Method for preparing biocontrol engineering bacteria by using trichoderma harzianum as dsrna carrier

Abstract

This invention belongs to the field of gene technology for disease control. This invention provides a method for preparing biocontrol engineered bacteria using *Trichoderma harzianum* as a dsRNA vector, comprising the following steps: determining the target gene based on the pathogenic fungus; using the target gene as an arm sequence, and centering on the VdTublin intron sequence of the *Verticillium dahliae* endogenous gene, connecting two identical arm sequences to both ends of the intron in a forward and reverse direction, respectively, to construct the target sequence; and transferring the target sequence into *Trichoderma harzianum* using ATMT to obtain engineered *Trichoderma harzianum* bacteria. The engineered strain obtained by this invention has a good inhibitory effect on the growth and pathogenicity of the corresponding fungus.

Description

Technical Field

[0001] This invention belongs to the field of crop disease control gene technology. Background Technology

[0002] RNA interference (RNAi) is a highly conserved gene expression regulation mechanism in eukaryotic cells, triggered by the production of small RNAs (sRNAs) from double-stranded RNA (dsRNA). RNAi can regulate gene expression at both the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels, exhibiting high efficiency and specific targeting.

[0003] dsRNAs that artificially target genes related to the growth, development, or pathogenicity of pathogens are designed and transferred into target crops or silenced through in vitro spraying to achieve disease control. For example, host-induced gene silence (HIGS) technology, proposed in 2010, inhibits the pathogen's pathogenicity by expressing dsRNAs targeting pathogens in host plants. However, many crops lack mature transformation technologies, and in vitro sprayed dsRNAs are unstable in the environment and cannot provide a continuous supply of dsRNAs. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing biocontrol engineered bacteria that avoids crop transgenics and overcomes the many limitations of in vitro spraying of dsRNA. This method utilizes the widely used biocontrol fungus *Trichoderma harzianum* to produce dsRNA targeting the endogenous gene of the pathogenic fungus, thereby inhibiting the growth of the pathogenic fungus. The method includes the following steps: determining the target gene based on the pathogenic fungus; using the target gene as an arm sequence, and using the endogenous gene of *Verticillium dahliae*... VdTublin Using an intron sequence as the center, two identical arm sequences were linked to the two ends of the intron in the forward and reverse directions, respectively, to construct a dsRNA sequence; the dsRNA sequence was then transferred into Trichoderma harzianum using ATMT to obtain the engineered Trichoderma harzianum.

[0005] In a specific embodiment of the present invention, the specific sequence information of the arm sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.4 in the sequence list.

[0006] In a specific embodiment of the present invention, the VdTublin The intron sequence is shown as SEQ ID NO.3 in the sequence listing.

[0007] In a specific embodiment of the present invention, the engineered Trichoderma harzianum strain is Trichoderma harzianum strain Th-dspmt1-1, Trichoderma harzianum strain Th-dspmt1-2, or Trichoderma harzianum strain Th-dspmt2.

[0008] In a specific embodiment of the present invention, the pathogenic bacterium is *Verticillium dahliae* (…). Verticillium dahliae ) and Fusarium oxysporum ( Fusarium oxysporum ).

[0009] The engineered strains obtained by this invention have the effect of inhibiting the gene expression of pathogenic fungi, thereby affecting their pathogenicity and controlling diseases. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of dsRNA construction.

[0011] Figure 2 These are images showing the colony morphology of Th-dsGFP and the results of Southern blot analysis.

[0012] Figure 3 This is a graph showing the fluorescence intensity of GFP co-cultured with V592-GFP and Th-dsGFP.

[0013] Figure 4 The images show the results of Northern blot (left) and Western blot (right) of co-culturing V592-GFP and Th-dsGFP mRNA.

[0014] Figure 5 These are images showing the colony morphology of Th-dspmt and the results of Southern blot analysis.

[0015] Figure 6 It is a graph showing changes at the transcriptional and translational levels.

[0016] Figure 7 These are experimental diagrams of the inhibition zones of Th-dspmt and V592.

[0017] Figure 8 This is a statistical chart of the biomass within the stem of cotton plants co-inoculated with Th-dspmt and V592.

[0018] Figure 9 This is a graph showing the disease incidence results of cotton plants co-inoculated with Th-dspmt and V592.

