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CRISPR/Cpf1 vector suitable for deep-sea fungi FS140 as well as construction method and application of CRISPR/Cpf1 vector

A construction method, cpf1 technology, applied in the direction of microorganism-based methods, introduction of foreign genetic material using vectors, application, etc., can solve problems such as gene knockout of deep-sea fungi that have not been seen yet

Active Publication Date: 2021-05-07
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 development of CRISPR / Cpf1 is conducive to breaking through and overcoming some limitations in the application of CRISPR / Cas9, so it is called a new generation of CRISPR. Due to the lack of corresponding vectors, the CRISPR / Cas9 system is rare in filamentous fungi and has not been seen yet. Gene knockout in deep-sea fungi using the CRISPR / Cpf1 system is reported

Method used

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  • CRISPR/Cpf1 vector suitable for deep-sea fungi FS140 as well as construction method and application of CRISPR/Cpf1 vector
  • CRISPR/Cpf1 vector suitable for deep-sea fungi FS140 as well as construction method and application of CRISPR/Cpf1 vector
  • CRISPR/Cpf1 vector suitable for deep-sea fungi FS140 as well as construction method and application of CRISPR/Cpf1 vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of targeted gliotoxin biosynthesis gene knockout vector

[0043] On the basis of the pFC332 plasmid, pFC332 was transformed, and PacI and BglII sites were used for double enzyme digestion, and the hygromycin resistance gene was replaced by the wisterin resistance gene; the Cpf1 protein was replaced by Cas9; a sequence was inserted: SNR52 Promoter (BglII): crRNA: SUP4 terminator (PacI) (which contains the SNR52 promoter, crRNA targeting sequence backbone gene and its terminator), enabling the plasmid to stably express Cpf1 protein and transcribe crRNA simultaneously after transformation into protoplasts (The plasmid was named pFC332-cbx-TEF1-Cpf1-crRNA).

[0044] The primers designed for the construction of the vector are shown in Table 1, and the GliK-pFC332-cbx-TEF1-Cpf1-crRNA-1 vector was constructed by restriction enzyme ligation and homologous recombination. The construction method is as follows:

[0045] Table 1 Primer Sequence

[0046] ...

Embodiment 2

[0053]Example 2 The method for introducing exogenous genes into G.pallida FS140 protoplasts is as follows:

[0054] 1) Pick an appropriate amount of G. pallida FS140 mycelium and inoculate it in 200 mL of PDB liquid medium, and culture it at 30° C. and 180 r / min for 3-4 days. Select 2 g (wet weight) of well-grown bacterium balls in a 50 mL centrifuge tube and wash twice with PBS buffer to fully wash away the residual PDB medium. Weigh 0.15g lyase and dissolve in 20mL KC buffer (0.6M KCl, 0.05MCaCl 2 ), and filtered with a 0.22 μm filter membrane, added to the washed bacterial balls, and placed in a shaker at 28°C and 80r / min for about 3 hours to lyse. Filter the lysate with a 200-mesh filter, filter the hyphae, and then filter again with 6 layers of lens tissue, centrifuge at 4°C and 6500×g for 10 minutes, and discard the supernatant. Add 5mL of KC buffer, gently blow and mix the precipitate with a pipette gun, centrifuge at 4000×g for 5min at 4°C, and discard the supernatan...

Embodiment 3

[0060] 将Glik基因(核苷酸序列为:atgaccatacaactccctcacacacctgacagagaagaaggccctggtgc ttctcctgcatgcaagttcaatgcaatccagacattccggtggttatgggacctagtcatcccaactagcgaccttactc aagaaaccggtcgatatcctccgaagacgacgatcgagagacggcgcgcatcaaccacagatagatcgctcgataaggac gaatacctagctgagaaggtcgcaggtcatcaggtcgaagaacatgtccccccggagaagaccgtcctctacctcgcgta cggctcgaatctggccgcggagaccttcctgggcaagcgaggcattaggcctctatcccagatcaacgttgtcgttcccg gcctacgactaactttcgaccttcctgggttaccatacgttgagccatgcttcgcagcgactcggcactggactcataca ccaagagtaacacaaacagaaggaaatggaagtaatgaaggggtcgatgcagaagtgttggagaattcgtccctcgtgcc acaggagaagaacgacatgcctctcgtcggcgtcgtgtatgaggtcactgtcgccgattatgccaagataatagccacag agggtggtgggcgcggataccgagacgtcgttgtcgattgctatccttttcccaaatcatacagtcccaccgatccggtc cctgagtgccccgaaaccaaacccttcaagtcccacaccctcctctctccagccgacgacgcagtctcgggtctcctggc cgcagggaagtcataccgacccgtccgacccaatcctggctacgcccagccctccgcttga)插入到pET-30a载体的酶切位点NdeI和XhoI之间的位置,获得pET30a-GliK,转化进大肠杆菌 BL21(DE3) competent cells were screened for positive clones to obtain Escheri...

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Abstract

The invention discloses a CRISPR / Cpf1 vector applicable to deep sea fungi FS140 as well as a construction method and application of the CRISPR / Cpf1 vector. According to the invention, the CRISPR / Cpf1 technology is utilized for the first time to construct a gene knockout system suitable for the deep-sea fungus G.pallida FS140, so that genetic engineering transformation of the G.pallida FS140 is promoted, and a molecular biology foundation is laid for clarification of a biosynthesis mechanism of G.pallida FS140 gliotoxin and acquisition of more gliotoxin derivatives with remarkable biological activity.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a CRISPR / Cpf1 vector construction and transformation method suitable for the deep-sea fungus G. pallida FS140. Background technique [0002] The previous research group has isolated about 20 gliotoxin compounds from the deep-sea fungus G.pallida FS140, including gliotoxin dimer compounds with rare structures, most of which have good Anti-tumor activity and anti-α-glucosidase activity, so it has the prospect of being developed as a drug lead compound. On this basis, the gliotoxin biosynthesis gene cluster was predicted by genome sequencing of G. pallida FS140, and the biochemical function verification of key gliotoxin biosynthesis functional genes was carried out in vitro. Due to the large genome sequence of non-model fungal organisms, fewer selective resistance selection markers, low gene editing efficiency, and long growth cycle and other constraints, it has a gre...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/66C12N15/65C12N15/64C12N15/55C12N15/54C12N1/15C12R1/645
CPCC12N15/80C12N15/65C12N9/22C12N9/1029
Inventor 李赛妮陈玉婵章卫民朱牧孜叶伟刘洪新刘昭明张维阳
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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