Trichoderma reesei engineered strain and application thereof

Abstract

The application belongs to the technical field of crop disease prevention and control genes. The application provides a Trichoderma harzianum engineering strain, the Trichoderma harzianum engineering strain is a strain Th-dspmt1-1 or Th-dspmt1-2; the Trichoderma harzianum engineering strain Th-dspmt1-1 is obtained by introducing a dsRNA sequence dspmt1-1 targeting a pathogenic fungus growth and development related gene into Trichoderma harzianum; the target dsRNA sequence dspmt1-1 is obtained by connecting two pieces of dspmt-1 arm sequences to both ends of an intron sequence in a forward direction and a reverse direction respectively; the sequence of the dspmt-1 arm sequence is shown in SEQ ID NO. 1 in the sequence table; the Trichoderma harzianum engineering strain Th-dspmt1-2 is obtained by introducing a dsRNA sequence dspmt1-2 targeting a pathogenic fungus growth and development related gene into Trichoderma harzianum; the target dsRNA sequence dspmt1-2 is obtained by connecting two pieces of dspmt-2 arm sequences to both ends of an intron sequence in a forward direction and a reverse direction respectively; the sequence of the dspmt-2 arm sequence is shown in SEQ ID NO. 2 in the sequence table; the sequence of the intron sequence is shown in SEQ ID NO. 3 in the sequence table. The Trichoderma harzianum engineering strain provided by the application can effectively inhibit the gene expression of Lecanicillium attenuatum, and then resist cotton verticillium wilt.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop disease prevention and control genes. BACKGROUND

[0002] RNA interference (RNAi) is a highly conserved gene expression regulation mechanism in eukaryotic cells, which is triggered by small RNA (sRNA) produced by double strain RNA (dsRNA). RNAi can regulate gene expression at the transcriptional level (transcriptional gene silencing, TGS) and the post-transcriptional level (post-transcriptional gene silencing, PTGS), and has the characteristics of high efficiency and specific targeting.

[0003] Artificially designed dsRNA targeting genes related to growth and development or pathogenicity of pathogenic bacteria is introduced into target crops or sprayed in vitro to silence target genes, so as to achieve the purpose of disease prevention and control. For example, the host-induced gene silencing (HIGS) technology proposed in 2010, that is, by expressing dsRNA targeting pathogenic bacteria in host plants, the pathogenicity of pathogenic bacteria to plants is inhibited. However, some crops lack mature transformation systems and have long transformation cycles, which limits the popularization and application of HIGS technology. The biocontrol strains screened from nature have unstable biocontrol effect. SUMMARY

[0004] Therefore, the application provides a Trichoderma harzianum engineering strain, which is a strain Th-dspmt1-1 or Th-dspmt1-2. The Trichoderma harzianum engineering strain Th-dspmt1-1 is obtained by introducing a dsRNA sequence dspmt1-1 targeting a gene related to growth and development of pathogenic fungi into Trichoderma harzianum. The target dsRNA sequence dspmt1-1 is obtained by connecting two dspmt-1 arm sequences to both ends of an intron sequence in forward and reverse directions, respectively. The sequence of the dspmt-1 arm sequence is shown in SEQ ID NO. 1 in the sequence table. The Trichoderma harzianum engineering strain Th-dspmt1-2 is obtained by introducing a dsRNA sequence dspmt1-2 targeting a gene related to growth and development of pathogenic fungi into Trichoderma harzianum. The target dsRNA sequence dspmt1-2 is obtained by connecting two dspmt-2 arm sequences to both ends of an intron sequence in forward and reverse directions, respectively. The sequence of the dspmt-2 arm sequence is shown in SEQ ID NO. 2 in the sequence table. The sequence of the intron sequence is shown in SEQ ID NO. 3 in the sequence table.

[0005] The Trichoderma harzianum engineering strain provided by the application can effectively inhibit the expression of growth and development related genes of Plenomyces ilicinus, and further resist cotton verticillium wilt. BRIEF DESCRIPTION OF DRAWINGS

[0006] Figure 1 is a connection diagram of the target arm gene sequence.

