CN110016520B

CN110016520B - Banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant and small RNA thereof

Info

Publication number: CN110016520B
Application number: CN201910362416.0A
Authority: CN
Inventors: 张欣; 曾凡云; 彭军; 漆艳香; 谢培兰; 谢艺贤
Original assignee: CATAS Environment and Plant Protection Institute
Current assignee: CATAS Environment and Plant Protection Institute
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2020-01-03
Anticipated expiration: 2039-04-30
Also published as: CN110016520A

Abstract

The invention discloses a banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant and a small RNA thereof. By adopting a homologous replacement principle, the gene deletion mutants of the banana fusarium oxysporum including delta ago1, delta ago2 and delta ago1/2 are constructed, abiotic stress test and pathogenicity analysis are carried out, and the results show that the delta ago2 deletion mutant is more sensitive and inhibited in growth after being treated by a fluorescent brightener CFW, and MgCl is adopted₂After treatment, the growth of the Δ ago1 mutant, the Δ ago2 mutant, and the Δ ago1/2 mutant were inhibited. The pathogenicity of the delta ago1 mutant is enhanced, and the pathogenicity of the delta ago2 mutant and the delta ago1/2 mutant are weakened; in addition, small RNA of each mutant is obtained by combining with deep sequencing of small RNA, so that theoretical and technical supports are provided for further analyzing pathogenic mechanisms of banana vascular wilt, developing prevention and control measures of RNA sources and the like.

Description

Banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant and small RNA thereof

Technical Field

The invention relates to a banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant and a small RNA thereof, belonging to the technical field of bioengineering.

Background

Small-molecule RNA is a type of 21-24nt non-coding RNA widely existing in eukaryotes, and comprises miRNA (microRNA) and siRNA (small interfering RNA), and can be combined with a ribozyme complex containing Argouaute (AGO) protein to form an RNA-induced silencing complex RISC (RNA-induced silencing complex), and sequence-specific post-transcriptional gene silencing regulation is caused by cutting target mRNA or translation inhibition. The AGO protein is an important component in RISC complex and plays an important role in small RNA-mediated gene silencing. The AGO protein is 700-1200 amino acid residues in full length and comprises four domains, namely an N-terminal domain, a PAZ domain, an MID domain and a PIWI domain. After 21-24nt small-fragment siRNAs containing 5 'end phosphate group, 3' end hydroxyl group and 2nt nucleotide protrusion are generated by Dice cutting the dsRNA, the siRNAs are combined with AGO protein containing PAZ functional domain to form RNA-induced silencing complex, and the complex has sequence-specific endonuclease activity and can specifically degrade target mRNA homologous with siRNA.

RNA silencing (rnaselencing) or gene silencing (gene silencing) is a highly conserved, sequence-specific RNA degradation mechanism that is widely present in eukaryotes such as plants, animals, nematodes, and fungi. RNA gene silencing is a post-transcriptional silencing (PTGS) phenomenon found in Caenorhabditis elegans (Fire et al, 1998) by double-stranded RNA (dsRNA) in a sequence-specific manner. After introducing a homologous fragment of the endogenous gene al-1 of Neurospora crassa (Neurospora crassa) by Romano and Macino, the expression of albino-1 gene was found to be suppressed, and they called RNA resting (quelling) (Romano and Macino, 1992). Subsequently, researchers obtained a series of quelling loss-of-function mutant strains, and studies of these mutants demonstrated that the quelling pathway was associated with siRNAs (Cogoni and Macino, 1997). Among them, QDE-1 encodes a cell-dependent RNA polymerase (RdRP), which is an important component necessary for RNAi in other eukaryotes (Cogoni and Macino, 1999). In addition, the QDE-2 gene encodes an Argonaute protein containing piwi-PAZ structural domain, is a homologous protein of caenorhabditis elegans RDE-1 (RNAi-specificity-1), and is one of important conserved elements in eukaryotic RNA silencing pathways. The protein encoded by QDE-3 belongs to the RecQ family of DNA helicases and is involved in homologous recombination, DNA replication and DNA repair (Cogoni and Macino, 1997).

Fungi also encode mirnas similar to plant mirnas, named microRNA-like small RNAs (milRNAs). The biosynthesis pathway of the milRNAs is greatly different from the biological pathway of miRNA of plants and animals, the length of the milRNAs found in Neurospora crassa (N.crassa) is generally 19-25nt, the milRNAs have hairpin structure, the formed milRNAs have the preference of 5' terminal U, and can be specifically combined with Argonaute (AGO) protein to play a function. There are four distinct pathways for the biogenesis of milRNAs in Neurospora crassa, which are produced by different combinations of Dicers, QDE-2, QIP effector proteins for exonuclease activity, and mitochondrial ribosomal large subunit MRPL3 protein (MRPL 3) with RNase III domain, including a Dicer-independent path for producing mil RNA (disRNAs) (Lee et al, 2010). Although the length of the RNA precursor sequence transcribed from the intergenic region varies in the four pathways, it differs from most plant and animal miRNA-dependent RNApolymerase II transcriptions, and the milRNA of Neurospora crassa is mainly transcribed by Pol III (Yang et al, 2013).

