Trichoderma harzianum engineered strain Th-dspmt2 and its application

Abstract

This invention belongs to the field of crop disease control gene technology. This invention provides an engineered Trichoderma harzianum strain Th-dspmt2, which is obtained by transferring a dsRNA targeting genes related to the growth and development of pathogenic fungi into Trichoderma harzianum. The target dsRNA sequence dspmt2 is obtained by linking two dspmt-3 arm sequences, one forward and one reverse, to both ends of an intron sequence. The sequence of the dspmt-3 arm is shown in SEQ ID NO.2 of the sequence listing; the sequence of the intron is shown in SEQ ID NO.1 of the sequence listing. The engineered Trichoderma harzianum strain Th-dspmt2 provided by this invention can effectively inhibit gene expression in Fusarium oxysporum, thereby inhibiting its growth.

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-stranded 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 fungi 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 fungi in host plants, the pathogenicity of pathogenic fungi 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 Th-dspmt2, which is obtained by introducing a dsRNA sequence dspmt2 targeting a growth and development related gene of a pathogenic fungus into Trichoderma harzianum; the target dsRNA sequence dspmt2 is obtained by connecting two dspmt-3 arm sequences in forward and reverse directions to both ends of an intron sequence; the sequence of the dspmt-3 arm sequence is shown in SEQ ID NO. 2 in the sequence table; and the sequence of the intron sequence is shown in SEQ ID NO. 1 in the sequence table.

[0005] The Trichoderma harzianum engineering strain Th-dspmt2 provided by the application can effectively inhibit the expression of Fusarium oxysporum genes and further inhibit the growth of Fusarium oxysporum. BRIEF DESCRIPTION OF DRAWINGS

[0006] Figure 1 is a schematic diagram of dsRNA construction.

[0007] Figure 2These are images showing the colony morphology of Th-dsGFP and the results of Southern blot analysis.

[0008] Figure 3 This is a graph showing the fluorescence intensity of GFP co-cultured with V592-GFP and Th-dsGFP.

[0009] Figure 4 These are the results of Northern blot (left) and Western blot (right) of co-cultured mRNA with V592-GFP and Th-dsGFP.

[0010] Figure 5 These are experimental diagrams showing the colony morphology and inhibition zones of different subspecies of Fusarium oxysporum. Detailed Implementation

[0011] 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.

[0012] 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.

[0013] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0014] Example 1

[0015] Constructing target dsRNA sequences

[0016] Using the 150-300bp transcribed region of the target gene from Fusarium oxysporum as an arm, and centering on the 100-200bp intron of the endogenous gene from Verticillium dahliae, two identical arm sequences were linked to the two ends of the intron, one forward and one reverse, respectively, to construct a dsRNA sequence.

[0017] The specific structure is as follows: Figure 1 As shown, the intron sequence of the VdTublin endogenous gene of Verticillium dahliae in the middle remains unchanged, but the connection direction of the gene sequences of the same target arms at both ends is changed.

[0018] 1. dspmt2 of Fusarium oxysporum Fopmt2 as a target

[0019] A 180bp arm sequence was selected from the transcriptional region of the Fusarium oxysporum Fopmt2 (FOXG_11176) gene and named dspmt-3. Using the 148bp intron sequence in the endogenous gene VdTublin (VDAG_10074) of Verticillium dahlia 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 named dspmt2.

[0020] The dspmt-3 arm sequence (180bp) is shown in SEQ ID NO.2 in the sequence listing.

[0021] The VdTublin intron sequence (148 bp) is shown in SEQ ID NO.1 of the sequence listing.

[0022] Table 1. Information on arm sequences and intron sequences

[0023]

[0024] Example 2

[0025] Engineered strains and preparation methods

[0026] 1. Th-dsGFP inhibits the fluorescence intensity of V592-GFP.

[0027] GFP was expressed in Verticillium dahliae V592 and dsRNA targeting GFP was expressed in the biocontrol bacterium Trichoderma harzianum Th. After co-culturing the two, microscopic observation revealed a significant decrease in the fluorescence intensity of V592-GFP. Molecular hybridization experiments showed that the expression level of GFP did not change significantly at the transcriptional level, but the accumulation level at the translational level decreased significantly.

