CN103173483B - Using Candida glycerolgenesis feature 5.8S sequences as the pURGAP vector constructions of integration site and its application - Google Patents
Using Candida glycerolgenesis feature 5.8S sequences as the pURGAP vector constructions of integration site and its application Download PDFInfo
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Abstract
Structure and its application the invention discloses a kind of Candida glycerolgenesis integration vector pURGAP that can be cloned with homologous recombination.Constructed pURGAP carriers include yeast Candida glycerinogenes by structure with 5.8S rDNA sequence SEQ NO.2, yeast Candida glycerinogenes feature 5.8S sequence SEQ NO.1 sites can be integrated in, constructed pURGAP carriers also include osmotic pressure promoter PCgGAP, zeocin resistance markers and Escherichia coli height copy all elements.Compared with other integration vectors, pURGAP is remarkably improved integration vector transformation efficiency, and exogenous protein expression intensity is remarkably reinforced using feature 5.8S rDNA sequences as integration site.Foreign gene or promoter effectively can be carried out integrant expression by constructed pURGAP in Candida glycerolgenesis, are the integration efficiencies for improving expression vector, accelerated a kind of important method of industrial strain Candida glycerolgenesis modified recombinant speed.
Description
Technical field
Industrial yeast is converted the present invention relates to a kind of integration vector containing expression system and by the integrating expression vector
One Candida glycerolgenesis of cell；It is related to protein expression genetic engineering field, especially with Candida glycerolgenesis conduct
The research field of cell factory expression alien gene belongs to microbiology, genetic engineering and biology field.
Background technology
Candida glycerolgenesis (Candida glycerolgenesis) is one plant of tool that China possesses independent intellectual property right
There is the industrial strain of excellent fermenting property, once obtain national invention second prize, it can be in the hypertonic of 55% glucose or 15%NaCl
Normal growth breeding on pressure culture medium thoroughly, has the characteristics of resistance to hypertonic, target product high yield, high conversion, big production intensity.
There are two significant properties, i.e. osmophilic strain (superelevation thick mash fermentation saves extraction energy consumption) and Portugal for C.glycerinogenes tools
Grape glycometabolism approach is complete, be one can genetic modification, the good application prospect having potential economized mode bacterial strain, to it
It carries out genetic improvement and approach is engineered just must be set up efficient genetic conversion system.
Although common laboratory yeast, the method for transformation such as Saccharomyces Cerevisiae in S .cerevisiae is very ripe,
These methods are undesirable for the genetic modification of industrial yeast strain, serious to hinder using genetic engineering means to producing glycerine vacation silk
Yeast metabolism approach carries out purposive transformation so as to improve the performance of Candida glycerolgenesis, the main reason is that the industry ferment
The genetic background complexity of mother strains is various.Therefore, this is just needed according to the characteristics of industrial yeast strain itself, such as cell wall knot
Structure, physio-biochemical characteristics, genetic background etc., it is established that corresponding transformation system.
Realization sets up corresponding transformation system this target and first has to obtain effective carrier, and builds saccharomyces integrated
One important process of carrier is to obtain integration site.To establish effective Candida glycerolgenesis conversion system, present invention choosing
Candida glycerolgenesis 5.8S rDNA characteristic sequences are selected as homologous recombination integration site, structure is suitable for producing glycerine vacation silk ferment
The integration vector of mother's conversion, establishes a set of efficient, simple Candida glycerolgenesis conversion system.
Invention content
For having no that Candida glycerolgenesis 5.8S rDNA sequences are applied in Candida glycerolgenesis expression vector at present
Present situation.The present invention is whole using the feature 5.8S rDNA sequence SEQ NO1 structure Candida glycerolgenesis of Candida glycerolgenesis
It closes expression vector as shown in Figure 1, obtains a kind of carrier that can significantly improve carrier transformation efficiency.
