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Traceless gene editing method for trichoderma fungi

A gene and Trichoderma technology, which is applied in the field of Trichoderma without traces in the field of gene cycle editing, can solve the problems of complex internal mechanism and inability to explain the comprehensive results of interaction of multi-gene products.

Active Publication Date: 2018-08-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the internal mechanism of different functions of Trichoderma is very complex, which is the result of the participation and joint regulation of multiple genes.
The above-mentioned genetic manipulation based on hygromycin B can only be carried out on a single gene in the Trichoderma genome. In many cases, the study of the traits and functions of a single gene is far from being able to explain the comprehensive results of the interaction of multiple gene products (promoting growth, antagonism, straw utilization, etc.)

Method used

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  • Traceless gene editing method for trichoderma fungi
  • Traceless gene editing method for trichoderma fungi
  • Traceless gene editing method for trichoderma fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Implementation 1: Construction of auxotrophic strain NJAU4742-Δura3 traceless mutant of Trichoderma Guizhou NJAU4742 (1) Construction of knockout fragment

[0045] In the process of seamless knockout of ura3, the presence or absence of the gene itself is used as a screening marker for secondary homologous recombination, so the construction of the knockout fragment is slightly different. The knockout fragment consists of 4 different parts: upstream fragment + downstream fragment + HygB resistance gene expression box + gene fragment. Among them, the HygB resistance gene expression cassette can be transcribed and translated into hygromycin B phosphotransferase, making the original strain NJAU4742 resistant to hygromycin B. After the second homologous recombination, the HygB resistance gene expression cassette and ura3 Gene sequences will be deleted together, so 5-FOA can be used for reverse screening to obtain a traceless knockout mutant of the ura3 gene. In summary, in t...

Embodiment 2

[0053] Implementation 2: The traceless knockout of the xyr1 gene, which regulates lignocellulosic degrading enzymes in Trichoderma Guizhou NJAU4742

[0054] Trichoderma strain: Guizhou Trichoderma NJAU4742-Δura3 (NJAU4742-Δura3)

[0055] (1) Construction of knockout fragments

[0056] Knockout fragment amplification primers are: upstream fragment amplification primer x1_upF: GGCCTTGAAACGGTATGTCGA (SEQ ID NO.11) and x1_upR: CGTACACCACCATCACAGGGATATCAATAGGAGATGGCTGAACTGTGTG (SEQ ID NO.12); downstream fragment amplification primer x1_downF: CACACAGTTCAGCCATCTCCTATTGATATCCCCTGTGATGTQGTACG 和x1_downR:GCCATATTGATGTAAGGTAGCTCTCCTCACTTCCGCTTCACATAGACC(SEQ ID NO.14);U3表达框扩增引物U3box_F:GAGAGCTACCTTACATCAATATGGCGCGCAGATGTAGCGGTACATG(SEQ ID NO.15)和U3box_R:GGTACTATGGCTTAGATGGAATACCCCGTATTCTCTGCCCTTGTTGC(SEQ ID NO.16);基因片段扩增引物x1_geneF:GGGTATTCCATCTAAGCCATAGTACCCTTCTTCAGCCCTTGATCCACAC(SEQ ID NO. 17) and x1_geneR: CCTTGATTCACACGCAAATGTTCC (SEQ ID NO. 18). The final concentration of the knockout...

Embodiment 3

[0064] Implementation 3: Trichoderma Guizhou NJAU4742 carbon source inhibition regulatory gene cre1 traceless knockout and ura3 gene complement Trichoderma strain: Guizhou Trichoderma NJAU4742-Δura3-Δxyr1 (NJAU4742-Δura3-Δxyr1)

[0065] (1) Construction of homologous knockout fragments

[0066] The knockout fragment is: cre1 gene upstream fragment + U3 expression box + cre1 gene downstream fragment. The amplification primers are: upstream fragment amplification primer c1_upF: GGTGGGCAAAAAGGAACCTGG (SEQ ID NO.21) and c1_upR: GCCATATTGATGTAAGGTAGCTCTCAGAGATTATCCGCTGGTGGAGTG (SEQ ID NO.22); U3 expression box amplification primer U3box_F: GAGAGCTACCTTACATCAATATGGCGCGCAGATNOGTAGCGGTACATG (SEQ.2) U3box_R: GGTACTATGGCTTAGATGGAATACCCCGTATTCTCTGCCCTTGTTGC (SEQ ID NO. 24); downstream fragment amplification primer c1_downF: GGGTATTCCATCTAAGCCATAGTACCGCGCCTCGAATGACTTGATGAC (SEQ ID NO. 25) and c1_downR: GCCCTACGAGAATGTCGGTTC (SEQ ID NO. 26). The final concentration of the knockout fragmen...

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Abstract

The invention relates to a traceless gene editing method for trichoderma fungi. According to the method, the traceless knockout mutant of the trichoderma guizhouense ura3 gene is obtained through twotimes of homologous recombination events and in combination with the resistance screening strategy of hygromycin B and the lethal strategy of 5-FOA; based on the mutant, the knockout fragment containing the ura3 gene expression cassette is inserted at the position of the target gene through the homologous recombination method, and the mutant having the first-time homologous recombination is screened by utilizing the nutrition defect feature of the ura3 traceless mutant; after the second-time homologous recombination, the ura3 gene and the target gene are removed through recombination, at the time, inverse screening is carried out by utilizing the lethal feature of 5-FOA, and thus the traceless mutant with the target gene completely deleted is obtained. The system is optimized sufficiently,the homologous recombination ratio is 15% or above, the single-gene traceless operating period is shortened to be within 15 days, meanwhile, the traceless overexpression of the target gene can be realized, and no exogenous fragments are introduced during the process.

Description

technical field [0001] The invention belongs to the technical field of applied microorganisms, and relates to a gene cycle editing method of Trichoderma untraceable. Background technique [0002] Microbial organic fertilizer has the functions of promoting plant growth, preventing and controlling soil-borne diseases, and can replace or partially replace chemical fertilizers. Its wide application in agriculture is in line with the national policy of "weight loss and drug reduction". The most important components of microbial organic fertilizers are active strains, mainly including Bacillus in bacterial classification and Trichoderma filamentous fungi in fungal classification. Long-term studies have shown that Trichoderma filamentous fungi have the functions of promoting plant growth (cucumber, corn, banana, watermelon, etc.), inducing plant exogenous immunity, enhancing plant system resistance, and preventing and controlling soil-borne diseases. It has various functions and i...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/65C12N15/90C12N1/15C12R1/885
CPCC07K14/37C12N9/88C12N15/65C12N15/80C12N15/902C12Y401/01023
Inventor 沈其荣缪有志黄启为梅慧玲夏燕维
Owner NANJING AGRICULTURAL UNIVERSITY
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