CN117757813A - Penicillium digitatum transcription factor PDIDSM_85260 and application thereof - Google Patents

Abstract

The invention relates to a finger penicillium transcription factor PDIDSM_85260 and application thereof. The nucleotide sequence of the finger-shaped penicillium transcription factor PDIDSM-85260 is shown as SEQ ID No.1, and the amino acid sequence of the protein encoded by the finger-shaped penicillium transcription factor PDIDSM-85260 gene is shown as SEQ ID No. 2. The invention utilizes PDIDSM 85260 gene over-expression strain to biologically transform and obtain the alpha-terpineol with the yield of 1870-2798 mg L ^‑1 The method is reported to have higher yield in the process of preparing the alpha-terpineol by directly utilizing microorganisms to convert at present, and solves the problem of low yield in the process of producing the alpha-terpineol by biological conversion of limonene.

Description

Penicillium digitatum transcription factor PDIDSM_85260 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a finger penicillium transcription factor PDIDSM_85260 and application thereof.

Background

The spice essence is an important auxiliary material in the industries of food, daily chemicals, medicines, tobacco, agriculture and the like, and is closely related to the aspects of improving the living standard of people, developing related industries, improving society, promoting internal needs, consuming and the like. Thus, fragrances and perfumes are an integral part of the human population. With the enhancement of health consciousness of people, natural flavors are increasingly widely used. Fragrances produced by bioconversion of microorganisms have been considered natural, which can convert limonene, a byproduct of citrus processing, to produce fragrances of higher aroma, such as alpha-terpineol, for higher application value. However, at present, the research on key genes/enzymes in the process of producing alpha-terpineol by biological conversion of limonene at home and abroad remains in a presumption stage, the related research is very few, the conversion mechanism is still not clear, the components of a conversion product are complex, the yield is low, and the industrial application is still limited to a certain extent. Therefore, the research on the molecular mechanism of producing alpha-terpineol by converting limonene through microorganisms has important promotion effect on the development of essence and perfume and industrial production.

The invention application publication No. CN104630284A discloses a method for producing natural perfume alpha-terpineol by a microbiological method, which converts limonene into alpha-terpineol by a microbiological fermentation method, but the yield of the alpha-terpineol is lower.

Disclosure of Invention

The invention aims to provide a finger-shaped penicillium transcription factor PDIDSM_85260 so as to solve the technical problem that the yield of alpha-terpineol is low when the alpha-terpineol is produced by limonene conversion.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a finger penicillium transcription factor PDIDSM_85260 has a nucleotide sequence shown in SEQ ID No. 1.

Use of the penicillium digitatum transcription factor pdidsm_85260 for promoting the microbial conversion process of limonene, thereby increasing the yield of alpha-terpineol.

Further, the overexpression vector of the PDMSM 85260 gene is transformed into a host strain, and the penicillium digitatum recombinant strain is constructed by using a host cell-mediated method.

Further, the host strain is agrobacterium tumefaciens.

Further, the preparation method of the penicillium digitatum recombinant strain comprises the following steps:

step 1, constructing an overexpression vector of a PDIDSM_85260 gene;

step 2, converting an overexpression vector of the PDIDSM_85260 gene into a host strain to obtain a host strain engineering bacterium;

and step 3, transforming the penicillium digitatum by utilizing the engineering bacteria of the host strain in the step 2, screening to obtain a positive transformant, and detecting the strain with the rising PDIDSM_85260 gene expression quantity to obtain the over-expression strain.

Further, the preparation method of the positive transformant in the step 3 comprises the following steps:

step 1, adding penicillium digitatum mycelium into host strain engineering bacterial liquid for co-culture to obtain co-cultured mycelium blocks;

step 2, spreading the co-cultured mycelium blocks in a flat plate containing hygromycin and cephalosporin, and culturing in an incubator until obvious extension of the mycelium begins to appear to obtain a resistant colony;

and 3, performing expansion culture on the resistant colonies.

Further, the preparation method of the pdidsm_85260 gene overexpression vector comprises the following steps:

step 1, amplifying a penicillium GPDA promoter gene fragment by using penicillium digitatum DSM 62840DNA as a template and using PdGPDAF and PdGPDAR as primers; the nucleotide sequence of the PdGPDA F is shown as SEQ ID No.3, and the nucleotide sequence of the PdGPDAR is shown as SEQ ID No. 4;

step 2, amplifying a transcription factor PDIDSM_85260 gene fragment by using penicillium digitatum DSM 62840DNA as a template and GPDA-85260F and GPDA-85260R as primers; the nucleotide sequence of the GPDA-85260F is shown as SEQ ID No.5, and the nucleotide sequence of the GPDA-85260R is shown as SEQ ID No. 6;

and 3, connecting the penicillium GPDA promoter gene fragment in the step 1 and the transcription factor PDIDSM_85260 gene fragment in the step 2 with a linearization vector pCAMBIA1303-TrpC-Hygro to form an over-expression vector of the transcription factor PDIDSM_85260 gene.