The present invention provides the recombination tables using C.glycerinogenes5.8S rDNA sequence SEQ NO1 as integration site
Up to the structure of carrier, the specific steps of integrating expression vector are built：
The clone of Candida glycerolgenesis part rDNA 1. (ribosome DNA, rDNA) sequence：Primer rdna1,
The sequence of rdna2 amplification Candida glycerolgenesis feature 5.8S rDNA, feature 5.8S rDNA are shown in SEQ NO1.
rdna1：ACGGAGCTCGAAACTCCGT CGTGC
rdna2：ACGAAGCTTTGCTTAAGTTCAGCGG；Sequence is shown in SEQ NO1.
2. structure (pUC18 is as basic framework) such as attached drawing 2 of integration vector；
(1) the PCR feature 5.8S rDNA obtained are connected to through Sac I and Hind III on pUC18, obtain cloning vector
pUC-5.8S rDNA；
(2) the promoter P for obtaining PCRCgGAPUsing connecting pUC- after restriction enzyme Sac II and Nco I digestions
5.8SrDNA obtains carrier pUC-5.8S rDNA-PCgGAP；
(3) the zeocin resistant genes for obtaining PCR after restriction enzyme Sal I and sph I digestions using being connected to
Carrier pUC-5.8S rDNA-PCgGAPTo get to carrier pURGAP；
3. Candida glycerolgenesis is converted with the Homologous integration carrier of linearisation, with positive gram of zeocin resistant panels screening
Longzi, and detect conversion positive clone molecule with PCR.
4. synergy is analyzed.The positive transformants daughter colony of confirmation is counted, calculates integration efficiency.With it is other homologous
Integration vector is compared, and is improved with the integration vector pURGAP integration efficiencies of the feature 5.8S rDNA sequence constructs shown in SEQ NO2
To the 300% of other methods.
5. the integration vector of structure is applied to expression of the foreign gene in Candida glycerolgenesis by the present invention, expression carries
Body pURGAP can efficient expression alien gene, the acquisition of high expression recombinant bacterial strain can be accelerated.
By implementing the specific technical solution of the present invention, following advantageous effect is realized：Feature 5.8S is contained by structure
The integration vector pURGAP of rDNA sequence SEQ NO2 improves the integration efficiency of expression vector, compared with other Homologous integration carriers
It is improved with the integration vector pURGAP integration efficiencies of the feature 5.8S rDNA sequence constructs shown in SEQ NO2 to other methods
300%, and foreign gene is realized in C.glycerolgenesis successful expressions, for important industrial strain is further transformed
Candida glycerolgenesis provide a kind of effective method.
Specific embodiment
Following embodiment can make the technical staff of this professional skill field more fully understand the present invention, but the present invention
It is not limited to following embodiments.
Strain used in the present invention：Candida glycerolgenesis Candida glycerolgenesis CCTCC M
93018、E.coliDH5a。
Embodiment 1：PCR amplification Candida glycerolgenesis 5.8S rDNA sequences
Candida glycerolgenesis 5.8S sequences are expanded with primer rdna1, rdna2：
rdna1：ACGGAGCTCGAAACTCCGT CGTGC
rdna2：ACGAAGCTTTGCTTAAGTTCAGCGG；Sequence is shown in SEQ NO1.
Embodiment 2：The structure of recombination and integration expression vector pURGAP
The recombinant expression carrier (pUC18 is as basic framework) of structure is referring to attached drawing 2：
(1) the PCR feature 5.8S rDNA obtained are connected to through Sac I and Hind III on pUC18, obtain cloning vector
pUC-5.8S rDNA；
(2) the promoter P for obtaining PCRCgGAPUsing connecting pUC- after restriction enzyme Sac II and Nco I digestions
5.8SrDNA obtains carrier pUC-5.8S rDNA-PCgGAP；
(3) the zeocin resistant genes for obtaining PCR after restriction enzyme Sal I and sph I digestions using being connected to
Carrier pUC-5.8S rDNA-PCgGAPTo get to carrier pURGAP；
Embodiment 3：The conversion and screening of recombinant expression carrier
Matter is expanded by the recombinant expression carrier pURGAP recombinant plasmids thermal shock method Transformed E .coli DH5a of structure
Grain.Plasmid is extracted, is linearized with HindIII.C.glycerinogenes is converted using protoplast transformation, with 0.7M's
NaCl is coated with the YEPD resistances regenerated plate containing zeocin as homeo-osmosis agent (1M sorbierites are regeneration stabilizer).
The positive transformants daughter colony of conversion is counted, takes the average value repeated three times, the transformation efficiency of pURGAP can reach 3 ×
102Cell/ng plasmids, and the integration of other feature 5.8S rDNA sequence constructs with shown in non-SEQ NO2 carries under similarity condition
Body transformation efficiency can only achieve 1 × 102Cell/ng plasmids, compared with other Homologous integration carriers with SEQ NO3 shown in feature
The integration vector integration efficiency of 5.8S rDNA sequence constructs is improved to the 300% of other methods.
Embodiment 4：Induction external source fluorescin is expressed in Candida glycerolgenesis
Fluorescence protein expression carrier pURGAP-gfp is built using fluorogene gfp, building process is referring to attached drawing 3.Institute's structure
It builds carrier pURGAP-gfp and is transferred to host Candida glycerolgenesis CCTCC M 93018, from containing 150ug/mL
The YEPD of zeocin picking positive colonies from tablet determine positive transformant using PCR, and positive transformant is inoculated in 10mL and contains
Have in the YEPD fluid nutrient mediums of 150ug/mL zeocin.30 DEG C of culture 20h, thalline were collected by centrifugation, transfers and contains 25% into 10mL
In the YEPD fluid nutrient mediums of glucose.30 DEG C are cultivated for 24 hours, shaking table revolution 200r/min.Thalline is obtained after centrifugation.
Fluorescence analysis is carried out to the thalline obtained using Olympus fluorescence microscopes.It can see green fluorescent protein
It is expressed in the height of host Candida glycerolgenesis CCTCC M 93018, compared with other Homologous integration carriers, with
The integration vector fluorescent protein expression intensity enhancing of feature 5.8S rDNA sequence constructs shown in SEQ NO2 is apparent (Fig. 4).
Claims (5)
1. a kind of recombinant expression carrier pURGAP, feature exist, the carrier pURGAP can be expressed to be a kind of using homologous recombination
The Candida glycerolgenesis integration vector of foreign gene, the carrier that sets out of the carrier pURGAP is pUC18, the carrier
PURGAP includes rDNA, osmotic pressure promoter PCgGAP, zeocin resistance markers, the sequence of the rDNA as shown in SEQ NO 1,
The carrier structure is as shown in Figure 1.
2. recombinant expression carrier pURGAP as described in claim 1, with feature 5.8S rDNA sequence SEQ NO 1 to integrate
Site, expanding primer rdna1, rdna2 used in 5.8S rDNA sequences is：
rdna1：ACGGAGCTCGAAACTCCGT CGTGC；
rdna2：ACGAAGCTTTGCTTAAGTTCAGCGG；
Using feature 5.8S rDNA sequence SEQ NO 2 as Homologous integration arm, transformation efficiency and the expression of integration vector can be improved
Intensity.
3. recombinant expression carrier pURGAP as described in claim 1, which is characterized in that the carrier pURGAP includes infiltration
Press promoter PCgGAP, zeocin resistance markers and Escherichia coli height copy all elements.
4. recombinant expression carrier pURGAP described in claim 1 is applied to the osmotic pressure regulation and control foreign gene of Candida glycerolgenesis
Expression, the screening and identification stablized expression or be overexpressed functional gene of functional gene.
It is engineered that 5. recombinant expression carrier pURGAP described in claim 1 is applied to Candida glycerolgenesis hypermetabolism.
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| CN107858361B (en) * | 2017-12-12 | 2020-02-18 | 江南大学 | Candida glycerinogenes heat shock protein gene CgHsp10 and application thereof |
| CN110656056B (en) * | 2019-10-31 | 2021-07-27 | 江南大学 | Construction method of pinene-producing engineering bacteria with high-concentration pinene tolerance |
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Non-Patent Citations (3)
| Title |
|---|
| Cheng Zhang 等.Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002‑5.《J Ind Microbiol Biotechnol》.2014,第42卷113–124. * |
| 产甘油假丝酵母产甘油关键酶基因的克隆、测序及植物表达载体的构建;赵有玺 等;《江西农业大学学报》;20051231;第27卷(第4期);491-495 * |
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