Pseudomonas syringae kiwifruit pathogenic variant gene knockout vector, strain, construction method of strain and application of strain in obtaining transgenic plants

Abstract

The invention relates to a pseudomonas syringae kiwifruit pathogenic variant gene knockout vector, a strain, a construction method of the strain and application of the strain in obtaining a transgenic plant. According to the invention, modification is carried out on the basis of an original CPRISPR / FnCas12a system, a genetic manipulation technology using Psa gene editing is constructed, gene knockout is successfully carried out on the three-type effector hopH1, plasmid elimination is realized, and subsequent gene supplementation or continuous knockout of other genes is facilitated. Through screening and verification, a knockout strain deltahopH1 of hopH1, a backfill strain C deltahopH1 of hopH1, a double knockout strain deltahopZ5 deltahopH1 of hopH1 and hopZ5, and a backfill strain C deltahopZ5 deltahopH1 of hopH1 and hopZ5 are obtained. Compared with the prior art, the method overcomes the defects that the knockout period of the pseudomonas syringae kiwifruit pathogenic variant gene is long and the operation is tedious.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene knockout vector and strain of Pseudomonas syringae pv. actinidia, and a construction method thereof, and application thereof in obtaining transgenic plants. Background Art

[0002] During the growth and development of plants, they face constant threats and challenges from various pathogens, including viruses, bacteria, and fungi. Pseudomonas syringae pv.actinidiae (Psa) is the pathogen that causes kiwifruit canker. Psa is a pathogen that poses a serious threat to kiwifruit, causing necrosis and shedding of kiwifruit leaf buds, shrunken and dry fruits, and vine death. In 2010, Psa infected approximately 92% of New Zealand's kiwifruit orchards, causing huge economic losses. Due to its impact on the kiwifruit industry, kiwifruit canker was included in the European and Mediterranean Plant Protection Organization (EPPO) alert list in 2019.

[0003] Many methods have been applied in the field of bacterial gene editing. Zinc finger nuclease (ZFN) technology is an early gene editing tool that uses zinc finger proteins to specifically recognize and cut DNA sequences. However, its application range is limited by the complex experimental design and high off-target rate. Based on ZFN, scientists have developed the more flexible and efficient transcription activator-like effector nuclease (TALEN) technology. TALENs are complexes composed of transcription activator-like effectors and DNA cleavage enzymes, which can more accurately recognize and cut DNA sequences. However, TALEN technology was quickly replaced by a groundbreaking clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. During the development process, more Cas proteins were applied to the field of gene editing. CRISPR / FnCas12a is widely used in gene editing and detection. Compared with CRISPR / Cas9, CRISPR / FnCas12a has a simpler structural design and a lower off-target rate. The CRISPR / Cas9 gRNA contains both crRNA and tracrRNA, while the CasFn12a gRNA consists solely of crRNA, which can self-process its pre-crRNA to form mature crRNA. Furthermore, the Cas9 gRNA is typically >100 bp, while the CasFn12a gRNA only requires 42-44 bp.

[0004] In the CRISPR / FnCas12a system, a short sgRNA recognizes DNA near the PAM sequence (TTTV) and promotes cleavage by guiding the FnCas12a protein, resulting in double-stranded DNA breaks (DSBs). DSBs can be repaired by non-homologous recombination repair proteins (NHEJ), in which the broken ends are directly joined without the need for a homologous template; this process can lead to insertions and deletions of the target DNA. CRISPR / FnCas12a-induced DSBs can be repaired by NHEJ proteins encoding Ku-like proteins (Ku) and ATP-dependent ligase D (LigD), but it is not clear whether NHEJ is required for Psa editing.

[0005] Chinese patent CN107446924B discloses a CRISPR-Cas9-based editing vector for the kiwifruit gene AcPDS, as well as its construction method and application. This vector enables efficient site-directed mutagenesis of the kiwifruit gene AcPDS, successfully inducing an albino phenotype in kiwifruit through Agrobacterium-mediated genetic transformation, establishing a technology for site-directed gene editing in kiwifruit. The research targets kiwifruit genes, aiming to achieve site-directed mutagenesis and obtain an albino phenotype.

