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Genetically engineered bacterium delta PlflbC of purpureocillium lavendulum with high sporulation quantity, and construction method and application of genetically engineered bacterium delta PlflbC

A technology of genetically engineered bacteria and construction methods, applied in the field of high spore-yielding P. violaceum genetically engineered bacteria ΔPlflbC and its construction and application, can solve problems such as the decline in Aspergillus sporulation and the stagnant research on spore-producing genetically engineered bacteria , to achieve the effect of increasing the number of spores produced and reducing the cost of use

Active Publication Date: 2019-11-08
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the existing research, the flbC gene is used as Aspergillus spore production regulation gene. By knocking out the flbC gene of Aspergillus, the spore production of Aspergillus is reduced, and the research on spore production genetically engineered bacteria has stagnated.

Method used

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  • Genetically engineered bacterium delta PlflbC of purpureocillium lavendulum with high sporulation quantity, and construction method and application of genetically engineered bacterium delta PlflbC
  • Genetically engineered bacterium delta PlflbC of purpureocillium lavendulum with high sporulation quantity, and construction method and application of genetically engineered bacterium delta PlflbC
  • Genetically engineered bacterium delta PlflbC of purpureocillium lavendulum with high sporulation quantity, and construction method and application of genetically engineered bacterium delta PlflbC

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: The construction method of the genetically engineered bacteria ΔPlflbC of the high spore-yielding Porphyridium violaceum, the specific steps are as follows:

[0047] (1) Construct the PlflbC gene knockout vector by using the OSCAR method: use the P. violaceum gene DNA as a template, clone the homologous recombination fragment at the 5' end of the PflbC gene with the primer flbC5F / flbC5R, and clone the homologous fragment at the 3' end with the primer flbC3F / flbC3R Recombinant fragment; the primer flbC5F / flbC5R is used to amplify the 1049bp fragment upstream of the flbC gene; the sequence of the primer flbC5F is ggggacagctttcttgtacaaagtggaaACCCGTGCCCAGTGAGA, the upstream fragment of the flbC gene, and the lowercase sequence is homologous to the att site of the plasmid; the sequence of the primer flbC5R is ggggactgcttttttgtacaaacttbflTCGTACGAT, the GTAATCCGTAC gene The upstream fragment, the lowercase sequence is homologous to the att site of the plasmid; the...

Embodiment 2

[0066] Embodiment 2: the construction method of the genetically engineered bacterium ΔPlflbC of P. violaceum of high sporulation yield, concrete steps are as follows:

[0067] (1) Construct the PlflbC gene knockout vector by using the OSCAR method: use the P. violaceum gene DNA as a template, clone the homologous recombination fragment at the 5' end of the PflbC gene with the primer flbC5F / flbC5R, and clone the homologous fragment at the 3' end with the primer flbC3F / flbC3R Recombinant fragment; the primer flbC5F / flbC5R is used to amplify the 1049bp fragment upstream of the flbC gene; the sequence of the primer flbC5F is ggggacagctttcttgtacaaagtggaaACCCGTGCCCAGTGAGA, the upstream fragment of the flbC gene, and the lowercase sequence is homologous to the att site of the plasmid; the sequence of the primer flbC5R is ggggactgcttttttgtacaaacttbflTCGTACGAT, the GTAATCCGTAC gene The upstream fragment, the lowercase sequence is homologous to the att site of the plasmid; the primer flb...

Embodiment 3

[0085] Embodiment 3: the construction method of the genetically engineered bacterium ΔPlflbC of P. violaceum of high sporulation yield, concrete steps are as follows:

[0086](1) Construct the PlflbC gene knockout vector by using the OSCAR method: use the P. violaceum gene DNA as a template, clone the homologous recombination fragment at the 5' end of the PflbC gene with the primer flbC5F / flbC5R, and clone the homologous fragment at the 3' end with the primer flbC3F / flbC3R Recombinant fragment; the primer flbC5F / flbC5R is used to amplify the 1049bp fragment upstream of the flbC gene; the sequence of the primer flbC5F is ggggacagctttcttgtacaaagtggaaACCCGTGCCCAGTGAGA, the upstream fragment of the flbC gene, and the lowercase sequence is homologous to the att site of the plasmid; the sequence of the primer flbC5R is ggggactgcttttttgtacaaacttbflTCGTACGAT, the GTAATCCGTAC gene The upstream fragment, the lowercase sequence is homologous to the att site of the plasmid; the primer flbC...

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Abstract

The invention discloses a genetically engineered bacterium delta PlflbC of purpureocillium lavendulum with the high sporulation quantity, and a construction method and application of the genetically engineered bacterium delta PlflbC, and belongs to the technical field of biological pesticides. The genetically engineered bacterium delta PlflbC is a PlflbC gene for knocking out the purpureocillium lavendulum; and the preservation number of the genetically engineered bacterium is CCTCC M 2019348, the sequence of the PlflbC gene is shown as SEQ ID NO.1, and the amino acid sequence of the PlflbC isshown as SEQ ID NO.2. A PlflbC gene knocking-out carrier is constructed through an OSCAR method; donor plasmid pA-sur(chlorimuron-ethyl resistant gene)-OSCAR, a receptor pPK2-OSCAR-GFP, a PlflbC gene5'end homologous recombination fragment and a PlflbC gene 3'end homologous recombination fragment are subjected to Gateway BP reaction to obtain the PlflbC gene knocking-out carrier; the PlflbC geneknocking-out carrier is transferred into agrobacterium to obtain the agrobacterium containing the PlflbC gene knocking-out carrier; the agrobacterium containing the PlflbC gene knocking-out carrier isconverted into the purpureocillium lavendulum; flbC transformants are screened, verification primers are sequentially adopted to verify primer flbC verification 5 / flbC verification 3, random primersare sequentially adopted to verify randomly inserted verification 5 / randomly inserted verification 3, and thus the genetically engineered bacterium delta PlflbC of the purpureocillium lavendulum withthe high sporulation quantity is obtained.

Description

technical field [0001] The invention relates to a genetic engineering bacterium ΔPlflbC of P. violaceum with high spore yield and its construction method and application, belonging to the technical field of biological pesticides. Background technique [0002] Plant parasitic nematodes have risen to become the second largest agricultural diseases next to fungal diseases, among which root-knot nematodes and cyst nematodes are the most prominent. [0003] The current methods of controlling plant parasitic nematodes mainly use chemical pesticides. However, the abuse of highly toxic chemical pesticides often produces negative effects such as endangering human health and polluting the environment. Therefore, almost all of the chemical pesticides used in the early days, including gramamifos, fenamiphos, methyl bromide, and aldicarb, were banned, and the incidence of root-knot nematode in vegetable bases was on the rise. Therefore, the research and development of safe and environm...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/90A01N63/04A01P5/00C12R1/645
CPCC07K14/37C12N15/80C12N15/902
Inventor 梁连铭凡海峰杨合玉陈迷邹成钢张克勤
Owner YUNNAN UNIV
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