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Filamentous fungi promoter and plasmid containing same

A filamentous fungus and promoter technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory positioning effect, and achieve the effect of easy positioning and high-efficiency expression

Inactive Publication Date: 2013-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention patent application with the publication number CN102154294A discloses a filamentous fungal promoter, terminator and plasmid containing them, but the expression and localization of the promoter-driven protein in Magnaporthe grisea are still not ideal, and more efficient methods need to be explored. , a general filamentous fungal protein expression system

Method used

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  • Filamentous fungi promoter and plasmid containing same
  • Filamentous fungi promoter and plasmid containing same
  • Filamentous fungi promoter and plasmid containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Obtaining the promoter sequence of Magnaporthe grisea H3

[0025] DNA was extracted from the mycelia of Magnaporthe grisea strain Guy11 (Fungal Genetics Stock Center, University of Missouri, Missouri, USA) and used as a template, primers were designed according to the genome database of Magnaporthe grisea 70-15 strain, and high-fidelity PCR method was used. The promoter sequence of the H3 gene was amplified.

[0026] Upstream primer a1: 5'-TTgaattcAGTCATGTTGATTGAGGTGTTGT-3'

[0027] Downstream primer a2: 5'-TGTCTAGACTTcccgggGATGGATCCGGCCATTGTGATTGATTTGTGATT-3',

[0028] An EcoRI site was introduced into the upstream primer; a BamHI-SmaI-XbaI site was introduced into the downstream primer.

[0029] The PCR system is: 1 μl of Magnaporthe grisea genomic DNA, 0.5 μl of high-fidelity DNA polymerase, 0.4 μl of dNTP (50 mM), 0.5 μl of upstream and downstream primers, 5 μl of 10x PCR buffer, and add water to 50 μl.

[0030] The PCR running conditions were: 94°C for...

Embodiment 2

[0032] Example 2 Construction of recombinant expression vector carrying H3 promoter and RP27 terminator

[0033] The DNA coding sequence of the RP27 terminator is shown in SEQ ID NO.2, and its obtaining method refers to the content disclosed in the invention patent application with publication number CN102154294A.

[0034] (1) Construction of recombinant expression vector pKD5

[0035] Using the PCR primers of the resistance gene SUR to obtain the SUR gene fragment from pCB1528 by PCR,

[0036] Upstream primer c1: 5'-GTGCCAACGCCACAGTGCC-3'

[0037] Downstream primer c2: 5'-GCGAATTCACTAGTGATTGTGAATCGTGAGAGCATGCAATTCCC-3',

[0038]Cloned into the pGEM-T vector, transformed Escherichia coli competent cell DH5α, picked the colony grown on the ampicillin plate for culture, extracted the plasmid, and identified it by enzyme digestion. The recombinant vector was then digested with XhoI and EcoRI, and the 2.8kb fragment (SUR gene fragment) was inserted into the XhoI and EcoRI sites...

Embodiment 3

[0054] Example 3 Strong expression of DsRED2 driven by H3 promoter in the mycelia of Magnaporthe grisea

[0055] Transformation of pKD5-RED vector into Magnaporthe grisea by ATMT method: The vector pKD5-RED was transformed into Agrobacterium strain AGL1 by freeze-thaw method. Then the ATMT method transforms in the germinated spores of Pyricularia oryzae: from the freshly cultivated LB plate (containing 50mg / ml kanamycin), select a single Agrobacterium colony to inoculate in 5ml LB liquid medium (containing 50mg / ml kanamycin) ), 200rpm / min, cultured overnight at 28°C; the next day, transfer 200-400μl culture solution to 5ml induction liquid medium (IM) containing 50mg / ml kanamycin, the OD value was about 0.15, cultured at 28°C for 5 ~6 hours to make OD600 reach 0.5~0.6; collection of conidia of Magnaporthe grisea: Wash the conidia of Magnaporthe grisea with 10ml sterilized distilled water from the CM plate cultivated for about 10 days, filter with three layers of mirror paper ...

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Abstract

The invention discloses a filamentous fungi promoter and a plasmid containing the same. The filamentous fungi promoter or a complementary strand thereof has a base sequence shown in SEQ ID (sequence identity) NO.1. The filamentous fungi promoter is a strong promoter, and can perform high-intensity promotion in a mycelium stage, a spore stage and an appressorium stage of pyricularia grisea. The gene of the promoter belongs to the gene, of which the expression intensity is stabilized within the first 100 regions inpyricularia grisea cells. The gene is a histone gene, and plays an important rolein maintaining the normal function of the cells. By utilizing the filamentous fungi promoter, a system that can express in filamentous fungi rapidly and efficiently can be built, so that the researchon the positioning of protein in pathogenic fungi cells as well as on the influence on pathogenic fungi caused by protein over-expression is facilitated.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a filamentous fungal promoter and a plasmid containing the promoter. Background technique [0002] Magnaporthe oryzae is a filamentous fungal model organism for studying phytopathogenic fungus-host plant interactions. It has a plant pathogenic infection cycle common to many pathogenic fungi, including spore production, Pathogenic processes such as the formation of dye plugs and the growth of invasive hyphae. Rice blast caused by Magnaporthe grisea is a devastating disease of rice worldwide. The rice yield loss caused by rice blast is between 10% and 30% every year in the world; the epidemic outbreak of rice blast fungus occurs almost every year in my country. Many developed countries are studying its pathogenic molecular mechanism, and expect to develop rice varieties with blast resistance characteristics through such research, as well as develop and design new antifungal dru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/80C12N1/15
Inventor 卢建平张莉林李海娇林福呈
Owner ZHEJIANG UNIV
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