[0006] Chinese patent CN113604490B discloses the kiwifruit canker susceptibility gene AcBXL1 and its applications. The AcBXL1 gene sequence was obtained through kiwifruit genome and transcriptome analysis, confirming that the gene promotes susceptibility to canker in kiwifruit. This gene can be used to generate transgenic plants susceptible to canker. Overexpression of the gene increases susceptibility to canker caused by Pseudomonas syringae pv. kiwifruit. The core of this technical solution is to utilize the expression of this gene in plants to alter their susceptibility. Psa gene editing is not involved. Summary of the Invention

[0007] In the prior art, the gene knockout cycle of Pseudomonas syringae pv. actinidia is long and the operation is cumbersome. To solve this technical problem, the present invention provides a gene knockout vector and strain of Pseudomonas syringae pv. actinidia and a method for constructing the same, as well as an application thereof in obtaining transgenic plants.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention first provides a method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit, comprising the following steps:

[0010] (1) According to the Psa M228 genome information on the NCBI website, crRNA primers for the target gene hopH1 were designed, namely crRNA1 hopH1 -F (sequence shown in SEQ ID No. 1) and crRNA1hopH1 -R (sequence as shown in SEQ ID No. 2), and through phosphorylation annealing to form a double-stranded crRNA with SapI sticky ends, i.e. crRNA hopH1 ;

[0011] (2) crRNA hopH1 The pHZB4-crRNA was formed by ligating it into pHZB4 after SapI digestion using T4 ligase. hopH1 .

[0012] The sequence of the Psa M228 genome is available at GenBank: CP032631, and the sequence information of the target gene hopH1 is available at GenBank: CN228 09540.

[0013] Wherein, the sequence of pHZB4 is shown as SEQ ID No.18.

[0014] In one embodiment of the present invention, the following steps are also included:

[0015] (3) pHZB4-crRNA hopH1 By BamHI digestion, the hopH1 , FnCas12a and NHEJ protein expression sequence fragments were ligated into pBBR1-MCS2 to form pBBR-B4-crRNA hopH1 .

[0016] Among them, FnCas12a and NHEJ proteins are both known proteins, and the fragments of their FnCas12a and NHEJ protein expression sequences are also known.

[0017] The sequence of pBBR1-MCS2 is shown as SEQ ID No. 19.

[0018] For pBBR-B4-crRNA hopH1 , the B4 inside is the abbreviation of pHZB4.

[0019] The present invention further provides a gene knockout vector of Pseudomonas syringae pv. kiwifruit prepared based on the above method, specifically crRNA hopH1 or pHZB4-crRNA hopH1 or pBBR-B4-crRNA hopH1 .

[0020] The present invention also provides another method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit, comprising the following steps:

[0021] (1) According to the Psa M228 genome information on the NCBI website, crRNA primers for the target gene hopZ5 were designed, namely crRNA hopZ5 -F (sequence as shown in SEQ ID No.3) and crRNA hopZ5 -R (sequence as shown in SEQ ID No.4), and through phosphorylation annealing to form a double-stranded crRNA with SapI sticky ends, i.e. crRNA hopZ5 ;

[0022] (2) Double-stranded crRNA hopZ5 The pHZB4-crRNA was formed by ligating it into pHZB4 after SapI digestion using T4 ligase. hopZ5 .

[0023] In one embodiment of the present invention, the following steps are also included:

[0024] (3) pHZB4-crRNA hopZ5 By BamHI digestion, the hopZ5 , FnCas12a and NHEJ protein expression sequence fragments were ligated into pBBR1-MCS2 to form pBBR-B4-crRNA hopZ5 .

[0025] The sequence of the Psa M228 genome is shown in GenBank: CP032631, wherein FnCas12a and NHEJ proteins are both known proteins, and fragments of their FnCas12a and NHEJ protein expression sequences are also known.

[0026] The present invention further provides a gene knockout vector of Pseudomonas syringae pv. kiwifruit prepared based on the above method, specifically crRNA hopZ5 or pHZB4-crRNA hopZ5 or pBBR-B4-crRNA hopZ5 .

