CN116286937A - Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier - Google Patents

Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier Download PDF

Info

Publication number
CN116286937A
CN116286937A CN202211166150.0A CN202211166150A CN116286937A CN 116286937 A CN116286937 A CN 116286937A CN 202211166150 A CN202211166150 A CN 202211166150A CN 116286937 A CN116286937 A CN 116286937A
Authority
CN
China
Prior art keywords
sequence
trichoderma harzianum
taking
dsrna
intron
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211166150.0A
Other languages
Chinese (zh)
Inventor
郭惠珊
赵建华
温汉光
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of CAS
Original Assignee
Institute of Microbiology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Microbiology of CAS filed Critical Institute of Microbiology of CAS
Priority to CN202211166150.0A priority Critical patent/CN116286937A/en
Publication of CN116286937A publication Critical patent/CN116286937A/en
Priority to PCT/CN2023/115863 priority patent/WO2024060950A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01109—Dolichyl-phosphate-mannose-protein mannosyltransferase (2.4.1.109)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Pest Control & Pesticides (AREA)
  • Environmental Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention belongs to the technical field of disease prevention and control genes. The invention provides a method for preparing biocontrol engineering bacteria by taking Trichoderma harzianum as a dsRNA carrier, which comprises the following steps: determining a target gene from a pathogenic fungus; taking a target gene as an arm sequence, taking an endogenous gene VdTublin intron sequence of the verticillium dahliae as a center, and respectively connecting two identical arm sequences to two ends of the intron in a forward direction and a reverse direction to construct a target sequence; and transferring the target sequence into Trichoderma harzianum by utilizing ATMT to obtain Trichoderma harzianum engineering bacteria. The engineering strain obtained by the invention has better effect of inhibiting the growth of corresponding fungi and pathogenicity thereof.