[0007] Figure 2 is a Th-dsGFP colony morphology and Southern blot result diagram.

[0008] Figure 3 is a V592-GFP and Th-dsGFP co-culture GFP fluorescence intensity observation diagram.

[0009] Figure 4 is a V592-GFP and Th-dsGFP co-culture mRNA Northern blot (left) and Western blot (right) result diagram.

[0010] Figure 5 is a Th-dspmt colony morphology and Southern blot result diagram.

[0011] Figure 6 is a change diagram at the transcription level and the translation level.

[0012] Figure 7 is a Th-dspmt and V592 inhibition zone experiment diagram.

[0013] Figure 8 is a Th-dspmt and V592 co-inoculation cotton stem biomass statistics diagram.

[0014] Figure 9 is a Th-dspmt and V592 co-inoculation cotton disease result diagram. DETAILED DESCRIPTION

[0015] The application will be further described in detail below in combination with specific embodiments, and the embodiments given are only for illustrating the application, and are not intended to limit the scope of the application.

[0016] The conventional experimental operations involved in the following examples are not described in detail; the specific steps of the unconventional experimental operations are described below.

[0017] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0018] Verticillium dahliae V592 (Feng-Gao, Bang-Jun Zhou, A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity. PLoS ONE 5(12): e15319.) in the following examples is publicly available from the Institute of Microbiology, Chinese Academy of Sciences.

[0019] Example 1

[0020] Construction of target dsRNA sequence

[0021] The 150-300 bp of the transcription region of the target gene of Verticillium dahliae was selected as the arm, and the 100-200 bp of the endogenous gene intron of Verticillium dahliae was selected as the center. Two identical arm sequences were respectively connected to the two ends of the intron in forward and reverse directions to construct a dsRNA sequence.

[0022] The specific structure is shown in Figure 1 The middle intron sequence of the endogenous gene VdTublin of Verticillium dahliae is unchanged, and the connection direction of the two identical target arm gene sequences at both ends is changed.

[0023] 1. dspmt of target Verticillium dahliae Vdpmt2

[0024] The dolichyl-phosphate-mannose-protein mannosyltransferase (PMT, the sequence of which is shown in SEQ ID NO. 4 in the sequence listing) gene Vdpmt2 (gene ID VDAG_03930) in Verticillium dahliae was selected as the target gene for subsequent experiments. The 210 bp coding region sequences of the upper gene transcription region were used as arm sequences and were named dspmt-1 and dspmt-2, respectively.

[0025] The 148 bp intron sequence of the endogenous gene VdTublin (VDAG_10074) of Verticillium dahliae was used as the center.

[0026] Two identical arm sequences were respectively connected to the two ends of the intron in forward and reverse directions to construct two dsRNAs, which were named dspmt1-1 and dspmt1-2, respectively.

[0027] dspmt-1 arm sequence (210 bp) as shown in SEQ ID NO. 1 in the sequence listing.

[0028] dspmt-2 arm sequence (210 bp) as shown in SEQ ID NO. 2 in the sequence listing.

[0029] VdTublin intron sequence (148 bp) as shown in SEQ ID NO. 3 in the sequence listing.

[0030] Table 1. Arm sequence and intron sequence information table

[0031]

[0032]

[0033] Example 2 Engineering strain and preparation method

[0034] 1. Th-dsGFP inhibits V592-GFP fluorescence intensity

[0035] After co-culturing GFP expressed in L. major V592 and dsRNA targeting GFP expressed in biocontrol fungus T. harzianum Th, it was found that the fluorescence intensity of V592-GFP decreased significantly under a microscope. Molecular hybridization experiments showed that the expression level of GFP at the transcription level did not change significantly, and the accumulation level at the translation level decreased significantly.

[0036] The specific experimental steps are as follows:

[0037] (1) dsGFP design:

[0038] The 500 bp transcription region of the GFP gene of L. major V592 was selected as the arm sequence, and the 148 bp intron of the endogenous gene VdTublin (VDAG_10074) of L. major was used as the loop. The two arm sequences were connected to the two ends of the intron in forward and reverse directions, respectively, to construct dsGFP.

