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Application of a yeast upstream activation element in filamentous fungi

A filamentous fungus, upstream technology, applied in the field of molecular biology, can solve problems such as unpredictable effects of transferring foreign genes to hosts

Active Publication Date: 2020-06-23
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the functional gene research of Saccharomyces cerevisiae has been very detailed, but the gene analysis of filamentous fungi is still at the sequencing stage. Therefore, when the sequence of the endogenous gene is not clear, the impact of the transfer of the exogenous gene on the host is even more unpredictable. predict

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  • Application of a yeast upstream activation element in filamentous fungi
  • Application of a yeast upstream activation element in filamentous fungi
  • Application of a yeast upstream activation element in filamentous fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Gene synthesis of UAS elements

[0035] The UAS elements were obtained from NCBI, and single-copy, two-copy and three-copy UAS elements were synthesized respectively. HindIII and SalI sites were added to the 5' and 3' ends of the sequence, and the target sequence was cloned into the pUC57 vector. The single-copy USA element sequence containing HindIII and SalI sites at both ends is shown in SEQ ID NO.1, and the double-copy USA element sequence containing HindIII and SalI sites at both ends is shown in SEQ ID NO.2, and both ends contain HindIII and the three-copy USA element sequence of the SalI site is shown in SEQ ID NO.3.

[0036] 2. Construction of expression vector

[0037] After obtaining the NA2 promoter (SEQ ID NO.4), terminator (SEQ ID NO.5), fungal amylase gene coding sequence (SEQ ID NO.6) from NCBI, the expression vector was constructed with pUC57 as the starting vector, and named for NA2-amdS ( figure 1 ), the sequence was synthesized by GenScript.

...

Embodiment 2

[0049] Example 2 Comparing the difference in the expression level of tandem copies of UAS

[0050] UAS2-NA2-amdS, UAS3-NA2-amdS vector transformation and enzyme activity identification methods are as described in Example 1.

[0051] Take 20 transformants each, and detect the enzyme activity of the shake flask samples, and the results are as follows: Figure 6 As shown, the expression level of three tandem UAS is about 20% higher than that of two tandem UAS.

Embodiment 3

[0052] Example 3 Effect of UAS cloning to terminator end on expression level.

[0053] UAS and two copies of UAS were cloned into the 3' end of the terminator of the NA2-amdS vector respectively, and the vectors were named NA2-UAS-amdS ( Figure 7 ), NA2-UAS2-amdS ( Figure 8 ). Vector transformation and enzyme activity identification methods are as described in Example 1.

[0054] Take 20 transformants each, and detect the enzyme activity of the shake flask sample, the results are shown in Table 2 and Figure 9 As shown, the results show that UAS cloning downstream of the terminator can still enhance the expression of the target protein fungal amylase.

[0055] Table 2

[0056]

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Abstract

The invention discloses application of yeast upstream activation element in filamentous fungi, and particularly relates to application of an upstream activation element UAS coming from brewer's yeast and shown as SEQ ID NO.1 in increasing expression amount of target protein. The upstream activation element UAS is preferably cloned to an upstream of a promoter or a downstream position of terminator while positions are not limited to the upstream and the downstream position. After the upstream activation element coming from the brewer's yeast is fused with the promoter, transformation level of a downstream coding protein sequence corresponding to the upstream activation element can be improved, so that the expression amount of the target protein is increased.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to the application of a yeast upstream activation element in filamentous fungi. Background technique [0002] Upstream transcriptional activation element UAS is a cis-acting element in molecular biology, which refers to a specific DNA sequence in a DNA molecule that has a transcriptional regulatory function, that is, a transcription factor DNA binding site and other regulatory motifs with special functions. These elements can be recognized and bound by specific transcription factors, thereby affecting gene expression activity. Usually, only specific promoters combined with cis-acting elements can perform corresponding transcriptional activation or transcriptional repression functions. Due to the differences in transcription factors between different species, cross-species cross-expression will generally cause cis-acting elements to be invalid. [0003] The combination of the yeast UAS...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/80C12R1/865
CPCC12N15/113C12N15/80C12N2830/704
Inventor 董亢徐昊珺李峰孙艳姜睿玲徐红
Owner NANJING BESTZYME BIO ENG CO LTD
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