Description

Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier
Technical Field
The invention belongs to the technical field of crop disease prevention and control genes.
Background
RNA interference (RNAi) is a highly conserved gene expression regulatory mechanism in eukaryotic cells that is triggered by the production of small RNAs (srnas) from double stranded RNAs (dsRNA). RNAi can regulate gene expression at the transcriptional level (transcriptional gene silencing, TGS) and the posttranscriptional level (post-transcriptional gene silencing, PTGS), and has the characteristics of high efficiency and specific targeting.
dsRNA of target pathogenic bacteria growth and development or pathogenicity related genes is artificially designed and transferred into target crops or sprayed in vitro to silence target genes, so that the aim of disease prevention and control is fulfilled. For example, the host-induced gene silencing (host-induced gene silence, HIGS) technique proposed in 2010, namely, by expressing dsRNA targeting pathogenic bacteria in host plants, thereby inhibiting the pathogenicity of pathogenic bacteria to plants. However, many crops lack mature transformation techniques and in vitro sprayed dsRNA is not environmentally stable and is not able to continue to provide dsRNA.
Disclosure of Invention
In view of the above, the invention provides a preparation method of biocontrol engineering bacteria, which can avoid crop transgenosis and get rid of various limitations of dsRNA in-vitro spraying. The method utilizes widely used biocontrol fungus Trichoderma harzianum to generate dsRNA targeting endogenous genes of pathogenic fungi to achieve the aim of inhibiting the growth of pathogenic fungi, and comprises the following steps: determining a target gene according to pathogenic fungi; taking a target gene as an arm sequence, taking an endogenous gene VdTublin intron sequence of the verticillium dahliae as a center, and respectively connecting two identical arm sequences to two ends of the intron in a forward direction and a reverse direction to construct a dsRNA sequence; and transferring the dsRNA sequence into Trichoderma harzianum by utilizing ATMT to obtain Trichoderma harzianum engineering bacteria.
In a specific embodiment of the invention, the specific sequence information of the arm sequence is shown as SEQ ID NO.1 in the sequence table, as SEQ ID NO.2 in the sequence table or as SEQ ID NO.4 in the sequence table.
In a specific embodiment of the invention, the sequence of the VdTublin intron is shown as SEQ ID NO.3 in a sequence table.
In the specific embodiment of the invention, the Trichoderma harzianum engineering bacteria are Trichoderma harzianum engineering bacteria Th-dspmt1-1, trichoderma harzianum engineering bacteria Th-dspmt1-2 or Trichoderma harzianum engineering bacteria Th-dspmt2.
In a specific embodiment of the invention, the pathogenic bacteria are verticillium dahliae (Verticillium dahliae) and fusarium oxysporum (Fusarium oxysporum).
The engineering strain obtained by the invention has the functions of inhibiting the gene expression of pathogenic fungi and further affecting the pathogenicity, prevention and control of diseases.
Drawings
FIG. 1 is a schematic diagram of dsRNA construction.
FIG. 2 is a diagram showing colony morphology of Th-dsGFP and Southern blot results.
FIG. 3 is a graph showing fluorescence intensity of V592-GFP and Th-dsGFP co-cultured GFP.
FIG. 4 is a graph showing the results of V592-GFP and Th-dsGFP co-culture mRNA Northern blot (left) and Western blot (right).
FIG. 5 is a plot of Th-dspmt colony morphology and Southern blot results.
FIG. 6 is a graph showing changes at the transcription level and translation level.
FIG. 7 is an experimental plot of Th-dspmt and V592 inhibition zone.
FIG. 8 is a graph of the biological statistics of Th-dspmt and V592 co-inoculated cotton stems.
FIG. 9 is a graph of the results of Th-dspmt and V592 co-inoculation of cotton.
FIG. 10 is an experimental plot of colony morphology and zone of inhibition of Fusarium oxysporum of different subspecies.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The general experimental procedure referred to in the examples below is not described in detail; the specific procedure is described in detail later.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Verticillium dahliae (Verticillium dahliae) V592 (Feng-Gao, bang-JunZhou, A.Glutaminic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenic.PLoS ONE 5 (12): e 15319.) in the examples described below is publicly available from the national academy of sciences of microbiology.
Example 1
Construction of target dsRNA sequences
The method comprises the steps of respectively selecting 150-300bp of target gene transcription regions of verticillium dahliae (Verticillium dahliae) and fusarium oxysporum (Fusarium oxysporum) as arms, taking 100-200bp of introns of endogenous genes of verticillium dahliae as the center, respectively connecting two identical arm sequences to two ends of the introns in forward and reverse directions, and constructing the target dsRNA sequences.
The specific structure is shown in figure 1, the intron sequence of the endogenous gene VdTublin of the verticillium dahliae is unchanged, and the connection direction of the sequences of the target arm genes with the same two ends is changed.
1. Dspmt of target verticillium dahliae Vdpmt2