The invention has the beneficial effects that:

the invention utilizes PDIDSM 85260 gene over-expression strain to biologically transform and obtain the alpha-terpineol with the yield of 1870-2798 mg L ^-1 The method is reported to have higher yield in the process of preparing the alpha-terpineol by directly utilizing microorganisms to convert at present, and solves the problem of low yield in the process of producing the alpha-terpineol by biological conversion of limonene.

Compared with the existing method, the method effectively reduces the transformation difficulty, has higher genetic stability, does not need to carry out additional stability analysis, and shortens the time for obtaining positive transformants to a great extent.

The plasmid used in the agrobacterium tumefaciens mediation is a pCAMBIA1303-TrpC-Hygro binary shuttle vector, the plasmid has hygromycin resistance genes, and hygromycin can be directly added into a PDA culture medium to screen target transformants.

The positive transformant obtained by co-culturing the agrobacterium tumefaciens containing the over-expression vector and the penicillium digitatum DSM 62840 and screening a resistance culture medium containing hygromycin has higher stability. The existing method needs to be carried out for 3-5 times in a plate without antibiotics and then cultured in a resistance plate to obtain the transformant, and the method does not need to carry out the process, thereby reducing the time for obtaining the transformant.

The invention utilizes transcriptome technology to analyze differential genes in different periods of limonene transformation, clones PDIDSM_85260 genes from penicillium digitatum DSM 62840, constructs an over-expression vector containing PDIDSM_85260 genes, transforms the PDIDSM_85260 gene over-expression vector into penicillium digitatum DSM 62840 by an agrobacterium-mediated genetic transformation method, detects the integration condition of the gene PDIDSM_85260 by PCR, verifies the expression quantity of the gene PDIDSM_85260 by fluorescent quantitative PCR to obtain an over-expression strain, and measures the alpha-terpineol yield by a solid phase microextraction-gas chromatography-mass spectrometry combined instrument (SPME-GC-MS), thus showing that the obtained over-expression strain can obviously promote the process of producing alpha-terpineol by microbial transformation of limonene.

The invention utilizes transcriptome technology to analyze the difference genes of different periods of limonene conversion, clones the transcription factors capable of regulating and controlling the limonene conversion process from the penicillium digitatum DSM 62840, and utilizes genetic engineering means to promote the limonene microbial conversion process, thereby improving the yield of alpha-terpineol and having important guiding significance for the large-scale production of the alpha-terpineol.

Drawings

FIG. 1A is an agarose gel electrophoresis of a PCR product (lane 1: amplified product of penicillium GPDA promoter fragment; lane 2: trans2kplus IIDNAmaror);

FIG. 1B is a agarose gel electrophoresis chart of PCR products (lane 1: PDIDSM_85260 amplification product; lane 2: trans2kplus IIDNAmaroker);

FIG. 1C is a colony PCR agarose gel electrophoresis (lanes 1-4:

the pCAMBIA1303-TrpC-Hygro-PdGPD-85260-Nos plasmid; lane 5: trans2 kplug dnamarker;

FIG. 1D is a diagram showing the construction of the overexpression vector pCAMBIA 1303-TrpC-Hygro-PdGPD-85260-Nos;

FIG. 2A shows the results of positive transformant verification (lane 1: overexpressing transformant; lane 2: P.digitalis wild type; lane 3: marker);

FIG. 2B shows the results of fluorescence quantitative analysis (WT: wild-type strain; OE: overexpressed strain; p <0.05, < p <0.01 compared to wild-type strain);

FIG. 3 shows the microbial transformation of limonene according to example 1 (WT: wild-type strain; OE: overexpressing strain; p <0.01 compared to wild-type strain);

FIG. 4 is a graph showing the yield of alpha-terpineol.

Detailed Description

The invention will be further described with reference to examples of embodiments of the invention and the accompanying drawings.

Example 1

Step one, construction of a PDIDSM 85260 gene-containing overexpression vector

1. Genomic DNA extraction

Penicillium digitatum DSM 62840DNA was extracted with reference to the tissue genomic DNA extraction kit instructions.

