Zinc finger protein zfp genes and uses thereof

Abstract

The application discloses a zinc finger protein ZFP gene and application thereof, and identifies the interaction protein ZFP of BmPGRP2 through yeast two-hybrid screening; the ZFP directly combines with the GACATAGATATT sequence on the PTEN promoter as a transcription factor, and inhibits the transcription of PTEN; the expression amount of PTEN can be significantly increased by knocking out the ZFP, and the proliferation of BmNPV is inhibited. Therefore, the ZFP is a negative regulation factor of PTEN, has important value in the research and application of the silkworm antiviral molecular breeding, and the antiviral ability of the silkworm can be improved by knocking out the gene through gene editing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of targeted knockout of zinc finger protein ZFP gene expression in the breeding of resistant silkworm varieties, and also to a method for preparing resistant silkworm varieties. Background Technology

[0002] The silkworm is a silk-producing insect of significant economic value, and sericulture is one of the main sources of income for farmers in my country. Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens threatening sericulture, causing severe economic losses every year. Identifying resistance target genes and cultivating resistant varieties through molecular breeding techniques are effective means of addressing the threat of silkworm diseases.

[0003] Chinese patent CN102433340A discloses the key target gene BmPGRP2 for silkworm resistance, finding that BmPGRP2 inhibits PTEN, activates the PI3K / Akt signaling pathway, thereby inhibiting apoptosis and promoting BmNPV proliferation. However, BmPGRP2 does not directly regulate PTEN.

[0004] Therefore, it is urgent to study which signal transduction molecules BmPGRP2 uses to regulate PTEN, which has important theoretical value and practical significance for the study of antiviral immune mechanisms in silkworms and the breeding of resistant varieties. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a zinc finger protein ZFP gene; a second objective of the present invention is to provide the application of targeted knockout of ZFP gene expression in the preparation of resistant silkworm varieties; a third objective of the present invention is to provide a method for preparing resistant silkworm varieties; and a fourth objective of the present invention is to provide the application of the ZFP gene in negatively regulating the promoter of the silkworm PTEN gene.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. Zinc finger protein ZFP gene, the sequence of which is shown in SEQ ID NO.1.

[0007] 2. Application of targeted knockout of ZFP gene expression in the breeding of resistant silkworm varieties, wherein the sequence of the ZFP gene is shown in SEQ ID NO.1.

[0008] Preferably, the targeted knockout of ZFP gene expression in this invention is performed using the CRISPR / Cas9 system.

[0009] Preferably, the gRNA site of the CRISPR / Cas9 system in this invention is shown in SEQ ID NO.12.

[0010] Preferably, the resistant strain of the present invention is resistant to silkworm nucleopolyhedrovirus.

[0011] A method for preparing a resistant silkworm variety involves knocking out the ZFP gene in silkworms, screening transgenic individuals, and obtaining a resistant silkworm variety.

[0012] Preferably, the ZFP gene knockout is performed by CRISPR-Cas9-mediated knockout of the silkworm ZFP gene, and the sequence of the ZFP gene is shown in SEQ ID NO.1.

[0013] Preferably, the CRISPR-Cas9-mediated knockout of the silkworm ZFP gene gRNA site is shown in SEQ ID NO. 12.

[0014] 4. The application of the ZFP gene in the negative regulation of the PTEN gene promoter in silkworm, characterized in that: the sequence of the ZFP gene is shown in SEQ ID NO.1.

[0015] Preferably, the key site for the binding of the ZFP gene to the PTEN promoter is GACATAGATATT.