[0027] The present invention further provides a method for preparing a gene knockout strain of Pseudomonas syringae pv. kiwifruit, comprising the following steps:

[0028] S1, the successfully constructed pBBR-B4-crRNA hopH1 The donor bacteria S17-1λpir were transformed by heat shock and the vector pBBR-B4-crRNA in S17-1λpir was transferred by biparental conjugation. hopH1The conjugate was transferred into the recipient bacteria M228 to obtain the conjugate, which was shaken in LB liquid medium in tetracycline for 1.5 h to induce the expression of FnCas12a protein; S2, the conjugate was spread on an LB resistance plate containing kanamycin, nalidixic acid and cefazolin, and cultured at 28 ° C for 3-5 days. The single colonies grown on the plate were verified by PCR products using two pairs of primers to screen for knockout of hopH1 and pBBR-B4-crRNA hopH1 For strains where the plasmid did not undergo self-cleavage, the specific knockout sequence was subsequently determined by Sanger sequencing;

[0029] S3, the pHM1B3-VD plasmid containing two crRNAs was digested with BamHI, one of which was a crRNA target gene oriV and the other was a crRNA target base LigD, and the expression sequence of FnCas12a was ligated into the pML-123 vector with a broad host range by T4 ligase to construct a Gm-resistant pML-B3free plasmid (pML-B3-F (shown in SEQ ID No.16) / pML-B3-R (shown in SEQ ID No.17) for PCR detection of the plasmid), and the pML-B3free plasmid was transferred into the donor bacteria S17-1λpir by heat shock transformation for elimination of the plasmid to obtain S17-1λpir containing the pML-B3free plasmid;

[0030] S4, S17-1λpir containing pML-B3free plasmid, and hopH1 knockout but still containing pBBR-B4-crRNA hopH1 The strain (i.e., the mutant with hopH1 knockout after the conjugate screening) was subjected to biparental conjugation and the strain with hopH1 knockout but not containing pBBR-B4-crRNA was screened. hopH1 The strain was named ΔhopH1 strain.

[0031] In one embodiment of the present invention, in step S1, the method of parental conjugation is as follows: S17-1λpir and Psa strains are shaken and cultured to OD 600 =1.5, take 2mL of bacterial solution from each: heart and collect the cells, wash them with sterile water 2-3 times, make the volume to 100μL, according to S17-1λpir:M228=1:10, mix well and take 100μL of bacterial solution on 0.22 sterile filter membrane LB plate, place it in a 28℃ incubator and culture for 12h, so that the vector pBBR-B4-crRNA in S17-1λpir hopH1 Transformed into recipient strain M228.

[0032] The donor strain S17-1λpir is a commonly used biomaterial and is available from Biyuntian Biotechnology. The recipient strain M228 is a commonly used biomaterial, and the genome sequence of Psa M228 is available in GenBank: CP032631.

[0033] In one embodiment of the present invention, the pHM1B3-VD plasmid is a known biological plasmid described in the document "CRISPR / FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates." The pHM1B3-VD plasmid is a genome editing tool specifically designed for efficient plasmid clearance and genome editing in bacteria. It contains two crRNAs, one targeting the oriV gene and the other targeting the LigD gene. Both oriV and LigD genes are located on pHZB4. This design enables the pHM1B3-VD plasmid to simultaneously clear any plasmid constructed by pHM1B4 and its own self-clearance.

[0034] Wherein, the sequence of pML-123 is shown as SEQ ID No.20.

[0035] The present invention further provides a Pseudomonas syringae pv. actinidia gene knockout strain prepared based on the above method, namely, a ΔhopH1 strain.

[0036] The present invention further provides a ΔhopZ5ΔhopH1 strain, i.e., a strain in which both the hopZ5 gene and the hopH1 gene are knocked out, wherein the specific method is as follows:

[0037] The ΔhopH1 strain was used as the background, and the corresponding vector was constructed and the ΔhopZ5ΔhopH1 strain was obtained by biparental conjugation.

[0038] The present invention further provides a gene knockout strain of Pseudomonas syringae pv. kiwifruit, the specific method is:

[0039] (1) Using primers hopH1-F / hopH1-R and hopZ5-F / hopH1-R, genomic DNA of Psa M228 was used to amplify the hopH1 and hopZ5-hopH1 sequences by PCR. The N-termini of hopH1 and hopZ5+hopH1 were labeled with a FLAG tag.