The small RNA plays an important role in mediating the pathogen-host interaction process, and viruses, fungi, bacteria and the like can inhibit the expression of host defense related genes by virtue of self-encoded miRNAs or milRNAs so as to promote the infection of the pathogen. Similarly, the siRNA coded by the pathogenic fungi can target host mRNA and enter plant cells to inhibit plant immune defense reaction by using small molecular RNA as an effector. Botrytis cinerea (Botrytis cinerea) can infect more than 200 plants, and the study of Weiberg et al (2013) shows that the Botrytis cinerea can deliver Bc-sRNAs into host cells to inhibit AGO1 function by specifically binding to AGO1, and silence host immune-related genes to promote pathogen infection. The AGO1 deletion mutant of arabidopsis thaliana has reduced sensitivity to botrytis cinerea due to loss of the AGO1 function; in contrast, the dcl1dcl2 double mutant of Botrytis cinerea completely lost infectivity due to the inability to process to produce Bc-sRNAs. Thus, the discovery that fungal pathogens promote pathogen infestation by inhibiting host defense responses by transferring pathogenic small RNA effectors into host cells illustrates this naturally occurring transboundary RNAi silencing phenomenon as a more advanced pathogenesis of pathogenic fungi (Weiberg et al, 2013; Wang et al, 2016). Subsequently, more studies have further demonstrated a mechanism of trans-border gene silencing. Wheat stripe rust (Pst) is one of the important diseases of wheat, and small RNA coded by the Pst is an important pathogenic factor. The Pst self-encoded microRNA-like RNA 1(Pst-milR1) only induces expression at infection, and targets the 1, 3-beta-glucanase (SM638) of the PR2 gene related to the wheat disease course through a cross-border silencing mechanism to promote infection. Silencing Pst-mil 1 expression in pathogenic bacteria reduces pathogenic virulence; conversely, knockout of the target PR2 gene in wheat enhances susceptibility of wheat to avirulent strains. Furthermore, Pst-milR1 was produced depending on DCLs processing (Wang et al, 2017). Verticillium dahliae (Verticillium dahliae) belongs to Verticillium fungi of Deuteromycotina, has a wide host range, and can cause cotton Verticillium wilt. The verticillium dahliae also codes small RNA, and recent research shows that VdmirRNA 1 coded by the verticillium dahliae is generated independently of AGOs and DCLs, and Vdmir 1 targets the 3' UTR region of Vdmhly 1 gene to regulate the expression of a target gene. Molecular regulation of VdmilR1-VdHy1 is not by way of target mRNA cleavage, but by way of transcriptional repression by increasing the methylation level of histone H3K9 (Jin et al, 2019).

Banana vascular wilt (Fusarium oxysporum f.sp. cubense, Foc) is a destructive soil-borne disease causing plant death by destroying banana vascular bundles, has strong pathogenic bacteria saprophytic capacity, can survive in soil for a long time, and belongs to typical latent infection. The plantain region in the world has found that there are 4 physiological races (other races may exist), of which the physiological Race1 (Race1) infects plantain (ABB group), canna (AAB group) and part of AAA group varieties; the physiological Race 2 (Race 2) only infects the hybrid triploid banana (ABB), and has less harm to the banana cultivar; the physiological Race 3 (Race 3) infects wild castanopsis, does not cause harm to banana cultivars, and is reported in many areas; the physiological race 4 comprises Tropical race 4 (Tropical race 4, TR4) and Subtropical race 4 (Subtropical race 4, ST4), and infects banana, canna and plantain, and the host range of the infection is larger than that of the physiological race 1. Early external symptoms are not obvious, and the symptoms are only expressed in the middle and later stages, so that huge economic losses are caused to banana industry and banana growers. The banana wilt causes serious harm and threat to the global banana industry, has wide influence and attracts general attention, but no effective prevention and treatment measures are available at present. Therefore, by utilizing the mechanism of the small RNA in the fusarium wilt-banana molecular interaction, a new theory and technical support is hopefully provided for preventing and controlling bananas.

Disclosure of Invention

The method predicts the funRNA (http:// funna. ricebast. snu. ac. kr) by bioinformatics, combines with functional domain analysis software SMART (simple modulated Architecture Research tool) (http:// smart. embl. de), determines that the banana fusarium wilt contains 2 AGO proteins, analyzes 2 AGOs genes of No. 4 physiological race of the banana fusarium wilt (TR4), and performs abiotic stress test and pathogenicity analysis on knockout mutants of the AGO genes; in addition, small RNA of each mutant is obtained by combining with deep sequencing of the small RNA, so that theoretical and technical supports are provided for further analyzing the pathogenic mechanism of the banana vascular wilt and developing prevention and control measures of RNA sources.

The technical scheme adopted by the invention is as follows:

a banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant is obtained by the following method:

1) constructing Foc4AGO1 gene deletion recombinant DNA fragment, adopting hygromycin resistance gene HPH, protoplast transformation to obtain Foc4AGO1 gene deletion mutant, namely delta AGO1 mutant; the AGO2 gene can be knocked out by the same method to construct a delta AGO2 mutant;

2) a Foc4AGO 1/2 double-gene deletion recombinant DNA fragment is constructed on the basis of a delta AGO1 mutant, and a neomycin NEO resistance gene is adopted to transform protoplasts to obtain a Foc4AGO 1/2 double-gene deletion mutant, namely a delta AGO1/2 mutant.