[0016] The beneficial effects of this invention are as follows: This invention provides the application of targeted knockout of ZFP gene expression in the breeding of resistant silkworm varieties. Through yeast two-hybrid screening, pull-down, and Co-IP detection, the interacting protein ZFP with BmPGRP2 was identified. As a transcription factor, ZFP directly binds to the GACATAGATATT sequence on the PTEN promoter, inhibiting PTEN transcription. Knocking out ZFP significantly upregulates PTEN expression and inhibits BmNPV proliferation. Therefore, ZFP is a negative regulator of PTEN and has important value in the research and application of molecular breeding for silkworm antiviral resistance. Knocking out this gene through gene editing can improve the antiviral ability of silkworms. Attached Figure Description

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Screening for BmPGRP2 interaction candidate proteins in yeast two-hybrid assays; Figure 2 To identify interacting proteins of BmPGRP2 using pull-down assays; Figure 3 To identify interacting proteins of BmPGRP2 for Co-IP; Figure 4 For the N-terminal bonding of ZFP and BmPGRP2; Figure 5 ZFP inhibits PTEN promoter activity; Figure 6 ZFP directly binds to the PTEN promoter; Figure 7 Incremental expression of ZFP promotes BmNPV proliferation; Figure 8 ZFP knockout inhibits BmNPV proliferation. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0019] Example 1: Screening and identification of BmPGRP2 interacting proteins (1) Total RNA was extracted from the midgut of silkworms to construct a nuclear yeast two-hybrid library. The BmPGRP2 gene was added to the basic vector pGBKT7 via homologous recombination to construct the bait plasmid pGBKT7-PGRP2. After sequencing verification, the cDNA yeast two-hybrid library was screened. Among the 34 initial positive clones, 21 were identified as activating the ADE2, HIS3, and MEL1 reporter genes. DNA sequencing and BLAST alignment analysis of these positive clone plasmids showed that they belonged to the coding genes of 21 different proteins. Further bioinformatics analysis revealed three candidate genes: ZFP (SEQ ID NO.1), BmAnnIX (SEQ ID NO.2), and BmSOD2 (SEQ ID NO.3). The full-length sequences of these three genes were cloned for plasmography verification and found that they all bound to BmPGRP2 ( ). Figure 1 ).

[0020] (2) In order to eliminate false positive results in yeast two-hybrid, ZFP with HA tag was added to the pSL1180[Hr3A4DsRedSV40] vector stored in our laboratory (see Chinese invention patent application number CN117016497A, referred to as 1180 empty vector) to construct the pSL1180-hr3-A4P-ZFP-SV40 expression vector (ZFPOE). The same method was used to construct the vector containing the AnnIXOE gene and the SOD2OE gene. ZFPOE, AnnIXOE, and SOD2OE were transfected into BmE cells, and total protein was collected as input. Pull-down assays were performed with recombinant protein GST-PGRP2-His (GST-PGRP2-His is induced by PGRP2 with GST and His tags linked into the PGEX-6P-1 prokaryotic expression vector) and GST control protein. Western blotting was then performed using HA, GST, and His antibodies. Results showed that AnnIX and SOD2 did not bind to GST-PGRP2-His, while ZFPOE bound to GST-PGRP2-His but not to GST. Figure 2 ).

[0021] (3) Construct the expression vector 1180-hr3-A4P-PGRP2-SV40 (PGRP2OE) of BmPGRP2 with a Flag tag (refer to the ZFPOE construction method). Co-transfect this vector with ZFPOE, AnnIXOE, and SOD2OE into BmE cells, and collect the cells to extract total protein as the input sample. Immunoprecipitation (IP) was performed using the Flag antibody. Then, the input and IP samples were analyzed by Western blotting using Flag and HA antibodies, respectively. The results showed that BmPGRP2 binds to ZFP but not to AnnIX and SOD2. Figure 3 Based on the above in vitro pull-down and in vivo Co-IP results, it is demonstrated that ZFP specifically binds to BmPGRP2.