[0040] (2) The cloned product T4 was ligated to pBBR1-MCS2 digested with HindIII to obtain pBBR-hopH1 and pBBR-hopZ5+hopH1 plasmids;

[0041] (3) The pBBR-hopH1 and pBBR-hopZ5+hopH1 plasmids were transferred into the ΔhopH1 and ΔhopH1ΔhopZ5 strains through biparental conjugation to obtain CΔhopH1 and CΔhopZ5ΔhopH1, respectively.

[0042] The present invention further provides the use of a gene knockout vector or strain of Pseudomonas syringae pv. actinidia in obtaining transgenic plants.

[0043] Compared with the prior art, the present invention is based on the original CPRISPR / FnCas12a system for transformation, constructs a genetic manipulation technology using Psa gene editing, and successfully performs gene knockout on the type III effector hopH1, and achieves the elimination of the plasmid, which is convenient for subsequent gene complementation or continued knockout of other genes. After screening and verification, the knockout strain ΔhopH1 of hopH1, the complementation strain CΔhopH1 of hopH1, the double knockout strain ΔhopZ5ΔhopH1 of hopH1 and hopZ5, and the complementation strain CΔhopZ5ΔhopH1 of hopH1 and hopZ5 were obtained.

[0044] The construction method of the knockout vector of the present invention is as follows: the crRNA targeting the target gene is constructed into the vector pHZB4, and then the expression sequence comprising crRNA, Cas12a protein and homologous recombination repair protein is integrated into the expression vector pBBR1-MCS2 through an enzyme digestion and ligation process to form a knockout vector. The knockout vector is transferred into the Psa strain via a biparental conjugation technique, and the strain in which gene editing occurs is screened by double-primer PCR and Sanger sequencing. Subsequently, the gene knockout strain and the complementing strain are obtained by plasmid elimination and backfilling of the corresponding gene for inoculation of kiwifruit for subsequent functional verification process.

[0045] The present invention overcomes the defects of long gene knockout period and complicated operation of Pseudomonas syringae pseudomonas actinidia pathogenic variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Schematic diagram of the process for obtaining HopH1 knockout strains;

[0047] Figure 2 : Schematic diagram of crRNAhopH1 targeting hopH1;

[0048] Figure 3 :pBBR-B4-crRNA hopH1 Schematic diagram of;

[0049] Figure 4 : crRNA targeting hopZ5 hopZ5 Schematic diagram of;

[0050] Figure 5 :pBBR-B4-crRNA hopZ5 Schematic diagram of;

[0051] Figure 6 :pBBR-B4-crRNA hopH1 Schematic diagram of enzyme digestion verification;

[0052] Figure 7 :pBBR-B4-crRNA hopZ5 Schematic diagram of enzyme digestion verification;

[0053] Figure 8 : Electrophoresis results of double primer detection of hopH1 knockout;

[0054] Figure 9 : Electrophoresis results of double primer detection of hopZ5 knockout based on △hopH1;

[0055] Figure 10 : Flowchart of plasmid elimination;

[0056] Figure 11 : Plasmid elimination pBBR-B4-crRNA hopH1 ;

[0057] Figure 12 : Construction of complementing plasmid;

[0058] Figure 13 : Psa strain inoculated into Walter kiwifruit. DETAILED DESCRIPTION

[0059] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] The primers used in the following examples are shown in Table 1.

[0061] Table 1 Primers used in the following examples

[0062]

[0063]

[0064] In the following examples, the kiwifruit varieties used were Actinidia chinensis (Hongyang), which is highly susceptible to canker, and Actinidia pubescens (Huate), which is highly resistant. Psa M228 (GenBank: CP032631) is a highly virulent biotype 3 strain isolated from Actinidia chinensis (Hongyang) grown in Meixian County, Shaanxi Province, China.

[0065] Psa and E. coli were grown in LB medium at 28°C and 37°C, respectively.

[0066] The final concentrations of antibiotics used in the experiment were (μg / ml): spectinomycin (Sp), 100; kanamycin (Km), 25; anhydrotetracycline (aTc), 100; nalidixic acid (Nal), 6; cefazolin (Cfz), 30; gentamicin (Gm), 30.

[0067] The culture medium used in the present invention is LB medium.

[0068] In the following examples, the competent cell preparation method was referred to the instructions of the Super Competent Cell Preparation Kit of Sangon Biotech (Shanghai) Co., Ltd.