More specifically:

step 1) the construction method of the delta ago1 mutant comprises the following steps:

first round PCR amplification:

left end LB: FOC4 genome DNA is used as a template, Foc4AGO1-LBCK and Foc4AGO1-HPH-LB-R are used as primers, and the size of an amplification product is 1993 bp;

right end RB: FOC4 genome DNA is used as a template, Foc4AGO1-HPH-RB-F and Foc4AGO1-RBCK are used as primers, and the size of an amplification product is 1988 bp;

hygromycin resistance gene HPH: vector plasmid DNA is used as a template, HYG-F and HYG-R are used as primers, and the size of an amplification product is 1376 bp;

second round of PCR amplification:

amplification of the left end LB + HP: the LB and hygromycin resistance gene HPH at the left end are used as templates, Foc4AGO1-LB-F and HYG-R1 are used as primers, and the size of an amplification product is 2415 bp;

right-end PH + RB amplification: right end RB and hygromycin resistance gene HPH are used as templates, HYG-F1 and FOC4AGO1-RB-R are used as primers, and the size of an amplification product is 2274 bp;

recovering the second round of PCR fragments, and introducing the second round of PCR fragments into protoplasts for transformation to obtain Foc4AGO1 gene deletion mutants, namely delta AGO1 mutants;

wherein, the sequences of Foc4AGO1-LBCK, Foc4AGO1-HPH-LB-R, Foc4AGO1-HPH-RB-F, Foc4AGO1-RBCK, Foc4AGO1-LB-F, HYG-R1, HYG-F1 and Foc4AGO1-RB-R are shown as SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 9, SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 11 in sequence.

More specifically:

step 1) the construction method of the delta ago2 mutant comprises the following steps:

first round PCR amplification:

left end LB: FOC4 genome DNA is used as a template, Foc4AGO2-LBCK and Foc4AGO2-HPH-LB-R are used as primers, and the size of an amplification product is 1840 bp;

right end RB: FOC4 genome DNA is used as a template, Foc4AGO2-HPH-RB-F and Foc4AGO2-RBCK are used as primers, and the size of an amplification product is 1900 bp;

second round of PCR amplification:

left end LB + HP: the LB and hygromycin resistance gene HPH at the left end are used as templates, Foc4AGO2-LB-F and HYG-R1 are used as primers, and the size of an amplification product is 2411 bp;

right end PH + RB: right end RB and hygromycin resistance gene HPH are used as templates, HYG-F1 and Foc4AGO2-RB-R are used as primers, and the size of an amplification product is 2134 bp;

wherein the sequences of Foc4AGO2-LBCK, Foc4AGO2-HPH-LB-R, Foc4AGO2-HPH-RB-F, Foc4AGO2-RBCK, Foc4AGO2-LB-F, HYG-R1, HYG-F1 and Foc4AGO2-RB-R are shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 15, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 17 in sequence.

More specifically:

first round PCR amplification:

left end LB: the delta AGO1 mutant genome DNA is used as a template, Foc4AGO2-NEO-LBCK and Foc4AGO2-NEO-LB-R are used as primers, and the size of an amplification product is 1918 bp;

right end RB: the delta AGO1 mutant genome DNA is used as a template, Foc4AGO2-NEO-RB-F and Foc4AGO2-NEO-RBCK are used as primers, and the size of an amplification product is 1913 bp;

neomycin NEO resistance gene: vector plasmid DNA is used as a template, NEO-F and NEO-R are used as primers, and the size of an amplification product is 1370 bp;

second round of PCR amplification:

left end LB + NE: the resistance genes of LB and neomycin NEO at the left end are taken as templates, Foc4AGO2-NEO-LB-F and NEO-R1 are taken as primers, and the size of an amplification product is 2530 bp;

right EO + RB: right end RB and neomycin NEO resistance gene are used as templates, NEO-F1 and Foc4AGO2-NEO-RB-R are used as primers, and the size of an amplification product is 2343 bp;

recovering the second round of PCR fragments, and introducing the recovered second round of PCR fragments into protoplasts for transformation to obtain Foc4AGO 1/2 double-gene deletion mutants, namely delta AGO1/2 mutants;

wherein, the sequences of Foc4AGO2-NEO-LBCK, Foc4AGO2-NEO-LB-R, Foc4AGO2-NEO-RB-F, Foc4AGO2-NEO-RBCK, Foc4AGO2-NEO-LB-F, NEO-R1, NEO-F1 and Foc4AGO2-NEO-RB-R are shown as SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 27, SEQ ID NO 21, SEQ ID NO 22 and SEQ ID NO 29 in sequence.

The invention further performs abiotic stress test and pathogenicity analysis on the obtained mutant, and the result shows that after the mutant is treated by the fluorescent brightener CFW, the delta ago2 deletion mutant is more sensitive and growth is inhibited, and MgCl is adopted₂After treatment, the growth of the Δ ago1 mutant, the Δ ago2 mutant, and the Δ ago1/2 mutant were inhibited. The mutant delta ago1 has enhanced virulence, and the mutant delta ago2 and the mutant delta ago1/2 have reduced virulence.

The invention also carries out deep sequencing on the delta ago1 mutant, the delta ago2 mutant and the delta ago1/2 mutant, and after comparison analysis, the delta ago1 mutant generates more specific small RNA of a sequence shown as any one of SEQ ID NO. 35-43 compared with Foc 4.