A gene Vdpmt2 (the gene ID of which is VDAG_03930) of long terpene-phosphomannoprotein mannosyltransferase (a sequence of which is shown as SEQ ID NO.5 in a sequence table) in verticillium dahliae is selected as a target gene for a subsequent experiment, and the sequences of the two 210bp coding regions are arm sequences which are named as dspmt-1 and dspmt-2 respectively.
The 148bp intron sequence in the verticillium dahliae endogenous gene VdTublin (VDAG_10074) is taken as the center.
Two identical arm sequences were forward and reverse ligated to both ends of the intron, respectively, to construct two dsRNAs, dspmt1-1 and dspmt1-2.
The dspmt-1 arm sequence (210 bp) is shown as SEQ ID NO.1 in the sequence Listing.
The dspmt-2 arm sequence (210 bp) is shown as SEQ ID NO.2 in the sequence Listing.
The VdTublin intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence table.
2. Dspmt2 of target fusarium oxysporum Fopmt2
180bp of fusarium oxysporum Fopmt2 (FOXG_ 11176) gene transcription region is selected as an arm sequence, named as dspmt-3, and two sections of identical arm sequences are respectively connected to two ends of an intron in forward and reverse directions by taking a 148bp intron sequence in a verticillium dahliae endogenous gene VdTublin (VDAG_10074) as a center, so as to construct a dsRNA sequence dspmt2.
The dspmt-3 arm sequence (180 bp) is shown as SEQ ID NO.4 in the sequence Listing.
The VdTublin intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence table.
TABLE 1 arm sequence and intron sequence information Table
Figure BDA0003855964690000041
Figure BDA0003855964690000051
Figure BDA0003855964690000061
Example 2
Engineering strain and preparation method thereof
Th-dsGFP inhibits V592-GFP fluorescence intensity
GFP is expressed in verticillium dahliae V592, dsRNA targeting GFP is expressed in biocontrol strain Trichoderma harzianum Th, after co-culture of the two dsRNA and dsRNA, the fluorescence intensity of V592-GFP is obviously reduced after observation under a microscope; molecular hybridization experiments show that GFP expression level is not obviously changed at the transcription level, and the accumulation level at the translation level is obviously reduced.
The specific experimental steps are as follows:
(1) dsGFP design:
the 500bp of GFP gene transcription region of verticillium dahliae V592 is selected as an arm sequence, the 148bp intron of the verticillium dahliae endogenous gene VdTublin (VDAG_10074) gene is used as a loop, and the two arm sequences are respectively connected to two ends of the intron in forward and reverse directions to construct dsGFP.
(2) And transferring dsGFP into the Trichoderma harzianum Th by using ATMT to obtain the Trichoderma harzianum engineering strain Th-dsGFP.
Transformants were then screened using PDA+G418 resistant plates and target gene copy numbers were detected by Southern blot. As shown in FIG. 2, southern blot detection showed that dsGFP was successfully transferred into the chassis strain, and only a single band was detected by HindIII single cleavage, confirming that the engineered strain dsRNA was a single copy insertion.
(3) After the Th and Th-dsGFP strains were co-cultured with GFP-expressing V592 (V592-GFP) in a search medium, the bacterial liquid was aspirated and the GFP fluorescence intensity in V592-GFP was observed under a laser scanning confocal microscope (Confocal laser scanning microscope, CLSM). As shown in FIG. 3, after 1.5 days and 3 days of co-culture with Th-dsGFP, the fluorescence intensity of GFP in V592-GFP was significantly reduced, and the effect of reducing the fluorescence intensity was more significant with the co-culture time.
(4) After the Th and Th-dsGFP strains were co-cultured with V592-GFP in PDB for 3 days, the changes in GFP transcripts and proteins were detected using Northern blot and Western blot, as shown in FIG. 4, which showed no significant difference in GFP transcript level expression levels (left panel), rRNA was loading control. Western blot detection shows that compared with Th, after V592-GFP is co-cultured with Th-dsGFP, the accumulation amount of GFP protein is obviously reduced. The above results demonstrate that Th-dsGFP inhibits translation of GFP protein in V592-GFP.
Th-dspmt inhibits V592 pathogenicity to cotton
1) After dsRNA (Th-dspmt) of the target verticillium dahliae V592 endogenous gene Vdpmt2 is expressed in biocontrol strain Trichoderma harzianum Th and co-cultured with the two, a molecular hybridization experiment shows that the expression quantity of the Vdpmt2 is not obviously changed at the transcription level, and the accumulation quantity of the Vdpmt2 is obviously reduced at the translation level; the experiment of the inhibition zone shows that Th-dspmt can effectively inhibit the growth of V592 to form an obvious inhibition zone; cotton inoculation experiments show that Th-dspmt can effectively reduce the colonization amount of V592 in cotton stems and reduce the pathogenicity of V592 to cotton.
The specific experimental steps are as follows:
(1) Searching a literature and combining with laboratory early-stage research, and selecting a long terpene phosphomannoprotein mannosyltransferase (dolichyl-phosphate-mannase-protein mannosyltransferase) gene Vdpmt2 (with a gene ID of VDAG_03930) in verticillium dahliae as a target gene of a subsequent experiment.