[0039] (2) dsGFP was transferred into T. harzianum Th using ATMT to obtain the engineering strain Th-dsGFP of T. harzianum.

[0040] Then, PDA+G418 resistance plates were used for transformant screening and the target gene copy number was detected by Southern blot. As shown in Figure 2 Southern blot detection showed that dsGFP was successfully transferred into the chassis strain, and Hind III single enzyme digestion only detected a single band, confirming that the engineering strain dsRNA was single copy insertion.

[0041] (3) After co-culturing Th and Th-dsGFP strains with GFP-expressing V592 (V592-GFP) in Czapek medium, the bacterial suspension was aspirated and the GFP silver light 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 pronounced with the extension of co-culturing time.

[0042] (4) After co-culturing Th and Th-dsGFP strains with V592-GFP in PDB for 3 days, the changes in GFP at the transcriptional and translational levels were detected using mRNA Northern blot and Western blot, respectively. Figure 4 As shown in the left figure, Northern blot analysis revealed no significant difference in GFP transcriptional expression levels, with rRNA serving as a loading control. Western blot analysis indicated that, compared to Th, co-culturing V592-GFP with Th-dsGFP significantly reduced the accumulation of GFP translational levels. These results suggest that Th-dsGFP inhibits the translation of GFP protein in V592-GFP.

[0043] 2. Th-dspmt inhibits the pathogenicity of V592 in cotton.

[0044] 1) dsRNA targeting the endogenous gene Vdpmt2 of Verticillium dahliae V592 (Th-dspmt) was expressed in the biocontrol bacterium Trichoderma harzianum (Th). After co-culturing the two, molecular hybridization experiments showed that the expression level of Vdpmt2 at the transcriptional level did not change significantly, but the accumulation at the translational 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.

[0045] The specific experimental steps are as follows:

[0046] (1) Based on literature review and previous laboratory research, the gene Vdpmt2 (gene ID VDAG_03930) of dolichyl-phosphate-mannose-protein mannosyltransferase in Verticillium dahliae was selected as the target gene for subsequent experiments.

[0047] The gene Vdpmt2 (VDAG_03930) of dolichyl-phosphate-mannose-protein mannosyltransferase from Verticillium dahliae was selected as the target gene for subsequent experiments. The two 210bp transcribed regions of the gene were selected as arm sequences and named dspmt-1 and dspmt-2, respectively.

[0048] The study focused on the 148bp intron sequence of the endogenous gene VdTublin (VDAG_10074) of Verticillium dahliae.

[0049] 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.

[0050] The dspmt-1 arm sequence (210 bp) is shown in SEQ ID NO.1 in the sequence listing.

[0051] The dspmt-2 arm sequence (210 bp) is shown in SEQ ID NO.2 in the sequence listing.

[0052] The VdTublin intron sequence (148 bp) is shown in SEQ ID NO.3 in the sequence listing.

[0053] (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.

[0054] Transformants were then screened using PDA+G418 resistance plates, and the target gene copy number was detected by Southern blot. Figure 5 As shown, Southern blot analysis indicated that dspmt was successfully transferred into the chassis strain, and that it was a multiple-copy insertion.

[0055] (3) After co-culturing Th and Th-dspmt strains with the Vdpmt2 knockout complement strain (Vd△pmt2 / Olic::3flag-pmt2) in PDB for 3 days, Northern blot and Western blot were used to detect changes in Vdpmt2 at the transcriptional and translational levels in Vd△pmt2 / Olic::3flag-pmt2. Figure 6As shown, Northern blot analysis indicated that the expression level of the Vdpmt2 gene did not change significantly at the transcriptional level. Western blot analysis showed that, compared with Th, after co-culturing VdΔpmt2 / Olic::3flag-pmt2 with Th-dspmt, the accumulation of Vdpmt2 protein at the translational level decreased significantly.