[0019] Figure 10 These are experimental diagrams showing the colony morphology and inhibition zones of different subspecies of Fusarium oxysporum. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0021] The routine experimental procedures involved in the following examples are not described in detail; the specific steps of the unconventional experimental procedures are described in detail below.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] Verticillium dahliae in the following examples ( Verticillium dahliae V592 (Feng-Gao, Bang-JunZhou, A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity. PLoS ONE 5(12): e15319.) is available to the public from the Institute of Microbiology, Chinese Academy of Sciences.

[0024] Example 1

[0025] Constructing target dsRNA sequences

[0026] Selected Verticillium dahliae ( Verticillium dahliae ) and Fusarium oxysporum ( Fusarium oxysporum Using the 150-300 bp transcribed region of the target gene as an arm, and centering on the 100-200 bp intron of the endogenous gene of Verticillium dahliae, two identical arm sequences were connected to the two ends of the intron in the forward and reverse directions, respectively, to construct the target dsRNA sequence.

[0027] The specific structure is as follows: Figure 1 As shown, the endogenous genes of *Verticillium intermedium*. VdTublin The intron sequence remains unchanged, but the connection direction of the gene sequences in the identical target arms at both ends is changed.

[0028] 1. Target: Verticillium dahliae Vdpmt2 dspmt

[0029] The gene of dolichyl-phosphate-mannose-protein mannosyltransferase (PMT, sequence shown in SEQ ID NO.5 of the sequence listing) from *Verticillium dahliae* was selected. Vdpmt2(Gene ID: VDAG_03930) is used as the target gene for subsequent experiments. The two 210 bp coding sequences above it are the arm sequences, named dspmt-1 and dspmt-2, respectively.

[0030] Endogenous genes of Verticillium dahliae VdTublin Centered on the 148 bp intron sequence of (VDAG_10074).

[0031] Two identical arm sequences were linked to both ends of an intron, one forward and one backward, to construct two dsRNAs, dspmt1-1 and dspmt1-2.

[0032] The dspmt-1 arm sequence (210 bp) is shown in SEQ ID NO.1 in the sequence listing.

[0033] The dspmt-2 arm sequence (210 bp) is shown in SEQ ID NO.2 in the sequence listing.

[0034] VdTublin The intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence listing.

[0035] 2. Target Fusarium oxysporum Fopmt2 dspmt2

[0036] Select Fusarium oxysporum Fopmt2 The 180 bp arm of the (FOXG_11176) gene transcription region is named dspmt-3, based on the endogenous gene of Verticillium dahliae. VdTublin Using the 148 bp intron sequence in (VDAG_10074) as the center, two identical arm sequences were connected to the two ends of the intron in the forward and reverse directions, respectively, to construct the dsRNA sequence dspmt2.

[0037] The dspmt-3 arm sequence (180 bp) is shown in SEQ ID NO.4 in the sequence listing.

[0038] VdTublin The intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence listing.

[0039] Table 1. Information on arm sequences and intron sequences.

[0040]

[0041]

[0042]

[0043] Example 2

[0044] Engineered strains and preparation methods

[0045] 1. Th-dsGFP inhibits V592-GFP fluorescence intensity

[0046] GFP was expressed in *Verticillium dahliae* V592, and dsRNA targeting GFP was expressed in the biocontrol bacterium *Trichoderma harzianum* Th. After co-culturing both, microscopic observation revealed a significant decrease in the fluorescence intensity of V592-GFP. Molecular hybridization experiments showed that... GFP There was no significant change in expression levels at the transcriptional level, but a significant decrease in accumulation at the translational level.

[0047] The specific experimental steps are as follows:

[0048] (1) dsGFP design.

[0049] Selected Verticillium dahliae V592 GFP The 500 bp arm sequence of the gene transcription region is used as the endogenous gene of Verticillium dahliae. VdTublin The (VDAG_10074) gene has a 148 bp intron as a loop. Two arm sequences are connected to the two ends of the intron in the forward and reverse directions, respectively, to construct dsGFP.

[0050] (2) dsGFP was introduced into Trichoderma harzianum Th using ATMT to obtain the Trichoderma harzianum engineered strain Th-dsGFP.

[0051] Then, transformants were screened using PDA+G418 resistance plates, and the target gene copy number was detected by Southern blot. For example... Figure 2 As shown, Southern blot analysis indicated that dsGFP was successfully transformed into the chassis strain, and Hind III single enzyme digestion detected only a single band, confirming that the engineered strain's dsRNA was a single copy insertion.