A gene Vdpmt2 (VDAG_03930) of mannoprotein mannosyltransferase (dolichyl-phosphate-mannase-protein mannosyltransferase) of the long terpene-based phospho-mannase in verticillium dahliae is selected as a target gene for subsequent experiments, and the two 210bp gene transcription regions are arm sequences, named as dspmt-1 and dspmt-2 respectively.
The 148bp intron sequence in the verticillium dahliae endogenous gene VdTublin (VDAG_10074) is taken as the center.
Two identical arm sequences were forward and reverse ligated to both ends of the intron sequence, respectively, to construct two target dsRNA sequences, dspmt1-1 and dspmt1-2.
The dspmt-1 arm sequence (210 bp) is shown as SEQ ID NO.1 in the sequence Listing.
The dspmt-2 arm sequence (210 bp) is shown as SEQ ID NO.2 in the sequence Listing.
The VdTublin intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence table.
(2) The two dsRNA sequences dspmt1-1 and dspmt1-2 are transferred into Trichoderma harzianum Th by using ATMT to obtain two Trichoderma harzianum engineering strains Th-dspmt1-1 and Th-dspmt1-2.
Transformant selection was performed using PDA+G418 resistant plates and target gene copy number was detected by Southern blot, as shown in FIG. 5, which indicated that dspmt was successfully transferred into the chassis strain and was inserted for multiple copies. .
(3) After the two strains Th and Th-dspmt were co-cultured with Vdpmt2 knockout make-up strain (VdΔpm2/Olic3:3 flag-pmt2) in PDB for 3 days, vdΔpm2/Olic3:3:3 Vdpmt2 was detected for changes in transcription and translation levels using Northern blot and Western blot. As shown in FIG. 6, northern blot detection shows that the expression level of the Vdpmt2 gene does not change obviously, and Western blot detection shows that compared with Th, vdΔpmt2/Olic is shown in the specification, vdΔpmt2-pmt2 and Th-dspmt are co-cultured and arranged in a rear row, and the accumulation amount of Vdpmt2 protein at the translation level is obviously reduced.
(4) And (3) bacteriostasis circle experiment:
v592 was dissolved in PDA and poured into a plate, after it solidified, th-dspmt1-1 and Th-dspmt1-2 bacterial cakes were gently placed on the plate, after 48 hours, the production of the zone of inhibition around the bacterial cake was observed, as shown in FIG. 7, th-dspmt could inhibit the growth of V592 around it, and an obvious zone of inhibition was produced.
(5) Th, th-dspmt1-1, th-dspmt1-2 and V592 were inoculated into cotton soil in different combinations after shaking in a search medium, cotton stems were taken at 14d, qPCR was used to detect differences in the amount of colonization of the internal V592 and observed for cotton disease at the later stage of the experiment, and the disease grade was counted. The five inoculation combinations include: mock, th, V592, V592+Th and V592+Th-dspmt. As shown in FIGS. 8, 9 and 10, th-dspmt reduced the colonization of V592 in the cotton stalk, thereby alleviating V592 pathogenicity in cotton.
2) After expressing dsRNA (Th-dspmt 2) targeting fusarium oxysporum endogenous gene Fopmt2 (FOXG_ 11176) in biocontrol strain Trichoderma harzianum Th and co-culturing the two, the experiment of the inhibition zone shows that Th-dspmt2 can effectively inhibit the growth of fusarium oxysporum of different subspecies to form an obvious inhibition zone.
The specific experimental steps are as follows:
(1) The literature was searched and combined with laboratory preliminary studies, the long terpene phosphomannoprotein mannosyltransferase (dolichyl-phosphate-mannose-protein mannosyltransferase) gene Fopmt2 (FOXG_ 11176) in Fusarium oxysporum (Fusarium oxysporum f.sp.lycopersici 4287) was selected as the target gene for subsequent experiments.
Fusarium oxysporum Fopmt2 (FOXG_ 11176) is selected as a target gene for subsequent experiments, and a 180bp gene transcription region on the gene is an arm sequence and is named as dspmt-3.
The 148bp intron sequence in the verticillium dahliae endogenous gene VdTublin (VDAG_10074) is taken as the center.
Two identical arm sequences were forward and reverse ligated to both ends of the intron, respectively, to construct the dsRNA sequence dspmt2.
The dspmt-3 arm sequence (180 bp) is shown as SEQ ID NO.4 in the sequence Listing.
The VdTublin intron sequence (148 bp) is shown as SEQ ID NO.3 in the sequence table.
(2) And transferring the dsRNA sequence dspmt2 into the Trichoderma harzianum Th by utilizing ATMT to obtain the Trichoderma harzianum engineering strain Th-dspmt2.
Transformants were then screened using pda+g418 resistance plates.
(4) And (3) bacteriostasis circle experiment:
different subspecies of fusarium oxysporum are dissolved in a PDA and poured into a flat plate, after the fusarium oxysporum is solidified, th and Th-dspmt2 bacterial cakes are gently placed on the flat plate, after 48 hours, the generation condition of a bacteriostasis zone around the bacterial cakes is observed, and as shown in figure 10, the Th-dspmt can inhibit the growth of the surrounding fusarium oxysporum, so that an obvious bacteriostasis zone is formed.
In the above examples, the information on different subspecies of fusarium oxysporum is shown in the table below:
Figure BDA0003855964690000091
Figure BDA0003855964690000101
the engineering bacteria of the invention avoid crop transgenosis, and get rid of the restriction of insufficient maturation of crop genetic transformation technology and long transformation period; overcomes the defect that dsRNA sprayed in vitro is unstable in the environment and can not be continuously supplied.