[0056] (4) Antibacterial zone test:

[0057] 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 significant inhibition zone.

[0058] (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 , 9 As shown, Th-dspmt can reduce the colonization of V592 on cotton stems, thereby mitigating the pathogenicity of V592 to cotton.

[0059] Example 3

[0060] cotton contamination experiment

[0061] 1. Spread activated Trichoderma harzianum and Verticillium dahliae on a PDA plate and incubate in the dark for 3 days.

[0062] 2. Scrape off pieces of Verticillium dahliae and place them in Czapek medium. Incubate at 26°C and 230 rpm for 3 days.

[0063] 3. Collect the bacterial culture by centrifugation at 8000 rpm in 50 mL centrifuge tubes, and resuspend the bacterial sample using ddH2O.

[0064] 4. Observe the bacterial concentration under a microscope using a hemocytometer and adjust it to 10. 7 CFU / cm 3 .

[0065] 5. Delint the wild-type cotton WC-CK seeds using 98% concentrated sulfuric acid, then rinse them with tap water and let them air dry. Before planting, soak them in 30% 84 disinfectant solution for 20 minutes, then rinse them three times with tap water and soak them in tap water overnight.

[0066] 6. With 10 6 The CFU / g concentration of the bacterial suspension was uniformly mixed into the soil. Six pots were sown for each treatment, with four cotton seeds sown in each pot. The pots were then incubated at 25°C under 16 hours of light followed by 8 hours of darkness. After 7 days, 0.5-0.6 g of MS nutrient solution was added to the soil. After 14 days, approximately 1-2 cm of the above-ground portion of the cotton stem was harvested to determine the V592 colonization level in the cotton stem.

[0067] 7. Disease severity statistics: After the cotton plants develop the disease, take photos and count the disease severity. The raw data is shown in the table below.

[0068] The pathogenicity levels are classified as follows:

[0069] Level 0 plants are healthy and show no symptoms;

[0070] 0.1%-25% of Grade 1 leaves are partially wilted;

[0071] 25%-50% of the leaves in grade 2 are partially wilted;

[0072] 50%-75% of the leaves in grade 3 are partially wilted;

[0073] 75%-100% of grade 4 leaves are partially wilted or die; the disease incidence rate for different grades = diseased leaves of different grades / total number of leaves.

[0074]

Claims

1. An engineered strain of Trichoderma harzianum, characterized in that, The engineered Trichoderma harzianum strains are strains Th-dspmt1-1 and Th-dspmt1-2; The *Trichoderma harzianum* engineered strain Th-dspmt1-1 was obtained by transforming the dsRNA sequence dspmt1-1, which targets genes related to the growth and development of pathogenic fungi, into *Trichoderma harzianum*. The dsRNA sequence dspmt1-1 was obtained by ligating two dspmt-1 arm sequences, one forward and one reverse, to both ends of an intron sequence, respectively. The dspmt-1 arm sequences are shown in SEQ ID NO.1 of the sequence listing. The *Trichoderma harzianum* engineered strain Th-dspmt1-2 was obtained by transforming the dsRNA sequence dspmt1-2, which targets genes related to the growth and development of pathogenic fungi, into *Trichoderma harzianum*. The dsRNA sequence dspmt1-2 was obtained by ligating two dspmt-2 arm sequences, one forward and one reverse, to both ends of an intron sequence. The dspmt-2 arm sequences are shown in SEQ ID NO.2 in the sequence listing. The intron sequence is shown as SEQ ID NO.3 in the sequence listing.

2. The use of the Trichoderma harzianum engineered strain according to claim 1 in the preparation of a fungal agent that inhibits the expression of mannosyltransferase of long-terpene phosphate mannosyltransferase by Verticillium dahliae, wherein the gene sequence of the long-terpene phosphate mannosyltransferase is shown in SEQ ID NO.4 in the sequence listing.

3. The application of the Trichoderma harzianum engineered strain according to claim 1 in the preparation of a cotton agent resistant to Verticillium dahliae.

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