CN-116286937-B - Method for preparing biocontrol engineering bacteria by taking trichoderma harzianum as dsRNA carrier

CN116286937BCN 116286937 BCN116286937 BCN 116286937BCN-116286937-B

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 of determining a target gene according to pathogenic fungi, taking the target gene as an arm sequence, taking an intronic sequence of an endogenous gene VdTublin of verticillium dahliae as a center, respectively connecting two identical arm sequences to two ends of the intronic in a forward and reverse direction to construct a target sequence, and transferring the target sequence into trichoderma harzianum by utilizing ATMT to obtain the trichoderma harzianum engineering bacteria. The engineering strain obtained by the invention has better effect of inhibiting the growth of corresponding fungi and pathogenicity thereof.

Inventors

GUO HUISHAN
ZHAO JIANHUA
WEN HANGUANG

Assignees

中国科学院微生物研究所

Dates

Publication Date: 20260505
Application Date: 20220921

Claims (2)

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; The target gene is an arm sequence, the endogenous gene VdTublin of the verticillium dahliae is taken as a center, two identical arm sequences are respectively connected to two ends of an intron in a forward direction and a reverse direction, and a dsRNA sequence is constructed, wherein the sequence of the arm sequence is shown as SEQ ID NO.1 in a sequence table, SEQ ID NO.2 in the sequence table or SEQ ID NO.4 in the sequence table, and the VdTublin intron sequence is shown as SEQ ID NO.3 in the sequence table; And transferring the dsRNA sequence into Trichoderma harzianum by utilizing ATMT to obtain Trichoderma harzianum engineering bacteria.
2. The method for preparing biocontrol engineering bacteria by using trichoderma harzianum as a dsRNA carrier according to claim 1, wherein the pathogenic fungus is verticillium dahliae (Verticillium dahliae) or fusarium oxysporum (Fusarium oxysporum).

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 (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 (double STRAIN RNA, 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, host-induced GENE SILENCE, HIGS technology 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 pathogenic fungi endogenous genes so as to achieve the aim of inhibiting the growth of pathogenic fungi, and comprises the following steps of determining target genes according to pathogenic fungi, taking the target genes as arm sequences, taking an intron sequence of the pathogenic fungi endogenous genes VdTublin as a center, connecting two identical arm sequences to two ends of the intron in a forward direction and a reverse direction respectively, constructing a dsRNA sequence, and transferring the dsRNA sequence into Trichoderma harzianum by utilizing ATMT so as 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 VdTublin intron sequence is shown as SEQ ID NO.3 of the sequence Listing. 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 the 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 diagram showing colony morphology of Th-dspmt 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 zones. FIG. 8 is a graph of biological statistics of Th-dspmt and V592 co-inoculated cotton stems. FIG. 9 is a graph showing 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 following examples are directed to conventional experimental procedures and non-conventional experimental procedures, and specific procedures are described in detail below. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The verticillium dahliae （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.) public in the examples described below is available from the national