[0052] (3) After co-culturing Th and Th-dsGFP strains with GFP-expressing V592 (V592-GFP) in Czapek medium, the bacterial culture was aspirated and the GFP fluorescence intensity in V592-GFP was observed under a confocal laser scanning microscope (CLSM). Figure 3 As shown, after co-culturing with Th-dsGFP for 1.5 days and 3 days, the fluorescence intensity of GFP in V592-GFP was significantly reduced, and the reduction in fluorescence intensity was more obvious with the extension of co-culturing time.

[0053] (4) After co-culturing Th and Th-dsGFP strains with V592-GFP in PDB for 3 days, the changes in GFP transcripts and proteins were detected by Northern blot and Western blot, such as... Figure 4 As shown in the left figure, Northern blot analysis revealed no significant difference in GFP transcriptional expression levels, with rRNA serving as the loading control. Western blot analysis indicated that, compared to Th, co-culturing V592-GFP with Th-dsGFP significantly reduced GFP protein accumulation. These results suggest that Th-dsGFP inhibits the translation of GFP protein in V592-GFP.

[0054] 2. Th-dspmt inhibits the pathogenicity of V592 in cotton.

[0055] 1) Expression of the endogenous gene targeting Verticillium dahliae V592 in the biocontrol bacterium Trichoderma harzianum Th Vdpmt2 The dsRNA (Th-dspmt) and the two were co-cultured, and molecular hybridization experiments showed that... Vdpmt2 At the transcriptional level, there was no significant change in expression, but at the translational level, the accumulation level decreased significantly. The inhibition zone experiment showed that Th-dspmt could effectively inhibit the growth of V592 and form a clear inhibition zone. The cotton inoculation experiment showed that Th-dspmt could effectively reduce the colonization of V592 in cotton stems and reduce the pathogenicity of V592 to cotton.

[0056] The specific experimental steps are as follows:

[0057] (1) Based on literature review and previous laboratory research, the gene of dolichyl-phosphate-mannose-protein mannosyltransferase from Verticillium dahliae was selected. Vdpmt2 (Gene ID: VDAG_03930) will be used as the target gene for subsequent experiments.

[0058] The gene for dolichyl-phosphate-mannose-protein mannosyltransferase from Verticillium dahliae was selected. Vdpmt2 (VDAG_03930) is the target gene for subsequent experiments. The two 210 bp gene transcription regions above it are arm sequences, named dspmt-1 and dspmt-2, respectively.

[0059] Endogenous genes of Verticillium dahliae VdTublin Centered on the 148 bp intron sequence of (VDAG_10074).

[0060] Two identical arm sequences were connected to the two ends of an intron sequence, one forward and one backward, respectively, to construct two target dsRNA sequences, dspmt1-1 and dspmt1-2.

[0061] The dspmt-1 arm sequence (210 bp) is shown in SEQ ID NO.1 in the sequence listing.

[0062] The dspmt-2 arm sequence (210 bp) is shown in SEQ ID NO.2 in the sequence listing.

[0063] VdTublin The intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence listing.

[0064] (2) Two dsRNA sequences, dspmt1-1 and dspmt1-2, were transformed into Trichoderma harzianum Th using ATMT to obtain two Trichoderma harzianum engineered strains, Th-dspmt1-1 and Th-dspmt1-2.

[0065] Transformants were screened using PDA+G418 resistance plates, and the copy number of the target gene was detected by Southern blot. Figure 5 As shown, Southern blot analysis indicates that dspmt was successfully transferred into the chassis strain, and that it was a multiple copy insertion.

[0066] (3) The two strains, Th and Th-dspmt, were respectively with Vdpmt2 Knockout and replacement strain (Vd) △pmt2 / Olic::3flag-pmt2) was co-cultured with PDB for 3 days, and Vd was detected by Northern blot and Western blot. △pmt2 / Olic::3flag-pmt2 Vdpmt2 Changes at the transcriptional and translational levels. For example... Figure 6 As shown, Northern blot analysis indicates Vdpmt2 No significant changes were observed in gene expression levels at the transcriptional level. Western blot analysis showed that, compared to Th, Vd... △pmt2 After co-culturing / Olic::3flag-pmt2 with Th-dspmt, the accumulation of Vdpmt2 protein at the translational level decreased significantly.