Compared with Foc4, the delta ago2 mutant produces a specific small RNA with a sequence shown as any one of SEQ ID NO. 44-47.

Compared with Foc4, the delta ago1/2 mutant generates specific small RNA with a sequence shown as any one of SEQ ID NO 48-57.

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

the invention adopts the principle of homologous replacement to construct banana vascular wilt delta AGO1, delta AGO2 and AGO1/2 gene deletion mutants, and carries out abiotic stress test and pathogenicity analysis on the AGO gene knockout mutants, and the result shows that the delta AGO2 mutant is more sensitive and growth is inhibited after being treated by a fluorescent brightener CFW (fluorescent whitening agent)₂After treatment, the growth of the Δ ago1 mutant, the Δ ago2 mutant, and the Δ ago1/2 mutant were inhibited. The pathogenicity of the delta ago1 mutant is enhanced, and the pathogenicity of the delta ago2 mutant and the delta ago1/2 mutant are weakened;

in addition, small RNA of each mutant is obtained by combining with deep sequencing of the small RNA, so that theoretical and technical supports are provided for further analyzing the pathogenic mechanism of the banana vascular wilt and developing prevention and control measures of RNA sources.

Drawings

FIG. 1 is a schematic representation of the Split-PCR gene knockout;

FIG. 2 is a schematic diagram showing single gene knockout of banana vascular wilt bacterium Foc4AGO and a PCR identification result of a positive transformant; (A) schematic diagram of AGO1 gene knockout; (B) is a graph of the results of PCR detection of the Δ ago1 mutant; (C) schematic diagram of AGO2 gene knockout; (D) is a graph of the results of PCR detection of the delta ago2 mutant;

FIG. 3 is a diagram showing the Δ ago1/2 double gene knockout and the PCR identification result of positive transformants; (A) schematic diagram of delta ago1/2 double gene knockout; (B) is a PCR detection result graph of the delta ago1/2 mutant;

FIG. 4 is a graph showing the results of analysis of the biological properties of Foc4AGO gene deletion mutants; (A) the growth conditions of banana vascular wilt Foc4 and delta ago1, delta ago2 and delta ago1/2 mutant PDA plates; (B) the growth rate measurement result graph of banana vascular wilt Foc4 and the delta ago1, delta ago2 and delta ago1/2 mutants is shown; (C) a functional domain analysis result chart of Foc4 AGOs; (D) the result chart of spore yield determination of banana vascular wilt Foc4 and mutants delta ago1, delta ago2 and delta ago1/2 is shown;

FIG. 5 is a graph showing the results of determination of abiotic stress sensitivity of mutants;

FIG. 6 is a graph showing the results of the virulence assay of the mutants; (A) foc4 and the aerial pathogenic phenotype of the mutant after the mutant is artificially inoculated with the banana by delta ago1, delta ago2 and delta ago 1/2; (B) foc4 and a cut-away view of the bulbs of the underground part after the mutants of delta ago1, delta ago2 and delta ago1/2 are inoculated with bananas; FIG. 6 shows the results of disease index analysis of Foc4 and Δ ago1, Δ ago2 and Δ ago1/2 mutants in the lower right panel.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example A Banana Fusarium Foc4 AGOs Gene knockout

The fusarium oxysporum f.sp.cubense contains two AGO protein genes of AGO1 and AGO2, a principle of homologous replacement is adopted in Foc4AGO single gene knockout, a DNA fragment of a resistance gene hygromycin B (HPH) gene is used for replacing a DNA fragment of a target gene Foc4AGO1 or Foc4AGO2, a schematic diagram of a Split-PCR gene knockout method is shown in figure 1, and primers used in a subsequent gene knockout step are marked in the same way as the schematic diagram.

EXAMPLE two Foc4AGO1 Gene deletion recombinant DNA fragment construction

The Split-PCR gene knockout method adopts two rounds of PCR amplification to recombine DNA fragments, and the first round of PCR amplification comprises three fragments: the DNA sequence of the upstream homology arm of the target gene, the full-length sequence of the resistance gene and the DNA sequence of the downstream homology arm of the target gene. And in the second round of PCR, the upstream and downstream homologous arm DNA sequences and the resistance gene DNA are respectively mixed to be used as PCR templates, and the upstream and downstream deletion recombinant DNA fragments are respectively amplified. Foc4AGO1 Gene sequence number Gene FOIG _01986, reference NCBI GenBank: EXM08967 genome sequence, design primer amplification AGO1 Gene upstream and downstream homology arm Gene sequence (figure 2-A, B). The method comprises the following specific steps:

first round PCR amplification:

left end LB: FOC4 genome DNA is used as a template, AGO1-LBCK/AGO1-HPH-LB-R is used as a primer, and the size of an amplification product is 1993 bp;

right end RB: FOC4 genome DNA is used as a template, AGO1-HPH-RB-F/AGO1-RBCK is used as a primer, and the size of an amplification product is 1988 bp;

hygromycin resistance gene HPH: HYG-F and HYG-R are used as primers, vector plasmid DNA is used as a template, and the size of an amplification product is 1376 bp;

PCR reaction procedure: 95 ℃ for 4min, 1 cycle; 94 ℃, 40s, 58 ℃, 40s, 72 ℃, 1min, 30s, 32 cycles; 72 ℃,10min, 1 cycle; hold at 16 ℃.

Second round of PCR amplification:

left-end recombinant DNA fragment (LB + HP): FOC4AGO1-LB-F and HYG-R1 are used as primers, LB + HPH is used as a template, and the size of an amplification product (LB1321+ HP1094) is 2415 bp;

right recombinant DNA fragment (PH + RB): HYG-F1 and FOC4AGO1-RB-R are used as primers, RB + HPH is used as a template, and the size of an amplification product (PH748+ RB1526) is 2274 bp;

PCR reaction procedure: 95 ℃ for 4min, 1 cycle; 94 ℃, 40s, 58 ℃, 1min 20s, 72 ℃, 2min30s, 32 cycles; 72 ℃,10min, 1 cycle; hold at 16 ℃.

The primer sequences are shown in Table 1. The second round of PCR fragments were recovered and introduced into protoplasts for homologous recombination gene knock-out (FIG. 2-A, B).

Example construction of a recombinant DNA fragment for deletion of the Gene tris Foc4AGO2

Foc4 the gene knockout method of AGO2 is the same as AGO1, and the primers and instructions are shown in Table 2. Foc4AGO2 Gene sequence number Gene FOIG _01246, reference GenBank: EXM11679 genome sequence, design primer amplification AGO2 Gene upper and lower homologous genome sequence (figure 2-C, D). The detailed operation method comprises the following steps:

first round PCR amplification:

the left end LB, FOC4AGO2-LBCK and FOC4AGO2-HPH-LB-R are primers, FOC4 genome DNA is a template, and the size of an amplification product is 1840 bp;

right end RB FOC4AGO2-HPH-RB-F and FOC4AGO2-RBCK are used as primers, FOC4 genome DNA is used as a template, and the size of an amplification product is 1900 bp;

HPH fragment: HYG-F and HYG-R are used as primers, vector plasmid DNA is used as a template, and the size of an amplification product is 1376 bp;

second round of PCR amplification:

left end (LB + HP): FOC4AGO2-LB-F and HYG-R1 are used as primers, LB + HPH is used as a template, and the size of an amplification product (LB1317+ HP1094) is 2411 bp;

right end (PH + RB): HYG-F1 and FOC4AGO2-RB-R are used as primers, RB + HPH is used as a template, and the size of an amplification product (PH748+ RB1386) is 2134 bp.

The detailed primer sequences and their descriptions are shown in Table 1. The second round of PCR fragments were recovered and introduced into protoplasts for homologous recombination gene knock-out (FIG. 2-C, D).

Example construction of four Foc4AGO 1/2 double Gene deletion recombinant DNA fragment

Foc4AGO 1/2 double gene knockout mutant is carried out on the basis of delta AGO1 mutant, neomycin NEO resistance gene is adopted, and the screening of delta AGO1/2 transformant is convenient (figure 3-A, B).

First round PCR amplification:

the left end LB, FOC4AGO2-NEO-LBCK and FOC4AGO2-NEO-LB-R are primers, delta AGO1 mutant genome DNA is a template, and the size of an amplification product is 1918 bp;

right end RB FOC4AGO2-NEO-RB-F and FOC4AGO2-NEO-RBCK are used as primers, delta AGO1 mutant genome DNA is used as a template, and the size of an amplification product is 1913 bp;

neomycin NEO resistance gene: NEO-F and NEO-R are used as primers, vector plasmid DNA is used as a template, and the size of an amplification product is 1370 bp;

second round of PCR amplification:

left end (LB + NE): FOC4AGO2-NEO-LB-F and NEO-R1 are primers, LB + NEO is a template, and the size of an amplification product (LB1630+ NE900) is 2530 bp;

right end (EO + RB): NEO-F1 and FOC4AGO2-NEO-RB-R, RB + NEO as templates, and the amplification product size (EO976+ RB1367)2343 bp.

The primer sequences are shown in Table 1. The second round of PCR fragments were recovered and introduced into protoplasts for homologous recombination gene knock-out (FIG. 3-A, B).

Example five PEG mediated transformation of protoplasts

1) Protoplast preparation

5-10 pieces of Foc4 mycelium pieces (cultured for 7-10 days, with size of 5mm) were added to 100ml of PDB, and shake-cultured at 28 deg.C and 150rpm for 4-5 days. Filtering with 3 layers of lens wiping paper, and collecting conidia in the filtrate. Adding 5-10ml of conidium solution into 200ml of PDB, and shaking at 28 deg.C and 150rpm for 12-18 h. The cultured broth was filtered through 4 layers of gauze and a suitable amount of 0.7M NaCl (8.18g NaCl, H)₂O to 200ml) solution, collecting fresh hyphae, and transferring the hyphae into a 50ml centrifuge tube. Adding 8-10ml of enzymolysis solution (the concentration of the storage solution is 20mg/ml, the working concentration is 10mg/ml, weighing the collapse enzyme, dissolving with 0.7M NaCl, and fixing the volume). And (4) vortexing to disperse the thalli and fully contact with the enzymolysis liquid, and carrying out warm bath for 3-4h at the temperature of 30 ℃ and the rpm of 80-100. After enzymolysis, adding 0.7M NaCl into a 50ml centrifuge tube to dilute the enzymolysis solution, reversing and uniformly mixing, filtering by three layers of sassafras paper, and washing the filter paper by a proper amount of 0.7M NaCl; the protoplasts were collected after centrifugation at 4000rpm for 15min at 4 ℃. With 10-20ml of STC (21.86g Sorbitol; 1.0ml of 1M Tris-HCl pH 7.5; 0.735g CaCl₂·2H₂O,H₂O to 100ml), the precipitate was dissolved sufficiently, centrifuged at 4000rpm for 15min, and the supernatant was discarded. Adding about 400 mul STC to fully dissolve the precipitate, wherein the amount of STC is determined according to the amount of the precipitate, and the concentration of the protoplast can reach 10⁷Pieces/ml, place them in 1.5ml centrifuge tubes.

2) Protoplast transformation

And (4) subpackaging the protoplast to ensure aseptic operation. Protoplasts (approximately 200. mu.l) were mixed with upstream recombinant DNA fragments (100. mu.l) and downstream recombinant DNA fragments (100. mu.l) in 1.5ml centrifuge tubes (gently swirled) and left for 20min at room temperature. The liquid in the small centrifuge tube was transferred to a tip centrifuge tube (50 ml marked with accurate graduations) and 400. mu.l PEG (30g PEG4000, 0.5ml 1M Tris-HCl pH 7.5, 0.735g CaCl) was added dropwise₂·2H₂O，H₂O to 50ml), cover with the lid and spin gently, ice-wash for 15 min. Adding 800 μ l PEG (dropwise adding at room temperature, not on ice), gently swirling, mixing, standing at room temperature for 20min, adding 2ml RB, culturing at 28 deg.C in incubator for 2 hr, and culturing at room temperatureRA medium (Glucose 10 g; KCl 0.52 g; MgSO 10 g) was added to a tip centrifuge tube₄·7H₂O 0.52g；KH₂PO₄ 0.25g；Sorbitol 218.5g(1.2M/L)；NaNO₃6g of a mixture; 1% Agar), making the volume to 50ml, transferring into 5 culture dishes, culturing in an incubator at 28 ℃ for 24h, covering a layer of PDA (50 mu g/ml containing hygromycin) on the surface of the culture medium, and culturing in the incubator.

EXAMPLE sixthly, identification of Gene deletion mutants

The genome DNA of the transformant is taken as a template, and four pairs of primers are utilized to respectively amplify an upstream recombinant DNA sequence, a downstream recombinant DNA sequence, a used resistance screening gene sequence and a target gene self-specificity sequence for identifying the transformant (see the design and the description of a knockout mutant primer in detail). Positive transformants should meet the following PCR amplification conditions: the upstream and downstream gene recombination DNA sequences are amplified positively, the resistance gene is PCR positive, and the target gene specific detection primer is amplified negatively. The PCR results (figure 2/figure 3) of the positive transformants detected by the four pairs of primers show that the upstream and downstream primer combination amplification products of the gene deletion mutant show that the upstream and downstream sequences of the transformants are replaced, and the target gene self-specific primers fail to amplify a band to show that the target gene sequence and the resistance gene sequence are subjected to homologous recombination and replacement.

TABLE 1 List of single and double knockout primer sequences for banana vascular wilt Foc4 AGOs genes

Experimental example biological and functional analysis of deletion mutant of banana vascular wilt

Test example 1 measurement of growth Rate

Foc4, delta ago1, delta ago2 and delta ago1/2 mutants cultured for 7d are taken, hypha blocks are punched by a 5mm puncher, the hypha blocks are inoculated in a PDA plate (phi is 60mm), the mutants are cultured in an inverted mode for 7d at 28 ℃, cross lines are drawn at the middle hypha cake positions of plates, two data are recorded in each plate, three data are repeated, the colony diameters are measured at 1dpi, 3dpi, 5dpi and 7 dpi respectively, the growth speeds of the colonies are measured, and data analysis is carried out by data measured at 5 dpi. After inoculation for 5d, the mutants of delta ago1, delta ago2 and delta ago1/2 grew over the whole plate, the colony diameter and the colony diameter of the wild strain were both 7.4cm, and the growth rate of the mutant was not significantly different from that of the wild strain Foc4 (FIG. 4-A, B).

The AGOs gene sequence of banana vascular wilt was introduced using online protein domain analysis software SMART (simple modulated Architecture Research tool) (http:// smart.embl. de), and functional domain analysis of Foc4 AGOs showed that AGO1 and AGO2 both contain DUF1785, PAZ and PIWI functional domains. DDH was conserved at Foc4AGO2, but in the PIWI domain of F G AGO1 and Foc4AGO1 of F.graminis, the third amino acid residue, histidine, was replaced by aspartic acid to DDD (FIG. 4-C).