[0069] The parent-child bonding method is:

[0070] The constructed plasmid was transferred into the donor strain E. coli S17-1λpir and cultured at 37°C and 220 rpm until the OD 600 =1.5, Psa was used as the recipient bacteria and cultured to OD 600 = 1.5. After incubation, the donor and recipient bacteria were washed 2-3 times with water and gently mixed in a ratio of S17-1λpir:Psa = 1:10. The mixture was plated onto 0.22μm sterile filter paper placed on an LB plate and incubated at 28°C for 12 hours. The ligation product after incubation was resuspended in sterile water and plated onto an LB plate containing the appropriate antibiotic.

[0071] The present invention will be further described in detail below through specific practical examples, but the specific application of the present invention is not limited to the genes described in the following examples, and can also be applied to the knockout of other genes of Pseudomonas syringae pv. actinidia.

[0072] Example 1

[0073] This example provides a gene knockout vector and strain for Pseudomonas syringae pv. kiwifruit, as well as a method for constructing the vector and the application thereof in obtaining transgenic plants. The specific contents include:

[0074] (1) crRNA hopH1 Acquisition

[0075] 1. Obtain the sequence of the gene to be knocked out, hopH1 (657 bp), of kiwifruit canker pathogen M228 through the NCBI website.

[0076] 2. Design crRNA based on the principle that the PAM sequence is TTTV, the crRNA length is 23bp, and the position is as close as possible to the middle of the gene hopH1hopH1 (https: / / chopchop.cbu.uib.no / )( Figure 2 ).

[0077] 3. Add AAA and GAT to the 5' end of the designed nucleotide sequence as the sticky end of SapI, respectively, crRNA hopH1 -F (sequence as shown in SEQ ID No.1) and crRNA hopH1 -R (sequence as shown in SEQ ID No. 2), crRNA hopH1 -F / crRNA hopH1 -R sequences were synthesized by Qingke Biotechnology Co., Ltd. (Beijing, China).

[0078] (2) pHZB4-crRNA hopH1 Construction

[0079] 1. Synthetic single-stranded nucleotide crRNA hopH1 -F, crRNA hopH1 -R is phosphorylated and annealed to form a double-stranded DNA sequence with SapI sticky ends, namely crRNA hopH1 .

[0080] 2. The crRNA hopH1 The sequence was ligated into pHZB4 digested with SapI using T4 ligase. The ligation product was heat-shock transformed into competent E. coli DH5α and plated on LB medium containing kanamycin. After incubation at 37°C for 12 hours, single colonies on the plate were picked for PCR verification. The primers were: crRNA1 hopH1 -F (shown in SEQ ID No. 1) / FnCas12a-R (shown in SEQ ID No. 5), the PCR product was subjected to electrophoresis, and the single colony with a product at 514 bp was the positive colony for preliminary verification. The positive colony was cultured overnight in LB liquid medium containing kanamycin, and the plasmid was extracted.

[0081] 3. The plasmid was subjected to Sanger sequencing by Boshang Biotechnology Shanghai Co., Ltd., and the sequencing primer was M13-47 (shown in SEQID No.6). The sequencing results detected crRNA hopH1 The plasmid present is the successfully constructed pHZB4-crRNA hopH1 .

[0082] (3) pBBR-B4-crRNA hopH1 The construction ( Figure 3 )

[0083] 1. pHZB4-crRNA hopH1The plasmid was digested with BamHI, and the fragment of about 8500 bp of the digestion product was recovered by gel electrophoresis.

[0084] 2. The recovered product was ligated into the digested pBBR1-MCS2 vector using T4 ligase. The ligation product was transformed into competent E. coli DH5α and plated on an LB plate containing kanamycin. After picking a single colony for PCR verification, the single colony with a band was shaken overnight in LB liquid medium containing kanamycin, the plasmid was extracted, and BamHI enzyme digestion was verified. The enzyme digestion product electrophoresis showed a band at 8531bp and 5144bp respectively. This plasmid was the successfully constructed plasmid pBBR-B4-crRNA hopH1 .

[0085] 3.pBBR-B4-crRNA hopH1 The plasmid was heat-shock transformed into S17-1λpir competent cells. After successful verification, the cells were used as donors for biparental conjugation with M228. The conjugation product was plated onto LB plates containing Km, Nal, and Cfz and incubated at 28°C for 5-7 days. Single colonies on the plates were selected for subsequent verification.

