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An ago gene deletion mutant of fusarium wilt of banana and its small rna

A deletion mutant and gene deletion technology, applied in DNA/RNA fragments, recombinant DNA technology, microorganisms, etc., can solve the problems of economic loss, harm and threat of banana industry and banana growers

Active Publication Date: 2020-01-03
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The external symptoms are not obvious in the early stage, and the symptoms appear in the middle and late stages. Therefore, it causes huge economic losses to the banana industry and banana growers
Banana Fusarium wilt has caused serious harm and threat to the global banana industry, has a wide range of effects and has attracted widespread attention, but there are no effective control measures at present

Method used

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  • An ago gene deletion mutant of fusarium wilt of banana and its small rna
  • An ago gene deletion mutant of fusarium wilt of banana and its small rna
  • An ago gene deletion mutant of fusarium wilt of banana and its small rna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Fusarium wilt Foc4 AGOs gene knockout

[0060] Fusarium wilt contains two AGO protein genes, AGO1 and AGO2, and the single gene knockout of Foc4 AGO adopts the principle of homologous replacement, replacing the DNA fragment of the target gene Foc4AGO1 or Foc4 AGO2 with the DNA fragment of the resistance gene hygromycin B (HPH) , figure 1 It is a schematic diagram of the Split-PCR gene knockout method, and the primers used in the subsequent gene knockout steps are consistent with the schematic marks.

Embodiment 2

[0061] Example 2 Construction of Foc4 AGO1 Gene Deletion Recombinant DNA Fragment

[0062]The split-PCR gene knockout method uses two rounds of PCR to amplify the recombinant DNA fragment, and the first round of PCR amplifies three fragments: the DNA sequence of the homology arm upstream of the target gene, the full-length sequence of the resistance gene, and the DNA of the homology arm downstream of the target gene sequence. In the second round of PCR, the DNA sequences of the upper and lower homology arms and the DNA of the resistance gene were mixed as PCR templates to amplify the upstream and downstream missing recombinant DNA fragments respectively. The gene sequence number of Foc4 AGO1 is Gene: FOIG_01986, referring to the genome sequence of NCBI GenBank: EXM08967, primers are designed to amplify the homology arm gene sequence of the upper and lower reaches of the AGO1 gene ( figure 2 -A,B). details as follows:

[0063] The first round of PCR amplification:

[0064]...

Embodiment 3

[0073] Example 3 Construction of Foc4 AGO2 Gene Deletion Recombinant DNA Fragment

[0074] The gene knockout method of Foc4 AGO2 is the same as that of AGO1, and the primers and descriptions used are shown in Table 2. The gene sequence number of Foc4 AGO2 Gene: FOIG_01246, referring to GenBank: EXM11679 genome sequence, designed primers to amplify the upper and lower homologous genome sequences of AGO2 gene ( figure 2 -C,D). The detailed operation method is as follows:

[0075] The first round of PCR amplification:

[0076] Left LB: FOC4AGO2-LBCK and FOC4AGO2-HPH-LB-R are primers, FOC4 genomic DNA is a template, and the size of the amplified product is 1840bp;

[0077] Right RB: FOC4AGO2-HPH-RB-F and FOC4AGO2-RBCK are primers, FOC4 genomic DNA is a template, and the size of the amplified product is 1900bp;

[0078] HPH fragment: HYG-F and HYG-R are primers, vector plasmid DNA is a template, and the size of the amplified product is 1376bp;

[0079] The second round of PCR...

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Abstract

The invention discloses an AGO gene deletion mutant of fusarium oxysporum No.4 physiological races, and microRNAs thereof. Based on the principle of homologous substitution, the deletion mutants of Delta ago1, Delta ago2 and Delta ago1 / 2 genes of fusarium oxysporum are constructed, and the abiotic stress test and pathogenicity analysis are carried out, the result shows that when treated with fluorescent brightener CFW, Delta ago2 deletion mutant is more sensitive and the growth is inhibited. When treated with MgCl2, the growth of Delta ago1 mutant, Delta ago2 mutant and Delta ago1 / 2 mutant isinhibited. The pathogenicity of the Delta ago1 mutant is enhanced, while that of the Delta ago2 mutant and the Delta ago1 / 2 mutant is weakened. In addition, mutant microRNAs are obtained by combiningthe microRNAs deep sequencing so as to provide theoretical and technical support for further analyzing the pathogenic mechanism of fusarium oxysporum, and developing prevention and control measures ofRNA sources.

Description

technical field [0001] The invention relates to an AGO gene deletion mutant of fusarium wilt of banana No. 4 physiological race and its small RNA, belonging to the technical field of bioengineering. Background technique [0002] Small molecule RNA is a kind of 21-24nt non-coding RNA widely present in eukaryotes, including miRNA (microRNA) and siRNA (small interfering RNA), which can bind to the ribozyme complex containing Argouaute (AGO) protein The RNA induced silencing complex RISC (RNA induced silencing complex) is formed, which leads to sequence-specific regulation of post-transcriptional gene silencing through cleaving target mRNA or translational inhibition. AGO protein is an important component in the RISC complex and plays an important role in small RNA-mediated gene silencing. The full length of AGO protein is 700-1200 amino acid residues, including four structural domains, which are N-terminal, PAZ, MID and PIWI domains. After Dice cleaves dsRNA to produce 21-24n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/113C12R1/645
CPCC12Q1/6895C12Q2600/156
Inventor 张欣曾凡云彭军漆艳香谢培兰谢艺贤
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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