Method and application for improving application efficiency of gene targeting technique in aspergillus terreus

A technology of gene targeting and Aspergillus terreus, applied in the field of genetic engineering, can solve the problems of low homologous recombination ability of Aspergillus terreus species, limited application of screening tags, etc.

Active Publication Date: 2015-09-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

[0006] In order to solve the technical problem that the homologous recombination ability of Aspergillus terreus is low and the number of screening tags is small, the application of gene targeting technology in the field of genetic transformation of Aspergillus terreus is limited. In the method of application efficiency, the technical scheme adopted is as follows:

Method used

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  • Method and application for improving application efficiency of gene targeting technique in aspergillus terreus
  • Method and application for improving application efficiency of gene targeting technique in aspergillus terreus
  • Method and application for improving application efficiency of gene targeting technique in aspergillus terreus

Examples

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Embodiment 1

[0068] Example 1 Analysis of integration probability of exogenous DNA homologous recombination in Aspergillus terreus CICC40205 wild-type strain

[0069] 1.1 Construction of Aspergillus terreus pyruvate decarboxylase gene pdc (ATET_04633) targeting element

[0070] Primers Updc-F (5'-agagggtggtatcattccgttg-3'), Updc-R (5'-ctttacgcttgcgatcccgaacccctgagtgagaaggaacatg-3'), Dpdc-F (5'-cctgggttcgcaaagataattgatccgaaacaacctcaacccg-3') were designed according to the information published in the Aspergillus terreus NIH2624 genome database Dpdc-R (5'-cgaggtgtgcgcaacaggcatgaatg-3'), using the genome of Aspergillus terreus strain CICC40205 as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using primers Updc-F / Updc- R can amplify the upstream sequence U-pdc of the pdc gene with a size of about 1.9kb, and use primers Dpdc-F / Dpdc-R to amplify the downstream sequence D-pdc of the pdc gene with a size of 2.1kb. Using pSGF957 as template, primer hp...

Embodiment 2

[0076] Example 2 Construction of NHEJ Pathway Deletion Genetic Engineering Strain At-Δku80 with ku80 Gene Knockout

[0077] 2.1 Construction of targeting elements for knocking out ku80

[0078] Primers Uku80-F (5'-gtcgtagctcttcttgccatc-3'), Uku80-R (5'-aatgggatcccgtaatcaattgccctcaatcaccatctcccttatc-3'), Dku80-F (5'-caagagcggctcatcgtcaccccattccggcctcgatgtgttttc-3')ggatg, c Dku80-R (5'-tccacgcggccatcaccgagc-3'), using the genome of Aspergillus terreus strain CICC40205 as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using primers Uku80-F / Uku80- R can amplify the upstream sequence U-ku80 of ku80 with a size of about 1.5kb, and use primers Dku80-F / Dku80-R to amplify the downstream sequence D-ku80 of ku80 with a size of 1.5kb. Using the plasmid pPTR II (TAKARA, Catalog No.: 3621) as a template, using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as primers to amplify with a size of about The 2.0kb ptrA f...

Embodiment 3

[0083] Example 3 Construction of NHEJ Pathway Deletion Genetic Engineering Strain At-Δlig4 with Knockout of lig4 Gene

[0084] 3.1 Construction of split-marker method to knock out the targeting element of lig4

[0085] Primers Ulig4-F(5'-cggctattctcacgcagctc-3'), Ulig4-R(5'-aatgggatcccgtaatcaattgccc atacagggtcatcctcggtc-3'), Dlig4-F(5'-caagagcggctcatcgtcaccccat AtataatgatagcttttgctC ), Dlig4-R (5'-cgtttgactgtcgcgacgtttcg-3'), using the genome of Aspergillus terreus strain CICC40205 as a template, using pfu DNA polymerase (Fermentas, Catalog No.: EP0501) for PCR amplification, using primer Ulig4-F / Ulig4-R can be amplified to obtain the upstream sequence U-lig4 of ku80 with a size of about 1.5kb, and the downstream sequence D-lig4 of lig4 with a size of 1.5kb can be amplified with primers Dlig4-F / Dlig4-R, after 1.0% Agarose gel electrophoresis detection and gel tapping recovery and purification. The U-lig4 fragment, D-lig4 and ptrA fragment (described in Example 2) were fused...

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Abstract

The invention discloses a method and application for improving application efficiency of a gene targeting technique in aspergillus terreus, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, by taking Aspergillus terreus as an initial bacterium, knocking off a ku80 gene or an lig4 gene so as to increase the exogenous DNA homologous recombination probability of a strain; secondly, establishing a pyrG gene deletion uracil auxotroph stain, establishing a inheritance conversion system based on a pyrG gene as a screening tag; and finally, cutting off the screening tag by using a Cre / LoxP specific binding site recombinant system, thereby obtaining a uracil auxotroph stain which can be applied to genetic modification again. By adopting the method disclosed by the invention, an efficient aspergillus terreus gene targeting platform can be established, the method has the advantages that high homologous recombination efficiency can be achieved, the bidirectional screening of the conversion system can be achieved, a screening tag cutting method is simple and feasible, the screening tag can be recycled, and the like, and basic support can be provided for efficient genetic modification of aspergillus terreus by using the gene targeting technique.

Description

technical field [0001] The invention relates to a method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus, belonging to the technical field of genetic engineering. Background technique [0002] Aspergillus terreus is an important filamentous fungus that produces a variety of valuable compounds, including organic acids, enzymes, lipids, and biologically active secondary metabolites. Among them, itaconic acid and lovastatin have been industrialized and have a large market. With the continuous development of molecular biology, it is of great significance to transform the production strains through metabolic engineering and improve the production level of the target product. [0003] Gene targeting technology is an important research method in modern molecular biology. It usually refers to the homologous recombination between a DNA fragment containing a known sequence and the genome of a recipient cell, integrating int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N1/15C12R1/66
Inventor 吕雪峰黄雪年李建军陈梅
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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