Test example 2 measurement of spore yield

Taking the mutants of banana vascular wilt Foc4, delta ago1, delta ago2 and delta ago1/2 cultured for 7d, punching a fungus cake by using a punch with the diameter of 5mm, transferring the fungus cake into a PDA plate with the diameter of 9cm, and culturing for 7d at 28 ℃. Using a puncher with the diameter of 5mm to pick 6 fungus cakes along the half of the bacterial colony, transferring the fungus cakes into a 2ml EP tube, adding 2 steel balls with the diameter of 5mm, placing the EP tube into a high-flux tissue grinder, vibrating the EP tube for 120s at the frequency of 50HZ, and adding 1mlddH into the ground fungus tissue₂O, shaking at 150rpm for 1h at room temperature to release the spores sufficiently. 200. mu.l of spore suspension was diluted 10-fold, and the diluted spore suspension was counted on a hemocytometer in triplicate for each strain to conduct spore production measurement (FIG. 4-D). The results show a reduction in the sporulation yield of Δ ago 1.

Test example 3 determination of Abiotic stress sensitivity

Regulating spore concentration of shake-cultured 3d banana vascular wilt Foc4 and delta ago1, delta ago2 and delta ago1/2 mutant spore suspension to 2 × 10 concentration with sterile water⁷、2×10⁶、2×10⁵、2×10⁴Each/ml, subpackaged into 1.5ml sterile EP tubes, and stored at 4 ℃ for later use. Adding abiotic stress factors into the MM culture medium respectively, and adjusting the concentrations to be: 1.0MGlucose, 200 μ g-ml CR、400μg/ml CFW、1.0M Sorbitol、0.7M NaCl、1.0M KCl、0.1M MgCl₂. MM plates (phi ═ 15cm) were prepared, and the bottom of the MM plates was scribed uniformly into 1.5X 1.5cm cells for use. 2.0. mu.l of each spore suspension with gradient concentration was dropped into a small cell of MM plate, and inverted culture was performed at 28 ℃ to observe abiotic stress sensitivity of deletion mutants.

The inhibition ratio (%) (control group colony diameter-test group colony diameter)/control group colony diameter × 100.

The results are shown in fig. 5, where the Δ ago2 deletion mutant was more sensitive and growth was somewhat inhibited after treatment with the fluorescent whitening agent CFW. Metal cation MgCl₂After treatment, the growth of the deletion mutants of delta ago1, delta ago2 and delta ago1/2 is inhibited, and other abiotic stress factors, including osmotic stress factors and cell wall integrity related stress factors, have no significant effect on the deletion mutants of delta ago1, delta ago2 and delta ago 1/2.

Test example 4 determination of virulence of mutant

Collecting Foc4 cultured for 7d, and mutant of delta ago1, delta ago2 and delta ago1/2, respectively, beating 3 pieces of mycelium with 5mm perforator, adding into 200ml PDB culture medium, shaking at 28 deg.C and 150rpm for 3d, filtering with 3 layers of mirror paper, collecting conidium solution, centrifuging at 5000rpm for 10min, adjusting spore suspension concentration to 2 × 10 with sterile water⁶One per ml. The pathogenicity of the strain is determined by adopting a pot root-damaging drenching method. Foc4 and the mutant were treated with 15 Brazilian banana seedlings, each of which was watered with 20mL of spore suspension, and 3 replicates with sterile water as a control. And (3) carrying out cultivation management according to a conventional method, longitudinally cutting Brazilian banana corms after 30 days, observing the browning degree of the corms and the yellowing condition of external leaves, referring to disease condition investigation grading standards such as Mohamed and the like, recording the disease level of each banana seedling and carrying out statistical analysis on disease condition indexes.

The disease index calculation formula is as follows:

disease index ∑ (disease-grade number of plants × representative value)/(total number of plants × representative value of the most serious disease-grade) × 100%

Pathogenicity assay results: negative control (water treatment), under the condition of not inoculating blight bacteria, the banana seedlings grow strongly and no blight disease occurs. A positive control is inoculated to the wild strain Foc4, the banana corm has obvious yellow brown symptom, and the disease index is 65.83; after inoculation of the delta ago1 mutant strain, the disease index 54.58 of the banana is higher than that of the wild strain Foc4, which indicates that the pathogenicity of the delta ago1 mutant strain is enhanced; the disease indexes of inoculated delta ago2 and delta ago1/2 mutants are reduced to 52.59 and 52.50 respectively, which are lower than that of wild strain Foc4 (figure 6).

TABLE 2 grading criteria for external symptoms and corm symptoms of banana vascular wilt

EXAMPLE seventhly, Small RNA sequencing of Fusarium oxysporum deletion mutants

1. Deletion mutant small RNA sequencing

Mycelia of Foc4 and delta ago1, delta ago2 and delta ago1/2 mutants cultured at 7d were taken for small RNA sequencing. After the extracted RNA is qualified, 1.5 mu g of RNA Sample is used as the initial amount of the RNA Sample, the volume is supplemented to 6 mu l by water, and the small RNA Sample Pre Kit is used for library construction. As the 5 'end of Small RNA has a phosphate group and the 3' end has a hydroxyl group, T4RNA Ligase 1 and T4RNA Ligase 2(truncated) are respectively connected with a linker at the 5 'end and the 5' end of Small RNA3, cDNA is synthesized by reverse transcription, PCR amplification is carried out, a target fragment is screened by adopting a gel separation technology, and the fragment obtained by gel cutting and recovery is a Small RNA library. HiSeq2500 was used for high throughput sequencing, with single-end (SE)50nt reads. Comparison analysis of small RNAs was performed with the Foc4 genome sequence ftp/ftp. NCBI. nlm. nih. gov/genes/all/GCA-000149955.2-ASM 14995v 2/GCA-000149955.2-ASM 14995v 2-genomic. fna. gz published by NCBI and Fol genome sequence ftp/ftp. ensimble. org/pub/release-20/fungi/fasta/fusarium _ oxysporum/dna/as reference sequences.

