CRISPR/Cas9 vector suitable for Dichotomomycescejpii FS110, construction method and application thereof

A technology of FS110 and construction method, applied in the fields of biochemistry and molecular biology, can solve problems that have not yet been seen, and achieve the effect of promoting development and utilization

Pending Publication Date: 2020-04-24
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR / Cas9 system has been widely used in the genome editing of eukaryotic cells such as mammalian cells, stem cells, and plants due to the advantages of high gene knockout efficiency, simple construction, and low cost. However, due to the lack of corresponding vectors, CRISPR The / Cas9 system is rare in filamentous fungi, and there is no report on gene knockout of deep-sea fungi using the CRISPR / Cas9 system

Method used

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  • CRISPR/Cas9 vector suitable for Dichotomomycescejpii FS110, construction method and application thereof
  • CRISPR/Cas9 vector suitable for Dichotomomycescejpii FS110, construction method and application thereof
  • CRISPR/Cas9 vector suitable for Dichotomomycescejpii FS110, construction method and application thereof

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Embodiment 1

[0032] Example 1: Construction of targeted gliotoxin biosynthesis gene knockout vector

[0033] On the basis of the pFC332 plasmid, pFC332 was subjected to double enzyme digestion at the PacI and BglII sites, and a sequence was inserted: 5SrRNA promoter-targeting sequence-sgRNA-sgRNA terminator (including the 5SrRNA promoter applicable in Eideria FS110 , the sgRNA targeting sequence backbone gene and its terminator that function in yeast cells), so that the plasmid can stably express Cas9 protein and transcribe sgRNA at the same time after being transformed into protoplasts (the plasmid is named pFC332-sgRNA).

[0034] The primers designed for constructing the vector are shown in Table 1, and the pFC332-sgRNA vector was constructed by restriction enzyme ligation. The construction method is as follows:

[0035] Table 1 Primer Sequence

[0036]

[0037]Using Eydleria FS110 genomic DNA as a template, and using primers 5SrRNA-promoter-F and 5SrRNA-promoter-R as primers before...

Embodiment 2

[0043] Knockout of gliotoxin biosynthesis genes in E. dermatella FS110:

[0044] The method of introducing exogenous gene into the protoplast of Eideria FS110 is as follows:

[0045] (1) Protoplasts of Eedleia FS110 prepared (for the specific preparation method refer to the inventor's patent number 201510540618.1, the name is: a patent of Eedleia FS110 protoplasts and its preparation method and transformation method) (1×10 8 / mL) were mixed with 2.5-5 μg G1-pFC332-sgRNA plasmid or O1-pFC332-sgRNA plasmid, and placed on ice for 5 minutes, then added 200 μL volume fraction of 30% PEG4000, placed at 30 ° C for 15 minutes, and then added 400 μL PEG4000, Place at 30°C for 15 minutes, then add 1.2mL of W5 solution to terminate the reaction, and finally add 4mL of WI buffer and place it on a shaker at 30°C at low speed for overnight incubation;

[0046] (2) After cooling the melted TB3 solid medium to room temperature, take 20 mL each time and mix gently with the overnight culture s...

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Abstract

The invention discloses a CRISPR / Cas9 vector suitable for Dichotomomycescejpii FS110, a construction method and application thereof. According to the invention, CRISPR / Cas9 technology is used for thefirst time to construct a recombinant Dichotomomycescejpii FS110 strain with gliotoxin biosynthesis gene knockout, and a CRISPR / CR9 gene knockout system suitable for deep-sea fungus Dichotomomycescejpii FS110 is established, so that the genetic engineering modification of Dichotomomycescejpii FS110 is promoted, and a molecular biological foundation is laid for elucidating a biosynthesis mechanismof the Dichotomomycescejpii FS110 gliotoxin and obtaining more gliotoxin derivatives with remarkable biological activity.

Description

technical field [0001] The invention belongs to the technical field of biochemistry and molecular biology, and in particular relates to a CRISPR / Cas9 vector suitable for E. dermatella FS110 and its construction method and application. Background technique [0002] The deep-sea fungus Dichotomyces cejpii (Dichotomyces cejpii) FS110 is an ascomycete from the deep sea, which can produce a large number of novel gliotoxins with anti-tumor activity. About 30 kinds of gliotoxin compounds have been isolated from the deep-sea fungus E. Tumor activity and anti-α-glucosidase activity, so it has the prospect of being developed as a drug lead compound. On this basis, the genome of E. dermatella FS110 was sequenced, the gliotoxin biosynthesis gene cluster was predicted, and the key biosynthesis function genes of gliotoxin biosynthesis were verified in vitro. However, at present, the gene knockout system of the deep-sea fungus Erderia has not been successfully established, so the biosynt...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/113C12N15/90C12N15/66C12N1/15C12R1/645
CPCC12N15/80C12N15/113C12N15/902C12N15/66C12N9/1088C12Y205/01018C12N2310/20
Inventor 叶伟黄自磊章卫民李赛妮朱牧孜孔亚丽刘桃妹刘珊
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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