Claims (5)

1. The method for preparing the biocontrol engineering bacteria by taking trichoderma harzianum as a dsRNA carrier is characterized by comprising the following steps of:
taking biocontrol bacteria Trichoderma harzianum as chassis bacteria;
determining a target gene from a pathogenic fungus;
taking a target gene as an arm sequence, taking an endogenous gene VdTublin intron sequence of the verticillium dahliae as a center, and respectively connecting two identical arm sequences to two ends of the intron in a forward direction and a reverse direction to construct a dsRNA sequence;
and transferring the dsRNA sequence into Trichoderma harzianum by utilizing ATMT to obtain Trichoderma harzianum engineering bacteria.
2. The method according to claim 1, wherein,
the sequence of the arm sequence is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4 in the sequence table.
3. The method according to claim 1, wherein the vdTublin intron sequence is shown in SEQ ID NO.3 of the sequence Listing.
4. The method according to claim 1, wherein the Trichoderma harzianum engineering strain is Trichoderma harzianum engineering strain Th-dspmt1-1, trichoderma harzianum engineering strain Th-dspmt1-2 or Trichoderma harzianum engineering strain Th-dspmt2.
5. The method for preparing trichoderma harzianum engineering bacteria containing dsRNA sequence according to claim 1, wherein the pathogenic bacteria are verticillium dahliae (Verticillium dahliae) and fusarium oxysporum (Fusarium oxysporum).
CN202211166150.0A 2022-09-21 2022-09-21 Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier Pending CN116286937A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202211166150.0A CN116286937A (en) 2022-09-21 2022-09-21 Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier
PCT/CN2023/115863 WO2024060950A1 (en) 2022-09-21 2023-08-30 Method for preparing biocontrol engineering microorganism using trichoderma harzianum as dsrna vector

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211166150.0A CN116286937A (en) 2022-09-21 2022-09-21 Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier

Publications (1)

Publication Number Publication Date
CN116286937A true CN116286937A (en) 2023-06-23

