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Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast

An expression vector and gene stacking technology, which is applied in the field of biochemistry and can solve the problems of multiple gene stacking cis-expressing yeast vectors, etc.

Inactive Publication Date: 2015-08-12
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the construction of multiple gene stacking cis-expression yeast vectors
[0005] In addition, the superimposition of multiple genes on the same carrier through two pairs of homologous enzymes has not been reported yet.

Method used

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  • Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast
  • Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast
  • Yeast vector for multigene stacking cis-form expression as well as construction method and application of yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of embodiment 1 pBDL6063 vector

[0040] 1. Experimental materials

[0041] Yeast expression vector pRS425 is a laboratory storage vector; restriction endonuclease and T4 DNA ligase were purchased from NEB Company; DNA polymerase and DNA marker were purchased from Beijing Quanshijin Biological Company; plasmid mini-extraction kit and gel recovery kit were purchased from From Tiangen Company; TOP 10 competent state was purchased from Kangwei Century Company; other reagents were domestic analytically pure products.

[0042] 2. Experimental methods and results

[0043] First, the promoter ADH2p and the terminator ADH2t of alcohol dehydrogenase ADH2 were added to the commercial yeast expression vector pRS425, and the two genes were preserved by our laboratory. First, the ADH2p gene was subjected to a double-enzyme digestion reaction with the corresponding restriction endonuclease, and the expression vector pRS425 was digested with the same endonuclease, ligat...

Embodiment 2

[0050] Example 2 Application of pBDL6063 vector to realize the biosynthesis of lasiodiplodin

[0051] 1. Experimental materials

[0052] The genes used for superimposed cis expression were all cloned in our laboratory (Table 2). All the other reagents are the same as in Example 1.

[0053] Table 2 Genes expressed in cis for overlay

[0054] gene name

Length (bp)

Features

source

LtLasS1

7173

reduced polyketide synthase

Lasiodiplodia theobromae

LtLasS2

6252

non-reduced polyketide synthase

Lasiodiplodia theobromae

LtOMT

1197

Oxymethyltransferase

Lasiodiplodia theobromae

OSMT

1202

Oxymethyltransferase

Hypomyces subiculosus

[0055] 2. Experimental methods and results

[0056] The fungal polyketide compound Lasiodiplodin can induce apoptosis and inhibit the growth of breast cancer cells, and has important medicinal value. It is also a plant toxin, human coagulation factor VII...

Embodiment 3

[0058] Example 3 Application of pBDL6063 carrier to realize the modification of lasiodiplodin

[0059] Some oxymethyltransferases have a wide range of substrates and do not require high substrate specificity. They are very suitable for modifying the molecular structure of various active compounds, increasing the diversity of compound libraries, and being used for the screening of lead drugs.

[0060] The present invention selects the oxygen methyltransferase HsOMT derived from the biosynthetic pathway of the fungal polyketide compound Hypothemycin, and first recombines the HsOMT gene into the plasmid pBDL6063 to obtain the recombinant plasmid pBDL6063-HsOMT. The pBDL6063-LtLasS1-LtLasS2-LtOMT plasmid was digested with Xho I and BamH I, and the vector was recovered; the pBDL6063-HsOMT plasmid was digested with Sal I and Bgl II, and the fragment was recovered. The above vector and fragment were ligated and routinely transformed into large intestine bacilli, positive clones were ...

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Abstract

The invention relates to a vector used for multigene stacking cis-form expression. The vector used for the multigene stacking cis-form expression is characterized by containing restriction enzyme cutting site sequences of two pairs of isocaudarners, and multiple genes can be stacked in one vector by virtue of the two pairs of isocaudarners. The construction method of the vector used for multigene stack cis-form expression comprises the step of respectively introducing corresponding restriction enzyme identification sequences at upstream of a promoter and downstream of a terminator through PCR site-specific mutagenesis. In the expression carrier, each gene carries an independent promoter and an independent terminator, an independent expression element is formed, then the isocaudarners are utilized for realizing stacking assembling of the independent element, and expression of each gene is relatively independent. The vector can conveniently and rapidly realize multigene stacking, namely only a target gene is respectively cloned to a polyclonal site of the expression carrier, and then the two pairs of isocaudarners are utilized for completing subsequent assembling.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a yeast vector for superimposed cis-expression of multiple genes. The invention also relates to a construction method of the vector, and the application of the vector in the aspect of superimposed cis-expression of multiple genes in synthetic biology . Background technique [0002] Yeast is one of the most commonly used genetic engineering host bacteria in the laboratory, which can be used to express eukaryotic exogenous genes. The commonly used yeast expression vector pRS425, the genotype is ori(f1)-lacZ-T7promoter-MCS(KpnI-SacI)-T3promoter-lacI-ori(pMB1)-ampR-ori(2micron)-LEU2, is Escherichia coli-yeast In the shuttle plasmid, the pMB1 replication origin site and the 2micron replication origin site respectively control the replication of the vector in Escherichia coli and yeast, and the vector contains an ampicillin resistance gene and a leucine nutritional marker, which ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81
Inventor 徐玉泉王晓婧张维刘航
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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