[0022] (4) BmPGRP2 was truncated into two parts: the C-terminus (including the PGRP domain) and the N-terminus. Flag-tagged BmPGRP2N (SEQ ID NO.4) and BmPGRP2C (SEQ ID NO.5) were constructed and ligated into the expression vector pSL1180[Hr3A4DsRedSV40] to obtain vectors pSL1180-hr3-A4P-PGRP2N-SV40 and pSL1180-hr3-A4P-PGRP2C-SV40 (abbreviated as PGRP2NOE and PGRP2COE). PGRP2OE, PGRP2NOE, and PGRP2COE were co-transfected with ZFPOE into BmE cells. Co-IP results showed that ZFP bound to BmPGRP2 and BmPGRP2N, but not to BmPGRP2C, indicating that ZFP binds to the N-terminus of BmPGRP2 (…). Figure 4 Bioinformatics analysis revealed that ZFP is a transcription factor.

[0023] Example 2: Regulation of the PTEN promoter by ZFP (1) Based on bioinformatics analysis, using the genomic DNA of *Bombyx mori* as a template, the promoter sequence (PTENP1) 1592 bp upstream of the translation start site (ATG) of the PTEN gene was cloned (SEQ ID NO.6). Then, two truncated promoters, PTENP2 (SEQ ID NO.7) and PTENP3 (SEQ ID NO.8), were constructed, with lengths of 1022 bp and 741 bp, respectively. These promoters were added to the PGL3-Basic vector, and PCR, enzyme digestion, and sequencing verified their correctness.

[0024] (2) Dual-luciferase assay results showed that PTENP1 and PTENP2 had high activity, while PTENP3 had almost no activity, indicating that the key active region of the PTEN promoter is located 1022-742 bp upstream of its ATG. Figure 5 A). In BmE cells transfected with PTENP1 and PTENP2, PGRP2OE, ZFPOE, and 1180 empty vector control were added, respectively. The results showed that BmPGRP2 and ZFP could significantly inhibit the activity of these two promoters. Figure 5 (B) indicates that the regulatory site of ZFP is located at 1022-742 bp of the PTEN promoter. Online prediction of this sequence revealed a potential ZFP binding site, GACATAGATATT, and a promoter mutated at this site, PTENP2M, was constructed. PTENP2 and PTENP2M were co-transfected with ZFPOE and 1180 empty vector controls, respectively. The results showed that PTENP2M activity was significantly higher than PTENP2, and its activity was not affected by ZFP. Figure 5These results indicate that ZFP targeting GACATAGATATT inhibits PTEN promoter activity.

[0025] (3) ZFP (SEQ ID NO.1) was double-digested with BamHI and NotI, then ligated into the PGEX-6P-1 prokaryotic expression vector. Recombinant protein GST-ZFP (SEQ ID NO.1) was prepared by induction of expression and protein purification. Figure 6 A). Using GACATAGATATT as the core site, biotin-labeled probes were designed and prepared: 5'-AATTAATAATGACATAGATATTACAAATCGAA-3' (SEQ ID NO. 9) and mutant probe 5'-AATTAATAATCCCCCCCCCCCCACAAATCGAA-3' (SEQ ID NO. 10). GST-ZFP and control GST proteins were incubated with the PTEN promoter probe. EMSA results showed that the probe did not bind to GST but bound to GST-ZFP. After adding 50-fold of unlabeled competing probe, this binding band disappeared. Neither GST-ZFP nor GST bound to the mutant probe. Figure 6 These results indicate that the key site for ZFP binding to the PTEN promoter is GACATAGATATT.

[0026] Example 3: Targeting ZFP to regulate BmNPV proliferation (1) ZFPOE and 1180 empty vectors were transfected into BmE cells, respectively. After 48 h, RNA was extracted and reverse transcribed into cDNA for qPCR detection. The results showed that ZFP expression was significantly increased in ZFPOE cells. Figure 7 (A), but PTEN expression was significantly reduced ( Figure 7 B). 48 h post-transfection, the virus was infected with BmNPV-GFP virus labeled with green fluorescent marker. DNA was extracted 48 h post-infection, and qPCR was performed using primers for the BmNPV virus GP41 gene and the silkworm housekeeping gene BmGAPDH. A significant increase in viral load was observed. Figure 7 (D). Fluorescence was observed 72 hours after viral infection, and it was found that the viral fluorescence in cells transfected with ZFPOE was significantly higher than that in the control (D). Figure 7 These results indicate that incremental expression of ZFP promotes the proliferation of BmNPV.

