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Method for efficiently modifying filamentous fungi and strain of modified filamentous fungi

A filamentous fungus, high-efficiency technology, applied in the fields of molecular biology and enzymology, can solve the problems that do not involve simultaneous interference, achieve the effect of reducing downstream isolation costs, fast transformation process, and reduced expression

Inactive Publication Date: 2014-01-08
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Novozymes' migrating RNAi technology does not involve simultaneous interference of multiple genes via a single RNAi construct

Method used

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  • Method for efficiently modifying filamentous fungi and strain of modified filamentous fungi
  • Method for efficiently modifying filamentous fungi and strain of modified filamentous fungi
  • Method for efficiently modifying filamentous fungi and strain of modified filamentous fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of RNA interference backbone vector pamdS-PgpdA-2ccdB-TagdA

[0042] 1.1 Extract the genome of Aspergillus niger (Aspergillus niger) CBS513.88.

[0043] The washed and dried Aspergillus niger CBS513.88 cells (American Type Culture Collection ATCC) were ground with liquid nitrogen, and the genomic DNA of Aspergillus niger was extracted with phenol chloroform.

[0044] 1.2 Construction of intermediate plasmid pHGW-ccdB-TagdA

[0045] Using Aspergillus niger CBS513.88 genomic DNA as a template, the fragment amplified by PCR with primers SEQ ID NO: 1 and SEQ ID NO: 2 was named TagdA. The TagdA fragment treated with SpeI and SacI was inserted into SpeI and SacI of the plasmid Phgw-ccdB to obtain pHGW-ccdB-TagdA. The plasmid was sent to Huada Gene Company for sequencing, and the result showed that the DNA sequence of the amplified glycosidase A terminator was SEQ ID NO.31.

[0046] 1.3 Construction of intermediate plasmid pSIMPLE18-intron8

[0047] Aspergillu...

Embodiment 2

[0059] Construction of plasmids containing single gene interference constructs

[0060] 2.1 Construction of single gene interference intermediate vectors pDONR221-long and pDONR221-short

[0061] During the construction of the single-gene interference plasmid, two pairs of primers were designed, and the Aspergillus niger CBS513.88 genomic DNA can be used as a template to amplify the glycoamylase coding region long through primers SEQ ID NO:9 and SEQ ID NO:10. The coding region sequence of the glucoamylase amplified by primers SEQ ID NO:11 and SEQ ID NO:12 is short.

[0062] The 5' primers of the two fragments all added the combination site for BP reaction, the forward primer added the attB1 site, and the reverse primer added the attB2 site. The amplified fragment and plasmid pDONR221 (purchased from Shanghai Yingwei Jieji) were acted on by BP Clonase II enzyme mix enzyme to perform BP reaction to generate two Entry vectors pDONR221-long and pDONR221-short. The long and short...

Embodiment 3

[0066] Construction of plasmids containing polygene interference constructs

[0067] 3.1 Amplify DNA fragments containing multiple gene coding region sequences

[0068] Using the Aspergillus niger genome as a template, the amylase amyA coding region fragment is amplified by primers SEQ ID NO:13 and SEQ ID NO:14. A fragment of the coding region of glucoamylase glaAB was amplified by primers SEQ ID NO:15 and SEQ ID NO:16. A fragment of the coding region of the prtT gene was amplified by SEQ ID NO:17 and SEQ ID NO:18. These three fragments contain overlapping regions. After purifying these fragments, the three fragments are fused together in a certain order by overlapping extension PCR technology. The sequence after the fusion of the three fragments is the sequence shown in SEQ ID NO:33.

[0069] 3.2 Construction of plasmid pamdS-PgpdA-Fragments-TagdA containing polygenic interference

[0070] The 5' end and 3' end of the three fused fragments are all added sites capable of ...

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Abstract

The invention discloses a method for efficiently modifying filamentous fungi and a strain of the modified filamentous fungi. The method is characterized by introducing a multi-gene interference constructing body and a target protein expression constructing body into filamentous fungus cells simultaneously, and then screening to obtain efficiently modified filamentous fungi, wherein the multi-gene interference constructing body comprises promoters, inverted repeat sequences and a terminator, wherein the promoters can initiate transcription in the filamentous fungus cells, the inverted repeat sequences are separated by introns and are connected together by coding sequences of 2-30 target genes in a mosaic manner, and the terminator can terminate transcription in the filamentous fungus cells; the target protein expression constructing body comprises promoters, coding sequences of target proteins and a terminator, wherein the promoters can initiate transcription in the filamentous fungus cells, and the terminator can terminate transcription in the filamentous fungus cells. The method and the strain have the advantages that the modification efficiency of the filamentous fungi is improved; the downstream separation and purification costs of the target proteins are reduced; the filamentous fungus cells modified through one-time transformation can be used for producing food safety level enzyme preparations.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and enzymology. The specific method is to transform filamentous fungi with DNA containing two elements of the polygene interference construct and the target protein expression construct, and screen 2-30 target genes that are disturbed and can efficiently express the transformants of the target protein. Background technique [0002] Filamentous fungi have a strong ability to secrete extracellular proteins and can be used to express many enzymes. (Kobayashi et al., 2007) Unmodified wild-type filamentous fungi secrete large amounts of extracellular hydrolytic enzymes during fermentation. The target protein and the hydrolase system secreted by filamentous fungi share the intracellular secretion system. This will competitively inhibit the various resources required for the secretion of the target protein and reduce the expression of the target protein (Yoon et al., 2010). At the same time...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12R1/66C12R1/80C12R1/785
Inventor 潘力何攀周斌吕扬勇王云艳
Owner SOUTH CHINA UNIV OF TECH
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