[0086] (4) Knockout verification

[0087] 1. Select the dual primers CRISPR-check-F / CRISPR-check-R and RV-M / pBBR-B4-check-R to perform PCR amplification on single colonies simultaneously to detect the knockout effect. CRISPR-check-F / CRISPR-check-R designed primers around hopH1 based on the genome sequence of M228 to detect the knockout status of hopH1. The electrophoresis results show that the single colony with a clear band at 2074bp is the wild type that has not been successfully knocked out. The colony with a band less than 2074bp is the knockout colony. The colony with no band means that the knockout sequence is outside the detection range of CRISPR-check-F / CRISPR-check-R. pBBR1-B4-crRNA hopH1 While FnCas12a in the plasmid performs the cleavage function on the strain genome, it may also perform the cleavage function on itself, making pBBR1-B4-crRNA hopH1 The plasmid is incomplete and the knockout effect is greatly reduced. RV-M / pBBR-B4-CHECK-R primer can be used to detect the integrity of the plasmid. The presence of a band at 576 bp indicates pBBR1-B4-crRNA hopH1 No self-cleavage occurs. The absence of bands indicates that the plasmid has undergone self-cleavage.

[0088] 2. Extract genomic DNA from the strain with intact plasmid and successful knockout, perform PCR amplification with CRISPR-check-F (shown in SEQ ID No.9) / CRISPR-check-R (shown in SEQ ID No.10) primers, and sequence the PCR product ( Figure 8 ).

[0089] (4) Plasmid elimination

[0090] 1. The knockout strain also contains the plasmid pBBR-B4-crRNA for knocking out hopH1 hopH1 To eliminate pBBR-B4-crRNA hopH1 Vector, use the elimination vector to eliminate the plasmid. pHM1B3-VD contains a crRNA array, which contains crRNAs for oriV replicon and LigD (Yan et al. 2023). pHM1B3-VD was digested with BamHI, and the sequence carrying the crRNA array was integrated into the broad-host vector pML-123 to obtain pML-B3free. ( Figure 1 )

[0091] 2. pML-B3free was heat-shocked and transferred into S17-1λpir.

[0092] 3. Transfer pML-B3free to the knockout plasmid pBBR-B4-crRNA by conjugation hopH1 In the strain, pBBR-B4-crRNA1 was eliminated hopH1 The ligation product was plated on LB agar plates containing Nal and Cfz to eliminate S17-1λpir, and then screened on LB plates containing and without Km. The sensitivity to Km indicated that the plasmid of the strain was successfully eliminated. Colony PCR was then performed using primers RV-M / pBBR-B4-check-R to verify that the plasmid was successfully eliminated. The electrophoresis results showed that there was no band at 576 bp, indicating that pBBR-B4-crRNA hopH1 After this process, the ΔhopH1 strain ( Figure 10 , Figure 11 ).

[0093] (5) Obtaining double knockout strains using the CRISPR / Cas12a system

[0094] 1. Using the ΔhopH1 strain as the starting strain, continue knocking out the hopZ5 gene and designing crRNA hopZ5 -F / crRNA hopZ5 -R( Figure 4 ).

[0095] 2. Construct knockout plasmid pBBR-B4-crRNA according to the above hopZ5 ( Figure 5 ) Heat shock transformed into S17-1λpir competent state.

[0096] 3. After conjugation transfer, the combined transformants were obtained, and two pairs of primers, Check-hopZ5-F (shown in SEQ ID No. 11) / Check-hopZ5-R (shown in SEQ ID No. 12) and RV-M (shown in SEQ ID No. 7) / pBBR-B4-CHECK-R (shown in SEQ ID No. 8), were designed to simultaneously detect the gene knockout of hopZ5 and pBBR-B4-crRNA hopZ5 Plasmid integrity ( Figure 9 ).

[0097] 4. Select a single colony with a knockout gene and intact plasmid to extract genomic DNA, and perform PCR using Check-hopZ5-F (shown in SEQ ID No. 11) / Check-hopZ5-R (shown in SEQ ID No. 12) as primers. The amplified product is sequenced to obtain specific sequence knockout information.