2. Amplification of target Gene

The penicillium digitatum DSM 62840DNA is used as a template, and the PdGPDAF and the PdGPDAR are used as primers to carry out PCR amplification to obtain a penicillium GPDA promoter gene segment, wherein the PCR amplification reaction is as follows: 98℃30s,98℃10s,57 ℃ (-1 ℃/cycle) 20s,72℃30s (5 cycle); 98℃for 10s,60℃for 20s,72℃for 30s (30 cycles), 72℃for 5min; and 1min at 12 ℃. The nucleotide sequence of the PdGPDAF is shown as SEQ ID No.3, and the nucleotide sequence of the PdGPDAR is shown as SEQ ID No. 4.

The transcription factor PDIDSM_85260 gene fragment is obtained by PCR amplification by taking the finger-shaped penicillium DSM 62840DNA as a template and GPDA-85260F and GPDA-85260R as primers, and the PCR amplification conditions are as follows: 98 ℃ for 30s;98℃for 10s,54 ℃ (-1 ℃/cycle) for 20s,72℃for 60s (5 cycle); 98℃10s,56℃20s,72℃60s (30 cycles); 72 ℃ for 5min; and 1min at 12 ℃. The nucleotide sequence of GPDA-85260F is shown as SEQ ID No.5, and the nucleotide sequence of GPDA-85260R is shown as SEQ ID No. 6.

After obtaining the gene fragment of the penicillium GPDA promoter and the gene fragment of the transcription factor PDIDSM_85260, agarose gel electrophoresis with the concentration of 1.5% is carried out, and the target fragment is recovered by a recovery kit after gel cutting. The PCR primer sequences are shown in Table 1.

TABLE 1PCR primer sequences

3. Linearized support preparation

The pCAMBIA1303-TrpC-Hygro vector backbone was digested with KpnI and SacI, and the digested system was reacted at 37℃for 1 hour and inactivated at 80℃for 10 minutes to give a linearized vector pCAMBIA1303-TrpC-Hygro, the digested system being shown in Table 2.

TABLE 2 enzyme digestion system

4. Gibson assembly

mu.L of plasmid pCAMBIA1303-TrpC-Hygro linearization fragment, penicillium GPDA promoter gene fragment and transcription factor PDIDSM 85260 gene fragment each 0.5. Mu.L and 5. Mu.L GibsonAssembly Master Mix (2X) were taken and ddH was added ₂ O is added to 10 mu L, and the mixture is reacted for 30min at 50 ℃ to obtain the overexpression vector of the PDIDSM-85260 gene.

5. Recombinant product conversion

The 10. Mu.LPDIDSM_ 85260 gene over-expression vector was added to 100. Mu.L of freshly thawed E.coli Trans1-T1 competent cells and gently mixed, heat-shocked at 42℃for 45s after 30min on ice, placed on ice for 2min, added to LB liquid medium, cultured at 37℃for 1h at 200rpm, centrifuged at 5000rpm for 5min, the supernatant was discarded, the cells were resuspended in the remaining medium, and the cells were incubated with a sterile coating bar containing kanamycin (50. Mu.g mL ^-1 ) The colony is obtained by lightly coating the colony on a flat plate of the strain, and inversely culturing the colony in an incubator at 37 ℃.

6. Recombinant product identification

mu.L of GPDA-85260F primer, 1. Mu.L of LYZ R primer, and 1. Mu.L of monoclonal colony on the recombinant reaction conversion plate were taken, 10. Mu.L2X Taq Plus Master Mix II (Dye Plus) and 7. Mu.L ddH ₂ O was identified by PCR. After the PCR product was obtained, agarose gel electrophoresis was performed at a concentration of 1.5%, and the target fragment was cut and recovered with a recovery kit. The nucleotide sequence of YZ R is shown as SEQ ID No. 11.

The PCR amplification reaction is as follows: 3min at 95 ℃;95℃20s,57℃20s,72℃2min,35cycle;72 ℃ for 5min; and 1min at 12 ℃.

Step two, agrobacterium-mediated pdidsm_85260 gene overexpression vector genetic transformation of penicillium digitatum DSM 62840 to obtain overexpression strain

1. Obtaining of overexpression vector agrobacterium tumefaciens engineering bacteria containing PDIDSM 85260 gene

(1) Taking the Agrobacterium tumefaciens AGL1 stored at the temperature of minus 80 ℃ and inserting the Agrobacterium tumefaciens AGL1 into ice when part of the Agrobacterium tumefaciens AGL is melted at room temperature and is in an ice water mixing state.