Family

ID=86836436

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211166150.0A Pending CN116286937A (en) 2022-09-21 2022-09-21 Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier

Country Status (2)

Country Link
CN (1) CN116286937A (en)
WO (1) WO2024060950A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024060950A1 (en) * 2022-09-21 2024-03-28 中国科学院微生物研究所 Method for preparing biocontrol engineering microorganism using trichoderma harzianum as dsrna vector

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104988139B (en) * 2015-05-19 2018-02-06 中国科学院微生物研究所 The method for cultivating the cotton of resisting verticillium
CN104928314B (en) * 2015-06-02 2017-09-22 石河子大学 Verticillium dahliae pathogen-relatedprotein VdpdaA1 purposes
CN106244614B (en) * 2016-08-20 2018-01-12 南京农业大学 The structure of Trichoderma harzianum engineered strain with strong parasitic broad-spectrum fungi and its application
CN113549635B (en) * 2021-09-18 2021-12-21 中国农业科学院生物技术研究所 Application of verticillium dahliae VdPRMT1 gene in improving disease resistance of crops or vegetables
CN116286937A (en) * 2022-09-21 2023-06-23 中国科学院微生物研究所 Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024060950A1 (en) * 2022-09-21 2024-03-28 中国科学院微生物研究所 Method for preparing biocontrol engineering microorganism using trichoderma harzianum as dsrna vector

Also Published As

Publication number Publication date
WO2024060950A1 (en) 2024-03-28

Similar Documents

Publication Publication Date Title
Hou et al. A Phytophthora effector suppresses trans-kingdom RNAi to promote disease susceptibility
Citovsky et al. Biological systems of the host cell involved in Agrobacterium infection
Park et al. Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii
Zhang et al. Two Phytophthora parasitica cysteine protease genes, PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors
WO2006070227A2 (en) Method for down-regulating gene expression in fungi
CN116286937A (en) Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier
CN110036114A (en) The itaconic acid of raising produces
Wu et al. Host-induced gene silencing of multiple pathogenic factors of Sclerotinia sclerotiorum confers resistance to Sclerotinia rot in Brassica napus
CN108623665A (en) Application of the GhHUB2 albumen in regulation and control cotton fiber length and intensity
CN111057654B (en) Cas9 gene knockout vector applicable to morinda officinalis endophytic fungus A761 and construction method and application thereof
CN103146743B (en) Method for improving currant tomato endogenous gene silencing efficiency by viruses through induction
CN109666690B (en) Method for over-expressing non-trace trichoderma fungus gene
Shen et al. Between-plant signaling
Hu et al. DNA methyltransferase implicated in the recovery of conidiation, through successive plant passages, in phenotypically degenerated Metarhizium
CN110016519B (en) Banana fusarium wilt bacterium No. 4 physiological race DCL gene deletion mutant and small RNA thereof
CN110982715B (en) High-spore-yield purple-spore-bacterium-gene engineering bacterium delta PlflbD and construction method and application thereof
CN109666592B (en) Fungal laccase expression strain and construction method and application thereof
Ouyang et al. Artificial trans-kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease
CN114107327B (en) Trichoderma viride high-temperature stress response key enzyme gene TvHSP70, recombinant expression vector, engineering bacteria and application thereof
CN116531488A (en) Application of transport protein TSPO and agonist and inhibitor thereof in related medicines of microsporidian bombycis
CN115976051A (en) Potato StRTP7 gene and application thereof in disease-resistant breeding
Jiang et al. Pectate lyase genes abundantly expressed during the infection regulate morphological development of Colletotrichum camelliae and CcPEL16 is required for full virulence to tea plants
CN116286813A (en) Trichoderma harzianum engineering strain Th-dspmt2 and application thereof
CN110468151B (en) Fusarium oxysporum TOR gene RNAi vector and method for preventing and treating potato dry rot and blight by combining fusarium oxysporum TOR gene RNAi vector with salicylic acid
Woo et al. Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination