CN102952800B - GAP (Candida Glycerinogenes) gene promoter and application thereof - Google Patents

Abstract

The invention discloses a GAP promoter gene and its application, belonging to the field of microbial molecular biology. The promoter sequence provided by the present invention is shown in SEQ ID NO.1, which is derived from Candida glycerologenus WL2002-5-59. The cloning of the gene and its promoter expands the gene resources for the study of microbial resistance to osmotic stress, and also lays a foundation for the molecular mechanism research of the high glycerol production of Candida glycerologenum. Applications containing the promoter sequence of the present invention, such as expression vectors, can be constructed as markers for screening cell transformation and for evaluating the efficiency of gene cloning. As a tool for gene knockout of Candida glycerologenicum, strain improvement is carried out to produce safer and more industrially applicable Candida glycerologenicum species.

Description

A kind of GAP gene promoter and its application

technical field

The present invention relates to a GAP gene promoter, especially a promoter derived from Candida glycerinogenes.

Background technique

Candida glycerolgenesis (CCTCC: M93018) is an industrial strain with excellent fermentation performance with independent intellectual property rights in my country. It was disclosed in the Chinese patent CN1070235C and announced on August 29, 2001. It has hypertonic resistance , High output, high conversion rate, and high production intensity, occupying a leading position in the world. It can normally grow and reproduce on the high osmotic pressure medium of 25% glucose or 5% NaCl. In laboratory scale fermentation, the yield of glycerol can reach 12%, even in industrial scale, the yield of glycerol can reach 10%. It is difficult to explain with the glycerol metabolism theory of Saccharomyces cerevisiae.

The final products of gene expression are RNA and protein. Any defect or disorder in the regulation of gene expression will have serious consequences for the organism. Therefore, the research on the mechanism of gene expression regulation is the focus and frontier of molecular biology research. The expression of protein-coding genes requires transcription and translation, each of which has different regulatory sites for gene expression. The regulation of the promoter plays a very important role in the transcription process. Therefore, it is of great significance to study the function of promoters for the study of gene expression regulation mechanism. Strong promoters are essential for efficient gene expression.

Promoter regions are essential for driving and regulating gene expression. These stretches of DNA sequence are usually found upstream of the gene's open reading frame. Key genetic elements in promoters include enhancer sites, silencer sites, upstream activator sequences, transcription factor binding sites, and RNA polymerase binding sites. These elements respond to the external environment of a given organism to regulate gene expression according to growth conditions. It is of great significance to obtain high-efficiency promoters.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new Candida glycerologenus gene and its promoter sequence, its nucleotide sequence is SEQ ID NO.1.

The above-mentioned polynucleotide sequence SEQ ID NO.1 is derived from the gene expression cassette of Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase.

Vectors containing the promoter, such as expression vectors, cloning vectors and shuttle vectors, are all within the protection scope of this patent.

The vector provided by the present invention can be applied to yeast expression system; screening of functional genes (gene cloned behind the promoter), identification and optimization; regulation of gene expression; stable expression of functional genes (gene cloned behind the promoter) or overexpression (Using multiple promoters or initiating stress mechanisms). The conventional applications of the above-mentioned promoters all belong to the protection scope of the present invention.

Verification of the expression strength of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene in Candida glycerovaciens:

3-Glyceraldehyde phosphate dehydrogenase gene promoter expression strength of the recombinant expression vector (Figure 1), the construction process is as follows:

(1) PCR cloning or adopting the method of chemical total synthesis will glycerologenic Candida CgGAP gene promoter P _CgGAP be connected to plasmid vector PYX212 (Regenberg, B., L.During-Olsen, MCKielland-Brandt and S.Holmberg, ( 1999)

Substrate specificity and gene expression of the amino-acid permeases in Saccharomyces cerevisiae.Curr.Genet.36:317-328.) to obtain the carrier PYX212-P _CgGAP ;

(2) connecting the green fluorescent protein gene GFP to the carrier PYX212-P _CgGAP to obtain the carrier PYX212-P _CgGAP -GFP;

(3) connecting the zeocin resistance gene to the carrier PYX212- _PCgGAP -GFP to obtain the carrier PYX212-zeocin- _PCgGAP -GFP;

Through the constructed recombinant expression vector PYX212-zeocin-P _CgGAP -GFP, the recombinant plasmid was transformed into Saccharomyces cerevisiae (ATCC: 4098 ^TM ) by heat shock method, and the YEPD-resistant plate containing zeocin was coated. Pick a single colony from the YEPD-resistant plate, transfer to YEPD liquid medium supplemented with zeocin (150ug/mL), extract the plasmid, and verify the positive transformant.

Pick positive clones from the plate and inoculate in 10mL YEPD liquid medium containing 150ug/mL zeocin. After culturing at 30°C for 20 hours, the cells were collected by centrifugation and transferred into 10 mL of YEPD liquid medium. Cultivate at 30°C for 24 hours, and the rotation speed of the shaker is 200r/min.

Fluorescence analysis was performed using an Olympus fluorescence microscope. The practicability and feasibility of the constructed integrated expression vector was confirmed by using the green fluorescent protein as a marker, and the expression intensity of the promoter of the Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase gene was further verified.

Beneficial effects of the present invention: the present invention provides a high-efficiency promoter sequence, which can be widely used in gene expression and functional gene screening research.

Description of drawings

Figure 1: PYX212-zeocin- _PCgGAP -GFP physical map;

Figure 2: Fluorescence verification diagram of the expression intensity of the GAP gene promoter of Candida glycerologenicum:

(A) Wild Saccharomyces cerevisiae was used as a control;

(B) recombinant Saccharomyces cerevisiae transformed with plasmid PYX212-zeocin-P _CgGAP -GFP;

Detailed ways

The present invention is further illustrated below by specific examples. These examples are only for illustrating the present invention and are not intended to limit the present invention.

Example 1: PCR amplification of the 3-phosphate glyceraldehyde dehydrogenase gene fragment of Candida glycerinogenes with degenerate primers

1. Design of degenerate primers

Search the sequences of related yeast and other eukaryotic organisms published in GeneBank, and find the amino acid sequences corresponding to two conserved regions by comparing and analyzing the amino acid sequences. IKVGINGF and YDNEYGYSTR are found, and a pair of degenerate primers are designed based on these two conserved regions

GAP1: TBRTB GGITCHGGYAAYTGGG

GAP2: RGCVAYVAYRTTYTTYARIGC

2. Extraction of Candida glycerigenes genomic DNA

Chromosomal DNA preparation of Candida glycerologenum (CCTCC: M93018) was carried out according to the reference [Technical Handbook of Yeast Genetics]. Measure the extracted DNA with DU640 nucleic acid and protein analyzer, judge the purity of the extracted DNA according to the ratio of A ₂₆₀ /A ₂₈₀ , calculate the concentration of the extracted DNA by using the OD value at 260nm, and at the same time, judge by agarose gel electrophoresis The quality of the template.

3.PCR amplification

A nucleic acid molecule with a fragment length of about 0.93kb was obtained by PCR. After gel recovery, TA cloning was performed according to the operation instructions of the pGEM-T-Easy (Promega) kit, and a sequence was obtained from the sequencing results.

Embodiment 2: Reverse primer PCR amplifies the full-length sequence of 3-phosphate glyceraldehyde dehydrogenase gene

1. Design of reverse PCR primers

Design a pair of reverse PCR primers according to the sequence results obtained by PCR amplification with degenerate primers

GAPI: CCTTATTTCCGTGTTCGTGTTATTAGTG

GAPF: CGCCTTCAATCAATTCTTCATAGAC

2. Preparation of PCR template

Genomic DNA of C. glycerinogenes was extracted according to the above method, and 5 μg of genomic DNA was digested with restriction enzyme Sac I respectively, and the effect of the digestion was checked by agarose electrophoresis. The digested product was extracted with equal volumes of 1:1 phenol and chloroform, then precipitated with 2.5 times the volume of absolute ethanol, and finally dissolved in 50uL ultrapure water. In the 200uL ligation system, add 0.1ug digested DNA, 20uL ligation buffer and 10U _T4 DNA ligase, make up the volume with water, and leave overnight at 16°C. After the ligation product was precipitated with absolute ethanol, it was dissolved in 25uL sterile ultrapure water for the next step of reverse PCR amplification.

3. Inverse PCR amplification

Reverse PCR amplification and sequencing results were spliced to obtain the full-length C. glycerinogenes gene sequence and promoter.

Example 3: C.glycerinogenes gene sequence information and homology analysis

The full-length nucleotide sequence of Candida glycerologenus of the present invention is shown in SEQ ID NO.1 for the detailed sequence, and the internal coding frame does not contain introns.

Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase gene sequence and its encoded protein were searched for nucleotide and protein homology with BLAST program and database, and it was found that Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase The enzyme gene has high homology with the glyceraldehyde 3-phosphate dehydrogenase gene of other yeasts at the protein level, and belongs to the same family protein.

Example 4: Structure and function of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida glycerogenes

The amino acids of the Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase gene were searched for the structural domain in the blocks database (http://www.blocks.fhcrc.org). The subcellular localization analysis of protein was carried out with online prediction software (http:/www.psort.nibb.ac.jp).

Example 5: Verification of the Expression Strength of the Glyceraldehyde 3-Phosphate Dehydrogenase Gene Promoter in Candida Glycerologenicum

See Figure 1 for the constructed recombinant expression vector and control vector. The specific construction process:

(1) PCR cloning of the Candida glycerol-producing GAP gene promoter P _CgGAP connected to the plasmid vector PYX212 (Regenberg, B., L.During-Olsen, MCKielland-Brandt and S.Holmberg, (1999) Substrate specificity and geneexpression of the amino-acid permeases in Saccharomyces cerevisiae.Curr.Genet.36: 317-328.) BamH I, Hind III, to obtain the carrier PYX212-P _CgGAP ;

(2) Synthesize the green fluorescent protein gene GFP according to the sequence announced by the plasmid vector pCAMBIA1302, and connect it to Hind III and Sac I of the carrier PYX212-P _CgGAP to obtain the carrier PYX212-P _CgGAP -GFP;

(3) The synthetic zeocin resistance gene according to the sequence announced by the plasmid vector pPGAPZb is connected to the EcoR I of the carrier PYX212- _PCgGAP -GFP to obtain the carrier PYX212-zeocin- _PCgGAP -GFP;

Embodiment 6: Transformation and screening of recombinant expression vector

Through the constructed recombinant expression vector PYX212-zeocin- _PCgGAP -GF, the recombinant plasmid was transformed into Saccharomyces cerevisiae by heat shock method, and the YEPD-resistant plate containing zeocin was coated. Pick a single colony from the YEPD-resistant plate, transfer to YEPD liquid medium supplemented with zeocin (150ug/mL), extract the plasmid, and verify the positive transformant.

Example 7: Yeast culture, induced expression and fluorescence analysis

Pick positive clones from the plate and inoculate in 10mL YEPD liquid medium containing 150ug/mL zeocin. After culturing at 30°C for 20 hours, the cells were collected by centrifugation and transferred into 10 mL of YEPD liquid medium. Cultivate at 30°C for 24 hours, and the rotation speed of the shaker is 200r/min.

Fluorescence analysis was performed using an Olympus fluorescence microscope. Green fluorescent protein is a very potential marker. Its endogenous fluorescent gene can efficiently emit clearly visible green light when excited by ultraviolet light or blue light and has stable fluorescence properties. Compared with existing markers, GFP has Incomparable advantages. Therefore, since gfp was discovered, it has been used as an ideal marker to monitor gene expression and protein localization in intact cells and tissues. The present invention uses green fluorescent protein as a marker to confirm the practicability and feasibility of the constructed integrated expression vector, and further verifies the expression intensity of the Candida glycerogenes 3-phosphate glyceraldehyde dehydrogenase gene promoter ( FIG. 2 ).

Claims (5)

1. a GAP gene promoter, is characterized in that deriving from product glycerin candida (Candida glycerinogenes) chromogene group; To produce glycerin candida chromogene group DNA as template, by adopting degenerated primer PCR and inverse PCR method to clone glyceraldehyde 3-phosphate dehydro-genase gene and promoter sequence thereof from produce glycerin candida genomic dna, the nucleotide sequence of described promotor is as shown in SEQ ID NO.1.

2. the carrier that contains promotor described in claim 1, is characterized in that, is expression vector, cloning vector or shuttle vectors.

3. promotor claimed in claim 1 is applied to yeast expression system.

4. promotor claimed in claim 1 is applied to the expression of regulatory gene.

5. promotor claimed in claim 1 is applied to the stably express of functional gene.