(2) 100 mu L of Agrobacterium tumefaciens AGL1 competent cells are respectively added with 0.1 mu g of PDIDSM-85260 gene overexpression vector plasmid DNA, the mixture is stirred by hands at the bottom of a tube and uniformly mixed, and then the mixture is placed on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min.

(3) 700. Mu.L of LB liquid medium without antibiotics was added and cultured with shaking at 28℃for 2-3 hours.

(4) Collecting bacteria by centrifuging at 6000rpm for 1min, collecting 100 μl supernatant, gently blowing, and coating the re-suspended bacteria mass on antibiotic-containing material (50 μg mL) ^-1 Kanamycin, 20 μg mL ^-1 Rifampicin) was placed upside down on LB plate and cultured in an incubator at 30℃for 2-3d.

(5) Positive clones were picked up to 10mL LB (50. Mu.g mL) ^-1 Kanamycin, 20 μg mL ^-1 Rifampicin) medium, shake culture at 30℃to OD ₆₀₀ The cells were collected by centrifugation at 0.6, and 10mL of PDB (acetosyringone 20mg L) ^-1 ，50μg mL ^-1 Kanamycin) was resuspended.

2. Agrobacterium tumefaciens-mediated PDIDSM 85260 gene conversion Penicillium digitatum DSM 62840

(1) Punching the penicillium digitatum and inoculating the penicillium digitatum into a PDA culture medium, and culturing in a 28 ℃ incubator until penicillium digitatum hyphae cover the whole plate;

(2) Fresh Penicillium digitatum mycelia were obtained by punching, and added to the fresh activated Agrobacterium tumefaciens AGL1 broth, and 0.1% Silwet-L77 was added to the medium to increase infection efficiency, and co-cultured at 25℃for 24 hours.

(3) The co-cultured mycelium blocks are spread until the mycelium blocks contain 100 mug mL after the residual agrobacterium tumefaciens is sucked on sterile filter paper ^-1 Hygromycin, 300 μg mL ^-1 In PDA plates of cephalosporin, an incubator at 28 ℃ is incubated until the hyphae begin to develop apparent extension.

(4) Transfer of resistant colonies to fresh containing 100. Mu.g mL ^-1 Hygromycin, 300 μg mL ^-1 The positive transformants were obtained by expansion culture on PDA plates of cephalosporin.

3. T-DNA integration verification

(1) And (3) taking part of resistance positive transformant hyphae, grinding the mycelia with liquid nitrogen, and extracting the genome DNA according to the operation instruction of the tissue genome DNA small-scale extraction kit.

(2) PCR amplification

mu.L of primer Hyg F, 1. Mu.L of primer Hyg R, 1. Mu.L of positive transformant genomic DNA, 10. Mu.L of 2X Taq Plus Master Mix II (Dye Plus) and 7. Mu.L of ddH were taken ₂ O was identified by PCR. After the PCR product was obtained, agarose gel electrophoresis was performed at a concentration of 1.5%, and the target fragment was cut and recovered with a recovery kit. The nucleotide sequence of Hyg F is shown as SEQ ID No.9, and the nucleotide sequence of Hyg R is shown as SEQ ID No. 10.

The PCR amplification reaction is as follows: 3min at 95 ℃;95℃20s,55℃20s,72℃60s,35 cycles; 72 ℃ for 5min; and 1min at 12 ℃.

4. Real-time fluorescent quantitative PCR detection

(1) The total RNA of the transformant was extracted by Trizol method.

(2) Reverse transcription into cDNA

Genomic DNA removal: mu.g of transformant RNA and 4. Mu.L of 4 XgDNAwiter Mix were taken and ddH was added ₂ O was added to 16. Mu.L, and the reaction was carried out at 42℃for 2min.

Reverse transcription reaction: to the above reaction mixture was directly added 4. Mu.L of 5X 4HiScript III RT SuperMix.

(3) Fluorescent quantitative PCR detection

mu.L 85260Forward, 1. Mu.L 85260Reverse, 1. Mu.L transformant cDNA, 22.5. Mu. L SYBR Green Master Mix, add ddH were taken ₂ O is added to 45 mu L, and fluorescent quantitative PCR detection is carried out to obtain the over-expression strain. GPDAForward and GPDAREVERSE were used as reference genes. The nucleotide sequence of 85260Forward is shown as SEQ ID No.7, and the nucleotide sequence of 85260Reverse is shown as SEQ ID No. 8. The nucleotide sequence of GPDAForward is shown as SEQ ID No.12, and the nucleotide sequence of GPDAREVERSE is shown as SEQ ID No. 13.

