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Filamentous fungus promoter, terminator and plasmid containing same

A technology of filamentous fungi and promoters, applied in the field of genetic engineering, can solve problems such as the lack of efficient protein expression systems for filamentous fungi

Inactive Publication Date: 2011-08-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, each gene research still needs to establish a gene expression system (such as NAR promoter; RP27 promoter), lack of efficient and general filamentous fungal protein expression system

Method used

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  • Filamentous fungus promoter, terminator and plasmid containing same
  • Filamentous fungus promoter, terminator and plasmid containing same
  • Filamentous fungus promoter, terminator and plasmid containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Obtaining the promoter sequence of Magnaporthe grisea SOD1.

[0023] DNA was extracted from the mycelium of Magnaporthe grisea strain Guy11 (Fungal Genetics Stock Center) and used as a template, primers were designed according to the genome database of Magnaporthe grisea strain 70-15, and the promoter sequence of the SOD1 gene was amplified by high-fidelity PCR .

[0024] Upstream primer a1: 5'-ATgaattcCGGTCATAACGCCAAGTTAATA-3';

[0025] Downstream primer a2: 5'-ATTCTAGACCCGGGGGATCCGACCATTTTGACGGTTGTTTGGTA-3'.

[0026] An EcoRI site was introduced into the upstream primer; a BamHI-SmaI-XbaI site was introduced into the downstream primer.

[0027] 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.

[0028] The PCR running conditions were: 94°C for 3 minutes, 35 cycles (94°C for 30 seconds, 58°C for...

Embodiment 2

[0030] Example 2 Obtaining the Magnaporthe grisea RP27 terminator sequence

[0031] DNA was extracted from the mycelium of Magnaporthe grisea strain Guy11 and used as a template, primers were designed according to the genome database of Magnaporthe grisea strain 70-15, and the terminator sequence of the RP27 gene was amplified by high-fidelity PCR.

[0032] Upstream primer b1: 5'-ATCTGCAGTAAGCGACACGCCATCACGATA-3';

[0033] Downstream primer b2: 5'-ATAAGCTTTGTTGAAATTACCAGCGATTCGA-3'.

[0034] A PstI site was introduced into the upstream primer; a HindIII site was introduced into the downstream primer. 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.

[0035] The PCR running conditions were: 94°C for 3 minutes, 35 cycles (94°C for 30 seconds, 60°C for 1 minute, 72°C for 30 seconds), 72°C for 10 minutes.

[0036] T...

Embodiment 3

[0037] Example 3 Construction of a recombinant expression vector carrying the SOD1 promoter and RP27 terminator

[0038] Construction of the recombinant expression vector pKD6: The SUR gene fragment was obtained from pCB1528 by PCR using the PCR primers of the resistance gene SUR.

[0039] Upstream primer c1: 5'-GTGCCAACGCCACAGTGCC-3'

[0040] Downstream primer c2: 5'-GCGAATTCACTAGTGATTGTGAATCGTGAGAGCATGCAATTCCC-3'

[0041]Cloned into the pGEM-T EASY vector, transformed Escherichia coli competent cell DH5α, picked the colonies 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 of the pCAMBIA1300 vector; the recombinant vector was transformed into E. coli competent cell DH5α, and the cells grown on the ampicillin plate were picked Colonies were cultured, plasmids were extracted...

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Abstract

The invention discloses a filamentous fungus promoter, a filamentous fungus terminator, and a plasmid containing the same. The filamentous fungus promoter has a base sequence represented by SEQ ID No.1, and the filamentous fungus terminator has a base sequence represented by SEQ ID No.2. The promoter of the invention is a strong promoter. The promoter has high-strength promotion effect in the hypha, spore and appresorium stages of pyricularia grisea. The promoter gene belongs to a gene of which the stability of the expression strength in cells of pyricularia grisea ranking in top 100. The gene is an important gene for removing active oxygen in cell and has a great significance for maintaining the normal operation of cells. The gene can be expressed in a large amount under the induction of active oxygen and is suitable for synthesizing a large amount of recombinant protein.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a filamentous fungal promoter, a terminator and a plasmid containing them. 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 antifung...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N1/15C12N15/63
Inventor 卢建平李海娇林福呈张丽琳
Owner ZHEJIANG UNIV
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