in the expression level of Drb7.1, and the nucleotide sequence of the gene corresponding to Drb7.1 is shown in SEQ ID NO:2; the interaction between P38 and Drb7.2 leads to a decrease in the expression level of Drb7.2, and the nucleotide sequence of the gene corresponding to Drb7.2 is shown in SEQ ID NO:

3.

4. The use according to claim 2, characterized in that The interaction between P38 and Drb7.1 or the interaction between P38 and Drb7.2 improves the growth ability of yeast in SD / -Leu / -Trp nutrient-deficient medium and / or SD / -Leu / -Trp / -His / -Ade nutrient-deficient medium.

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