[0098] 5. The ΔhopZ5ΔhopH1 double knockout mutant was then obtained by plasmid elimination.

[0099] (6) Construction of complement strains

[0100] 1. Using primers hopH1-F (shown in SEQ ID No. 13) / hopH1-R (shown in SEQ ID No. 14) and hopZ5-F (shown in SEQ ID No. 15) / hopH1-R (shown in SEQ ID No. 14), PCR amplification of the hopH1 and hopZ5-hopH1 sequences was performed using genomic DNA from Psa M228. FLAG tags were added to the N-termini of hopH1 and hopZ5+hopH1 for labeling.

[0101] 2. The cloned product T4 was connected to the HindIII-digested pBBR1-MCS2. The single colonies grown on the kanamycin LB plate were verified by PCR and plasmid HindIII digestion to obtain pBBR-hopH1 and pBBR-hopZ5+hopH1 plasmids ( Figure 12 ).

[0102] 3. The successfully constructed plasmid was heat-shocked and transformed into the competent cell line S17-1λpir.

[0103] 4. The pBBR-hopH1 and pBBR-hopZ5+hopH1 plasmids were transferred into the ΔhopH1 and ΔhopH1ΔhopZ5 strains through biparental conjugation to obtain CΔhopH1 and CΔhopZ5ΔhopH1, respectively.

[0104] (7) Pathogenicity determination

[0105] 1. Psa strains (ΔhopH, ΔhopZ5ΔhopH1, CΔhopH1 and CΔhopZ5ΔhopH1) were cultured overnight and transferred into 10 ml LB medium (Km+Nal+Cfz) at a ratio of 1:20. The culture was shaken until the OD 600 = 0.5-1.0. Wash once with PBS and resuspend in 10 ml PBS.

[0106] 2. Measure OD after resuspension 600 Add PBS and resuspended bacterial solution according to the proportion, and dilute the bacteria in each tube to OD 600 =0.5.

[0107] 3. Kiwifruit Leaves: Select healthy kiwifruit leaves from the Palm-sized Group. Surface disinfect them with 0.6% sodium hypochlorite solution for 10-15 minutes, then rinse three times with sterile water. Use a sterilized 10mm punch to punch holes, avoiding the main veins. For each treatment, use 30 leaf discs and filter three times at 0.06-0.08 kPa for 10 minutes each.

[0108] 4. Wash the filtered leaves three times with sterile water and absorb all surface moisture from the leaves with sterile filter paper. Place the leaf disc face up on a 0.8% water agar medium (absorb as much moisture as possible).

[0109] 5. Place in an artificial climate incubator with an L / D ratio of 16h / 8h, a day / night temperature of 16°C / 10°C, a humidity of 75%, and a brightness of 40%. After 5 days, record the disease progression and take photos.

[0110] 6. Select 3 leaf discs, add 1 ml of sterile water and grind into slurry (time 1 min (grind 55 s, rest 5 s), 55 Hz). The ground slurry is the stock solution, and it is diluted to 10 -1 -10 -8 , spot on LB plate. After drying, incubate at 28℃ for 48h. Record the bacterial growth log 10 cfu / cm 2 .

[0111] (Calculation formula = log 10 (dilution factor*number of bacteria / 5) / 3π).

[0112] The results of pathogenicity determination were as follows Figure 13 shown.

[0113] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit, characterized in that: The following steps are involved: (1) According to the Psa M228 genome information on the NCBI website, crRNA primers for the target gene hopH1 were designed, namely crRNA1 hopH1 -F and crRNA1 hopH1 -R, and annealed by phosphorylation to form a double-stranded crRNA with a SapI sticky end, namely crRNA hopH1 ; (2) crRNA hopH1 The pHZB4-crRNA was formed by ligating it into pHZB4 after SapI digestion using T4 ligase. hopH1 ; Among them, crRNA1 hopH1 -F sequence is shown in SEQ ID No.1, crRNA1 hopH1 -R sequence is shown in SEQ ID No. 2; The sequence of the Psa M228 genome is available at GenBank: CP032631, and the sequence information of the target gene hopH1 is available at GenBank: CN228 09540; The sequence of pHZB4 is shown as SEQ ID No.

18.