[0067] (4) Antibacterial zone test:

[0068] Dissolve V592 in PDA and pour into agar plates. After solidification, gently place Th, Th-dspmt1-1, and Th-dspmt1-2 mycelial cakes on the plates. Observe the formation of inhibition zones around the mycelial cakes after 48 hours. Figure 7 As shown, Th-dspmt can inhibit the growth of V592 around it, producing a distinct inhibition zone.

[0069] (5) After shaking culture of Th, Th-dspmt1-1, Th-dspmt1-2, and V592 in Chowsky medium, different combinations were inoculated into cotton soil. Cotton stems were harvested after 14 days, and the differences in V592 colonization within the stems were detected by qPCR. Cotton disease incidence was observed in the later stages of the experiment, and the disease severity was statistically analyzed. The five inoculation combinations included: Mock, Th, V592, V592+Th, and V592+Th-dspmt. Figure 8 and 9 As shown, Th-dspmt can reduce the colonization of V592 on cotton stems, thereby mitigating the pathogenicity of V592 to cotton.

[0070] 2) Expression of endogenous genes targeting Fusarium oxysporum in the biocontrol bacterium Trichoderma Th Fopmt2 When dsRNA (Th-dspmt2) of (FOXG_11176) was co-cultured with it, the inhibition zone experiment showed that Th-dspmt2 could effectively inhibit the growth of different subspecies of Fusarium oxysporum and form obvious inhibition zones.

[0071] The specific experimental steps are as follows:

[0072] (1) Based on literature review and previous laboratory research, the gene for dolichyl-phosphate-mannose-protein mannosyltransferase from Fusarium oxysporum f.sp. lycopersici 4287 was selected. Fopmt2 (FOXG_11176) was used as the target gene for subsequent experiments.

[0073] Select Fusarium oxysporum Fopmt2 (FOXG_11176) was used as the target gene for subsequent experiments. The 180 bp transcribed region of the gene was the arm sequence and named dspmt-3.

[0074] Endogenous genes of Verticillium dahliae VdTublin Centered on the 148 bp intron sequence of (VDAG_10074).

[0075] Two identical arm sequences were linked to both ends of an intron, one forward and one backward, to construct the dsRNA sequence dspmt2.

[0076] The dspmt-3 arm sequence (180 bp) is shown in SEQ ID NO.4 in the sequence listing.

[0077] VdTublin The intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence listing.

[0078] (2) The dsRNA sequence dspmt2 was transferred into Trichoderma harzianum Th using ATMT to obtain the Trichoderma harzianum engineered strain Th-dspmt2.

[0079] Transformants were then screened using PDA+G418 resistance plates.

[0080] (4) Antibacterial zone test:

[0081] Different subspecies of Fusarium oxysporum were dissolved in PDA and poured into plates. After solidification, Th and Th-dspmt2 mycelial cakes were gently placed on the plates. After 48 hours, the formation of inhibition zones around the mycelial cakes was observed. Figure 10 As shown, Th-dspmt can inhibit the growth of surrounding Fusarium oxysporum, forming a distinct inhibition zone.

[0082] The information on different subspecies of Fusarium oxysporum in the above embodiments is shown in the table below:

[0083]

[0084] The engineered bacteria of this invention avoid crop transgenics, overcome the limitations of immature crop genetic transformation technology and long transformation cycles; and overcome the disadvantages of in vitro spraying of dsRNA being unstable in the environment and unable to be continuously supplied.

Claims

1. A method for preparing biocontrol engineered bacteria using *Trichoderma harzianum* as a dsRNA vector, characterized in that, Includes the following steps: The biocontrol fungus *Trichoderma harzianum* was used as the substrate fungus; Target genes are determined based on pathogenic fungi; Using the target gene as the arm sequence, and the endogenous gene of Verticillium dahliae... VdTublin Using an intron sequence as the center, two identical arm sequences are connected to both ends of the intron, one forward and one backward, respectively, to construct a dsRNA sequence; the arm sequences are as shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.4 of the sequence listing; VdTublin The intron sequence is shown as SEQ ID NO.3 in the sequence listing; The dsRNA sequence was transferred into Trichoderma harzianum using ATMT to obtain engineered Trichoderma harzianum.

2. The method for preparing biocontrol engineered bacteria using *Trichoderma harzianum* as a dsRNA vector according to claim 1, characterized in that, The pathogenic fungus is *Verticillium dahliae* (… Verticillium dahliae ) or Fusarium oxysporum ( Fusarium oxysporum ).

Images