2. Small RNA sequencing data analysis method

Sequencing Using the designated Fusarium _ oxysporum as reference genomeThe results after alignment are as follows, the mature milRNA length is mainly concentrated in the range of 20nt to 24 nt. Typical miRNA base ratios were obtained by base bias analysis of mirnas. And carrying out statistics on the expression quantity of miRNA in each sample, and carrying out normalization processing on the expression quantity by using a TPM algorithm. The TPM normalization processing formula is as follows: TPM Readcount × 10⁶[ MappedReads ] (Table 3).

TABLE 3 miRNA prediction results summary table after alignment of small RNA sequencing data with reference genome

3. Results of small RNA sequencing data analysis

The Δ ago1 mutant produced more specific small RNAs as shown in table 4, the Δ ago2 mutant produced more specific small RNAs as shown in table 5, and the Δ ago1/2 mutant produced more specific small RNAs as shown in table 6, relative to wild-type Foc 4.

TABLE 4

TABLE 5

TABLE 6

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.

Sequence listing

<110> institute for environmental and plant protection of tropical agricultural academy of sciences in China

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gcgataaggg cagttgacaa 20

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aacagaaagc cacctctaac tcc 23

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gggtgaagtg atacaggagt ggt 23

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<210> 42

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<212> DNA/RNA

<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<212> DNA/RNA

<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<212> DNA/RNA

<213> banana vascular wilt bacteria (Fusarium oxysporum)

<400> 55

tgaattacat tgatcgtgac at 22

<210> 56

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<212> DNA/RNA

<213> banana vascular wilt bacteria (Fusarium oxysporum)

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<213> banana vascular wilt bacteria (Fusarium oxysporum)

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tgaagaagac gacgacgagg agcat 25

Claims

1. The banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant is characterized in that the banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutant is any one of the following 3 banana fusarium oxysporum No. 4 physiological race AGO gene deletion mutants: delta ago1 mutant, delta ago2 mutant, and delta ago1/2 mutant;

the delta ago1 mutant, the delta ago2 mutant and the delta ago1/2 mutant are obtained by the following methods:

1) constructing Foc4AGO1 gene deletion recombinant DNA fragment, replacing AGO1 gene with hygromycin resistance gene HPH, transforming protoplast to obtain Foc4AGO1 gene deletion mutant, namely delta AGO1 mutant; knocking out AGO2 gene by the same method to construct delta AGO2 mutant;

2) a Foc4AGO 1/2 double-gene deletion recombinant DNA fragment is constructed on the basis of a delta AGO1 mutant, an AGO2 gene is replaced by a neomycin NEO resistance gene, protoplast transformation is carried out, and a Foc4AGO 1/2 double-gene deletion mutant, namely a delta AGO1/2 mutant, is obtained.

2. The AGO gene deletion mutant of the number 4 physiological race of fusarium oxysporum f.sp.cubense of the banana according to claim 1, wherein the construction method of the delta AGO1 mutant in the step 1) comprises the following steps:

first round PCR amplification:

second round of PCR amplification:

left end LB + HP: the LB and hygromycin resistance gene HPH at the left end are used as templates, Foc4AGO1-LB-F and HYG-R1 are used as primers, and the size of an amplification product is 2415 bp;

right end PH + RB: right end RB and hygromycin resistance gene HPH are used as templates, HYG-F1 and FOC4AGO1-RB-R are used as primers, and the size of an amplification product is 2274 bp;

3. The AGO gene deletion mutant of the number 4 physiological race of fusarium oxysporum f.sp.cubense of the banana according to claim 1, wherein the construction method of the delta AGO2 mutant in the step 1) comprises the following steps:

first round PCR amplification:

second round of PCR amplification:

wherein the sequences of Foc4AGO2-LBCK, Foc4AGO2-HPH-LB-R, Foc4AGO2-HPH-RB-F, Foc4AGO2-RBCK, Foc4AGO2-LB-F, HYG-R1, HYG-F1 and Foc4AGO2-RB-R are shown as SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 15, SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 17 in sequence.

4. The physiological race AGO gene deletion mutant of banana vascular wilt bacteria No. 4, according to claim 1, characterized in that step 2):

first round PCR amplification:

right end RB: FOC4 genome DNA is used as a template, Foc4AGO2-NEO-RB-F and Foc4AGO2-NEO-RBCK are used as primers, and the size of an amplification product is 1913 bp;

second round of PCR amplification:

5. The AGO gene deletion mutant of the number 4 physiological race of fusarium oxysporum f.sp.cubense according to claim 1, characterized in that the delta AGO2 mutant is more sensitive and growth is inhibited after being treated by a fluorescent whitening agent CFW; MgCl₂After treatment, the growth of the Δ ago1 mutant, the Δ ago2 mutant, and the Δ ago1/2 mutant were inhibited.

6. The physiological race AGO gene deletion mutant of banana vascular wilt bacteria No. 4, as set forth in claim 1, characterized in that the Δ AGO1 mutant, Δ AGO2 mutant and Δ AGO1/2 mutant are attenuated in virulence.