2. The method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit according to claim 1, characterized in that: The following steps are also included: (3) pHZB4-crRNA hopH1 By BamHI digestion, the hopH1 , FnCas12a and NHEJ protein expression sequence fragments were ligated into pBBR1-MCS2 to form pBBR-B4-crRNA hopH1 ; The sequence of pBBR1-MCS2 is shown as SEQ ID No.

19.

3. A gene knockout vector for Pseudomonas syringae pv. kiwifruit, characterized in that: Prepared by the method of claim 1 or 2, pHZB4-crRNA hopH1 or pBBR-B4-crRNA hopH1 .

4. A method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit, characterized in that: The following steps are involved: (1) According to the Psa M228 genome information on the NCBI website, crRNA primers for the target gene hopZ5 were designed, namely crRNA hopZ5 -F and crRNA hopZ5 -R, and annealed by phosphorylation to form a double-stranded crRNA with a SapI sticky end, namely crRNA hopZ5 ; (2) Double-stranded crRNA hopZ5 The pHZB4-crRNA was formed by ligating it into pHZB4 after SapI digestion using T4 ligase. hopZ5 ; Among them, crRNA hopZ5 -F sequence is shown in SEQ ID No.3, crRNA hopZ5 The -R sequence is shown in SEQ ID No.

4.

5. The method for preparing a gene knockout vector of Pseudomonas syringae pv. kiwifruit according to claim 4, characterized in that: The following steps are also included: (3) pHZB4-crRNA hopZ5 By BamHI digestion, the hopZ5 , FnCas12a and NHEJ protein expression sequence fragments were ligated into pBBR1-MCS2 to form pBBR-B4-crRNA hopZ5 .

6. A gene knockout vector for Pseudomonas syringae pv. kiwifruit, characterized in that: Prepared by the method of claim 4 or 5, pHZB4-crRNA hopZ5 or pBBR-B4-crRNA hopZ5 .

7. A method for preparing a gene knockout strain of Pseudomonas syringae pv. kiwifruit, characterized in that: The following steps are involved: S1, the successfully constructed pBBR-B4-crRNA hopH1 The donor bacteria S17-1λpir were transformed by heat shock and the vector pBBR-B4-crRNA in S17-1λpir was transferred by biparental conjugation. hopH1 The conjugate was transformed into the recipient bacteria M228 to obtain the conjugate, which was shaken in LB liquid medium containing tetracycline for 1.5 h to induce the expression of FnCas12a protein; S2. Spread the conjugate on an LB resistance plate containing kanamycin, nalidixic acid, and cefazolin, and culture at 28°C for 3-5 days. Verify the PCR products of the single colonies grown on the plate using two pairs of primers to screen for knockout of hopH1 and pBBR-B4-crRNA. hopH1 For strains where the plasmid did not undergo self-cleavage, the specific knockout sequence was subsequently determined by Sanger sequencing; S3, the pHM1B3-VD plasmid containing two crRNAs was digested with BamHI, one of which was a crRNA target gene oriV and the other was a crRNA target gene LigD, and the expression sequence of FnCas12a was ligated into the pML-123 vector with a broad host range by T4 ligase to construct a Gm-resistant pML-B3free plasmid, which was then transferred into the donor bacteria S17-1λpir by heat shock transformation for plasmid elimination, thereby obtaining S17-1λpir containing the pML-B3free plasmid; S4, S17-1λpir containing pML-B3free plasmid, and hopH1 knockout but still containing pBBR-B4-crRNA hopH1 The strains were screened for hopH1 knockout but did not contain pBBR-B4-crRNA. hopH1 The strain was named ΔhopH1 strain. 8 . A Pseudomonas syringae pv. kiwifruit gene knockout strain prepared based on the method of claim 7 , namely, the ΔhopH1 strain.

9. A ΔhopZ5ΔhopH1 strain, characterized in that This is a strain in which both the hopZ5 gene and the hopH1 gene are knocked out. The specific preparation method is as follows: The ΔhopH1 strain was used as the background, and the corresponding vector was constructed and the ΔhopZ5ΔhopH1 strain was obtained by biparental conjugation.

10. Use of the Pseudomonas syringae pv. actinidiae gene knockout vector according to claim 3 or 6 or the strain according to claim 8 or 9 in obtaining transgenic plants.

Images