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Construction method for endophytic fungus efficient genetic system and application thereof

A technology of endophytic fungi and construction methods, which is applied in the field of construction of efficient genetic systems, can solve problems such as the establishment of high-efficiency genetic systems, and achieve the effects of low cost, high transformation efficiency, and simple operation

Active Publication Date: 2018-07-10
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Agrobacterium-mediated high-efficiency genetic system has not been established before

Method used

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  • Construction method for endophytic fungus efficient genetic system and application thereof
  • Construction method for endophytic fungus efficient genetic system and application thereof
  • Construction method for endophytic fungus efficient genetic system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the establishment of the genetic system of Dendrosporium

[0031] 1. Sensitivity test of Dendrosporium to antibiotics

[0032] Spread the wild-type spores of Dendrosporium evenly on PDA medium containing different concentrations of geneticin and culture them at 25°C for 7 days, and set the concentrations of geneticin as 0, 25, 50, 100, 150, and 200 μg / mL, respectively. Dendrosporium was tested for resistance to geneticin. It was found that (see attached figure 1 ): When the concentration of geneticin reached 100 μg / mL, Dendtium was completely unable to grow. Therefore, 100 μg / mL geneticin was selected as the selection pressure in this experiment.

[0033] 2. Transformation method of Agrobacterium dentifera

[0034] 1. Plasmid construction

[0035] Plasmid pFGL815N (purchased from addgene) contains T-DNA and is a commonly used vector in Agrobacterium transformation. Using primer 1+2 (see Table 1) Aspergillus nidulans (Aspergillus nidulans) gpdA promot...

Embodiment 2

[0057] Example 2: Establishment of a high expression system in vivo for Dentische sp.

[0058] 1. Plasmid construction

[0059] The promoter aurAp (see SEQ ID NO.26 for the sequence) of the polyketide synthase gene in the aurovertin synthesis gene cluster in Dendrodentium and three commonly used constitutive promoters in filamentous fungi: gpdAp (the sequence See SEQ ID NO. 27), tef1p (see SEQ ID NO. 28 for its sequence) and tubCp (see SEQ ID NO. 29 for its sequence) were respectively linked to AurC as test promoters.

[0060] The aurA promoter (aurAp) and the aurC terminator (aurCt) were amplified using primers 7 and 8, 9 and 10, respectively, based on the Genomic DNA of Dentische dentifera, and then integrated into pFGL-neoR digested by HindIII / SmaI to form the plasmid pFLG -aurAp. Similarly, primers 11+12, 13+14 and 15+16 were used to amplify the gpdA promoter (gpdAp), the tef1 promoter (tef1p) and the tubC promoter (tubCp) from the C. The pFLG-aurAp plasmid pFLG-aurAp w...

Embodiment 3

[0070] Example 3: Establishment of an expression system for red fluorescent protein in C. dentifera

[0071] 1. Plasmid construction

[0072] The construction process of the plasmid pFGL-tubCp-RFP is: tubCp and mcherry DNA (RFP) (see SEQ ID NO.34 for its sequence) are obtained from the Genome of Dendrobium and pFA6a-link-yoPA-mCherry by primers 15+22 and 23+24 respectively -Kan was amplified, and then integrated into the BamHI / HindIII double-digested pFGL-tubCp plasmid.

[0073] Table 3 Primers used to construct red fluorescent protein particles

[0074]

[0075] 2. High expression of RFP in C. dentifera

[0076] The above-mentioned plasmids were genetically transformed with Agrobacterium to Dentische dentifera, and the grown monoclonal hyphae were picked and observed under a fluorescent microscope. The results are shown in the attached Image 6 shown. Compared with the wild-type strain, the RFP-introduced mutant strain had a strong red fluorescence signal in the red fluo...

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Abstract

The invention provides a construction method for an endophytic fungus efficient genetic system. The method includes: (1) agrobacterium-mediated transformation suitable for calcarisporium arbuscula; (2) establishment of a calcarisporium arbuscula in-vivo high expression system; (3) establishment of a calcarisporium arbuscula in-vivo fluorescence expression system; and (4) establishment of a calcarisporium arbuscula gene directional knockout system. The endophytic fungus is calcarisporium arbuscula. According to the endophytic fungus calcarisporium arbuscula genetic system provided by the invention, the calcarisporium arbuscula in-vivo high expression system is established for the first time, and four strong promoters fitting calcarisporium arbuscula are screened out. The constructed endophytic fungus calcarisporium arbuscula genetic system can be applied to activation of recessive gene clusters, compound biosynthesis mechanisms and enzyme functional study. The method provided by the invention is reasonable in design, and compared with the existing protoplast transformation methods, the method provided by the invention has the characteristics of low cost, simple operation, short experiment cycle and high transformation efficiency, and provides materials for genetic engineering research and genetic modification of calcarisporium arbuscula.

Description

technical field [0001] The invention belongs to the genetic method in the field of biology, relates to a genetic operation system of endophytic fungi, in particular to a method for constructing an efficient genetic system and its application. Background technique [0002] Filamentous fungi are an important source of natural products. Calcarisporium arbuscula is an endophytic filamentous fungus of mushrooms, which can produce mitochondrial F0F1-ATP synthase inhibitor aurovertin series compounds, including aurovertin E, B(1), D( 2) etc. The study found that aurovertin B has good anti-breast cancer activity, and it is expected to become a candidate drug for clinical treatment of breast cancer. Aurovertins contain a unique 2,6-dioxabicyclooctane structure for which a unique biosynthetic pathway has been elucidated. Preliminary sequencing results revealed that there are 68 natural product biosynthesis gene clusters in C. dentatus, which have great development value. However, C...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/65
CPCC12N15/65C12N15/80
Inventor 毛旭明李永泉陈新爱曹菲程锦涛
Owner ZHEJIANG UNIV
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