A dcl gene deletion mutant of Fusarium wilt of banana and its small RNA in race 4

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

Active Publication Date: 2020-04-14
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • 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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  • A dcl gene deletion mutant of Fusarium wilt of banana and its small RNA in race 4
  • A dcl gene deletion mutant of Fusarium wilt of banana and its small RNA in race 4
  • A dcl gene deletion mutant of Fusarium wilt of banana and its small RNA in race 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Fusarium wilt Foc4 DCLs gene knockout

[0056] Fusarium wilt contains two DCL protein genes, DCL1 and DCL2, and the single gene knockout of Foc4 DCL adopts the principle of homologous replacement, replacing the DNA of the target gene Foc4 DCL1 or Foc4 DCL2 with the DNA fragment of the resistance gene hygromycin B (HPH) gene fragment, 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 those in the schematic diagram.

Embodiment 2

[0057] Example 2 Construction of Foc4 DCL1 Gene Deletion Recombinant DNA Fragment

[0058] 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 DCL1 is Gene: FOIG_04851, referring to the genome sequence of NCBI GenBank: JH658277.1, primers are designed to amplify the homology arm gene sequence of the upper and lower reaches of the DCL1 gene ( figure 2 -A,B). details as follows:

[0059] The first round of PCR amplification:

[00...

Embodiment 3

[0069] Example 3 Construction of Foc4 DCL2 Gene Deletion Recombinant DNA Fragment

[0070] The gene knockout method of Foc4 DCL2 is the same as that of DCL1, and the primers and descriptions used are shown in Table 2. The gene sequence number of Foc4 DCL2 Gene: FOIG_04495, referring to GenBank: JH658276.1 genome sequence, designed primers to amplify the upper and lower homologous genome sequences of DCL2 gene ( figure 2 -C,D). The detailed operation method is as follows:

[0071] The first round of PCR amplification:

[0072] Left LB: DCL2-LBCK and DCL2-HPH-LB-R are primers, FOC4 genomic DNA is a template, and the size of the amplified product is 1957bp;

[0073] RB at the right end: DCL2-HPH-RB-F and DCL2-RBCK are primers, FOC4 genomic DNA is a template, and the size of the amplified product is 1837bp;

[0074] HPH fragment: HYG-F and HYG-R are primers, vector plasmid DNA is a template, and the size of the amplified product is 1376bp;

[0075] The second round of PCR am...

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Abstract

The invention discloses a fusarium oxysporum No. 4 physiological race DCL gene deletion mutant and small RNA thereof. A homologous displacement principle is adopted, a fusarium oxysporum delta dcl1 gene deletion mutant, a fusarium oxysporum delta dcl2 gene deletion mutant and a fusarium oxysporum delta dcl1 / 2 gene deletion mutant are constructed, abiotic stress test and pathogenicity analysis areperformed, the result shows that after being treated with a fluorescent whitening agent CFW, the delta dcl1 mutant, the delta dcl2 mutant and the delta dcl1 / 2 mutant are more sensitive, and the growthis restrained; and after being treated with MgCl2, the delta dcl1 mutant, the delta dcl2 mutant and the delta dcl1 / 2 mutant are restrained in growth. When the pathogenicity of the deltadcl1 mutant isstrengthened, the pathogenicity of the deltadcl2 mutant and the pathogenicity of the deltadcl1 / 2 mutant are weakened. In addition, small RNA of each mutant is obtained through combining with small RNA depth sequencing, and a theoretic and technical support is provided for further analyzing the infective mechanism of the fusarium oxysporum and developing the preventing and controlling measures ofRNA sources.

Description

technical field [0001] The invention relates to a DCL gene deletion mutant of fusarium wilt of banana No. 4 physiological race and small RNA thereof, belonging to the technical field of bioengineering. Background technique [0002] Small RNAs, including miRNAs and siRNAs, are a class of small molecule non-coding RNAs with a length of 21-24 nt, which lead to sequence-specific post-transcriptional gene silencing regulation by cleaving target mRNA or transcriptional repression, all of which are regulated by nucleic acids similar to RNase III Endonuclease Dicer (or Dicer-like protein) processing. As a specific endoribonuclease, Dicer (DCL) participates in the small RNA biosynthesis pathway, and the mechanism of action of Dicer is divided into two steps: the first step, Dicer combines with dsRNA to form an enzyme-dsRNA complex, under the action of ATP The dsRNA is uncoiled and cut to produce 21-24nt small fragment siRNAs with 5' terminal phosphate group, 3' terminal hydroxyl gro...

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 彭军曾凡云张欣漆艳香谢培兰谢艺贤
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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