[0028] The specific experimental steps are as follows:

[0029] (1) dsGFP design:

[0030] The 500bp transcribed region of the GFP gene from Verticillium dahliae V592 was selected as an arm sequence, and the 148bp intron of the endogenous gene VdTublin (VDAG_10074) from Verticillium dahliae was used as a loop. The two arm sequences were connected to the two ends of the intron in the forward and reverse directions, respectively, to construct dsGFP.

[0031] (2) dsGFP was introduced into Trichoderma harzianum Th using ATMT to obtain the Trichoderma harzianum engineered strain Th-dsGFP.

[0032] 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.

[0033] (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 even more pronounced with the extension of co-culturing time.

[0034] (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 indicated no significant difference in GFP transcriptional expression levels, with rRNA serving as a loading control. Western blot analysis showed that, compared to Th, co-culturing V592-GFP with Th-dsGFP significantly reduced the accumulation of GFP translational levels. These results indicate that Th-dsGFP inhibits the translation of GFP protein in V592-GFP.

[0035] 2. Th-dspmt antibacterial properties

[0036] In the biocontrol bacterium Trichoderma harzianum Th, dsRNA (Th-dspmt2) targeting the endogenous gene Fopmt2 (FOXG_11176) of Fusarium oxysporum was expressed. After co-culturing the two, the inhibition zone experiment showed that Th-dspmt2 could effectively inhibit the growth of different subspecies of Fusarium oxysporum and form obvious inhibition zones.

[0037] The specific experimental steps are as follows:

[0038] (1) Based on literature review and previous laboratory research, the gene Fopmt2 (FOXG_11176) of dolichyl-phosphate-mannose-protein mannosyltransferase from Fusarium oxysporum f.sp.lycopersici 4287 was selected as the target gene for subsequent experiments.

[0039] Fusarium oxysporum Fopmt2 (FOXG_11176) was selected as the target gene for subsequent experiments. The 180bp arm sequence of its gene transcription region was named dspmt-3.

[0040] The study focused on the 148bp intron sequence of the endogenous gene VdTublin (VDAG_10074) of Verticillium dahlia.

[0041] Two identical arm sequences were linked to both ends of an intron, one forward and one backward, to construct a dsRNA sequence, named dspmt2.

[0042] The dspmt-3 arm sequence (210 bp) is shown in SEQ ID NO.2 in the sequence listing.

[0043] The VdTublin intron sequence (148 bp) is shown in SEQ ID NO.1 of the sequence listing.

[0044] (2) dspmt2 was transferred into Trichoderma harzianum Th using ATMT to obtain the Trichoderma harzianum engineered strain Th-dspmt2.

[0045] Transformants were then screened using PDA+G418 resistance plates.

[0046] (3) Antibacterial zone test:

[0047] Different subspecies of Fusarium oxysporum were dissolved in PDA and poured onto 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 5 As shown, the Th-dspmt2 strain can inhibit the growth of Fusarium oxysporum and form a distinct inhibition zone.

[0048] The information on different subspecies of Fusarium oxysporum in the above embodiments is shown in the table below:

[0049]

[0050]

Claims

1. A Trichoderma harzianum engineering strain Th-dspmt2, characterized in that, the strain Th-dspmt2 is obtained by transforming a dsRNA sequence dspmt2 targeting a pathogenic fungus growth and development related gene into Trichoderma harzianum; the dsRNA sequence dspmt2 is obtained by connecting two dspmt-3 arm sequences respectively in forward and reverse direction to both ends of an intron sequence; the sequence of the dspmt-3 arm sequence is shown in SEQ ID NO. 2 in the sequence listing; the sequence of the intron sequence is shown in SEQ ID NO. 1 in the sequence listing.

2. Use of the Trichoderma harzianum engineering strain Th-dspmt2 according to claim 1 in the preparation of a microbial agent for inhibiting the expression of Fusarium oxysporum.

Images