The PCR amplification procedure was: 50 ℃ for 2min;95℃for 5min,95℃for 15s,56℃for 20s,72℃for 40s,40 cycles.

Step three, a limonene microbial transformation experiment of a transcription factor PDIDSM_85260 gene overexpression strain

Preparation of spore suspension by 1-2 weeks of culture of wild strain and pdidsm_85260 gene overexpression strain slant, and concentration of spore suspension is adjusted to 1.5X10 ⁷ spores mL ^-1 . 1mL of the spore suspension was inoculated into 100 mM YB medium, and cultured at 24℃with 150r/min shaking for 48 hours. Then 840mg/L limonene (0.22 μm filter sterilization) was added for microbial transformation experiments. After 12h of conversion, the yield of the product α -terpineol was determined using SPME-GC-MS. The yield of the transformation product alpha-terpineol was 100% based on the wild type strain. FIG. 3 shows the result of overexpression of the PDIDSM 85260 gene in Penicillium digitatum DSM 62840, which positively regulates the transformation of limonene microorganisms to produce alpha terpineol.

The nucleotide sequence of the finger penicillium transcription factor PDIDSM-85260 is shown as SEQ ID No.1, and the amino acid sequence of the protein encoded by the finger penicillium transcription factor PDIDSM-85260 gene is shown as SEQ ID No. 2.

Claims (7)

1. The finger penicillium transcription factor PDIDSM-85260 is characterized in that the nucleotide sequence of the finger penicillium transcription factor PDIDSM-85260 is shown as SEQ ID No. 1.

2. Use of the penicillium digitatum transcription factor pdidsm_85260 as claimed in claim 1 for promoting a microbial conversion process of limonene, thereby increasing the yield of α -terpineol.

3. The use of the finger penicillium transcription factor pdidsm_85260 according to claim 2, comprising the steps of: the over-expression vector of the PDMSM 85260 gene is transformed into a host strain, and the host cell mediated method is utilized to construct the penicillium digitatum recombinant strain.

4. The use of the Penicillium digitatum transcription factor PDIDSM_85260 according to claim 3, wherein the host strain is Agrobacterium tumefaciens.

5. The use of the finger penicillium transcription factor pdidsm_85260 as claimed in claim 3, wherein the preparation method of the finger penicillium recombinant strain comprises the following steps:

step 1, constructing an overexpression vector of a PDIDSM_85260 gene;

step 2, converting an overexpression vector of the PDIDSM_85260 gene into a host strain to obtain a host strain engineering bacterium;

and step 3, transforming the penicillium digitatum by utilizing the engineering bacteria of the host strain in the step 2, screening to obtain a positive transformant, and detecting the strain with the rising PDIDSM_85260 gene expression quantity to obtain the over-expression strain.

6. The use of the finger penicillium transcription factor pdidsm_85260 according to claim 5, wherein the positive transformant in step 3 is prepared by the following method:

step 1, adding penicillium digitatum mycelium into host strain engineering bacterial liquid for co-culture to obtain co-cultured mycelium blocks;

step 2, spreading the co-cultured mycelium blocks into a flat plate containing hygromycin and cephalosporin, and culturing in an incubator until obvious extension of the mycelium begins to appear to obtain a resistant colony;

and 3, performing expansion culture on the resistant colonies.

7. The use of the finger penicillium transcription factor pdidsm_85260 as claimed in claim 5, wherein the preparation method of the overexpression vector of the pdidsm_85260 gene comprises the following steps:

step 1, amplifying a penicillium GPDA promoter gene fragment by using penicillium digitatum DSM 62840DNA as a template and using PdGPDAF and PdGPDAR as primers; the nucleotide sequence of the PdGPDA F is shown as SEQ ID No.3, and the nucleotide sequence of the PdGPDAR is shown as SEQ ID No. 4;

step 2, amplifying a transcription factor PDIDSM_85260 gene fragment by using penicillium digitatum DSM 62840DNA as a template and GPDA-85260F and GPDA-85260R as primers; the nucleotide sequence of the GPDA-85260F is shown as SEQ ID No.5, and the nucleotide sequence of the GPDA-85260R is shown as SEQ ID No. 6;

and 3, connecting the penicillium GPDA promoter gene fragment in the step 1 and the transcription factor PDIDSM_85260 gene fragment in the step 2 with a linearization vector pCAMBIA1303-TrpC-Hygro to form an over-expression vector of the transcription factor PDIDSM_85260 gene.