[0027] (2) The gRNA sites of ZFP were analyzed online using CRISPR / Cas9 target prediction software (https: / / cctop.cos.uni-heidelberg.de / ), and three gRNA sequences were designed. The specific sequences are as follows: ZFP-gRNA1: 5'-GCATTCGCGCGCGAAATGCC-3' (SEQ ID NO. 11); ZFP-gRNA2: 5'-CCGCACAGGACACTTTGCGA-3' (SEQ ID NO. 12); ZFP-gRNA3: 5'-GCACGGAACTGTCCCGAGGG-3' (SEQ ID NO. 13).

[0028] Three gRNAs were inserted into the base vector piggyBac [U6-TTTTT-hr3-hsp70-Cas9-SV40] (P10) preserved in this experiment (refer to Genome-wide CRISPR screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori) to construct gRNA vectors piggyBac [U6-ZFPgRNA1-TTTTT-hr3-hsp70-Cas9-SV40], piggyBac [U6-ZFPgRNA2-TTTTT-hr3-hsp70-Cas9-SV40], and piggyBac [U6-ZFPgRNA3-TTTTT-hr3-hsp70-Cas9-SV40], named sgZFP1, sgZFP2, and sgZFP3. Three gRNA vectors, sgZFP1 / 2 / 3, were transfected into BmE cells. After 48 hours, genomic DNA was extracted for PCR amplification and sequencing. The results showed a double peak only near the gRNA2 site. The PCR product was used for cloning and sequencing, which confirmed that ZFP cells had mutations involving deletions of one and two bases. Figure 8 A). BmE cells were transfected with plasmids sgZFP2 and control P10, respectively. RNA was extracted and analyzed by qPCR 48 h later. The results showed that PTEN levels in sgZFP2 cells were significantly increased (A). Figure 8 B). Cells were infected with BmNPV-GFP virus 48 h post-transfection, and DNA was extracted and analyzed by qPCR 48 h post-infection. The results showed a significant decrease in viral load in cells transfected with sgZFP2. Figure 8 (D), viral fluorescence was also significantly reduced ( Figure 8 These results indicate that ZFP knockout leads to increased PTEN expression, which in turn significantly enhances the antiviral ability of silkworms.

[0029] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The zinc finger protein ZFP gene, characterized by: The sequence of the ZFP gene is shown in SEQ ID NO.

1.

2. The application of targeted knockout of ZFP gene expression in the breeding of resistant silkworm varieties, characterized by: The sequence of the ZFP gene is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that: The targeted knockout of ZFP gene expression was performed using the CRISPR / Cas9 system.

4. The application according to claim 1, characterized in that: The gRNA site of the CRISPR / Cas9 system is shown in SEQ ID NO.

12.

5. The application according to claim 1, characterized in that: The resistant strain is resistant to silkworm nucleopolyhedrovirus.

6. A method for preparing a resistant silkworm variety, characterized in that: By knocking out the ZFP gene in silkworms and screening transgenic individuals, resistant varieties of silkworms can be obtained.

7. The preparation method according to claim 5, characterized in that: The ZFP gene knockout is achieved by CRISPR-Cas9-mediated knockout of the silkworm ZFP gene, and the sequence of the ZFP gene is shown in SEQ ID NO.

1.

8. The preparation method according to claim 5, characterized in that: The CRISPR-Cas9-mediated knockout of the silkworm ZFP gene gRNA site is shown in SEQ ID NO.

12.

9. The application of the ZFP gene in the negative regulation of the silkworm PTEN gene promoter, characterized by: The sequence of the ZFP gene is shown in SEQ ID NO.

1.

10. The application according to claim 8, characterized in that: The key site for the binding of the ZFP gene to the PTEN promoter is GACATAGATATT.

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