CN101302535A - A method for constructing a plasmid expressing xylulokinase gene - Google Patents

Abstract

A method for constructing a plasmid expressing xylulokinase gene, which relates to a method for constructing a plasmid vector. The method solves the problems that the xylose utilization rate of Saccharomyces cerevisiae modified by metabolic engineering is still low and the production of xylitol, a by-product of fermentation, is high. Construction method: first clone the KanR gene, XKS1 gene, ADH1 terminator fragment, and 2.2kb rDNA fragment in Saccharomyces cerevisiae; then recover and purify the gene fragments and connect them to the pGEMT Easy vector; A 2.2kb rDNA fragment was added to plasmid pRKT. The vector constructed by the method of the present invention can integrate a high copy of the XKS1 gene into the chromosomal genome of Saccharomyces cerevisiae, realize the over-stable expression of XKS1, dredge the xylose metabolic flow of Saccharomyces cerevisiae using xylose to produce ethanol, and improve the production of xylose Utilization rate reduces the yield of by-product xylitol.

Description

A method for constructing a plasmid expressing xylulokinase gene

technical field

The invention relates to a method for constructing a plasmid vector.

Background technique

Saccharomyces cerevisiae does not have the ability to metabolize xylose, so it cannot ferment plant fiber hydrolyzate to produce ethanol. At present, xylose metabolism pathways—xylose reductase-xylitol dehydrogenase (XR-XDH) pathway or xylose isomerase (XI) pathway—are artificially established in Saccharomyces cerevisiae by means of metabolic engineering. However, the utilization rate of xylose in Saccharomyces cerevisiae after metabolic engineering is still very low, and the production of xylitol, a by-product of fermentation, is high, which makes it difficult to increase the yield of ethanol.

Contents of the invention

The purpose of the present invention is to provide a plasmid expressing xylulokinase gene in order to solve the problem that the xylose utilization rate of Saccharomyces cerevisiae after metabolic engineering is still very low and the fermentation by-product xylitol production is high The construction method.

The construction method of the plasmid expressing the xylulokinase gene is carried out according to the following steps: 1. clone the KanR gene in the plasmid pKT0150; 2. clone the XKS1 gene of Saccharomyces cerevisiae; 3. clone the ADH1 terminator fragment in the plasmid pKT0150; 2.2kb rDNA fragment; 5. Recover and purify the gene fragments prepared in steps 1 to 4, and then ligate them with pGEMT Easy vector at 16°C for 10-12 hours to obtain pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and pGMT -rDNA; 6. Construct plasmid pR, and use Saccharomyces cerevisiae integration carrier p406ADH1 as the backbone to introduce the gene KanR with resistance marker; 7. Construct plasmid pRK, add XKS1 gene to plasmid pR; 8. Construct plasmid pRKT, insert ADH1 terminator fragment Add the plasmid pRK; 9. Add the 2.2kb rDNA fragment to the plasmid pRKT to obtain a plasmid expressing the xylulokinase gene; wherein the sequence of the upstream primer amplified in step 1 is 5'-TTCCATATGTCTGTTTAGCTTGCCTC-3', and the sequence of the downstream primer is 5' -CTTGACGTCTATCATCGATGAATTCGA-3'; the sequence of the upstream primer for amplification in step 2 is 5'-CGGACTAGTCTCAATCTTCAGCAAGCGAC-3', and the sequence of the downstream primer is 5'-CGCGTCGACAACGGGGAACAAAATGATG-3'; the sequence of the upstream primer for amplification in step 3 is 5'-CGCGTCGACATTTGTTACTGCTGCTGGTATT-3 ', the sequence of the downstream primer is 5'-CTCGGCCGCCCTGTTATTCCCTAGCGG-3'; the sequence of the upstream primer for amplification in step 4 is 5'-TTCCATATGGGAACCTCTAATCATTCGCT-3', and the sequence of the downstream primer is 5'-TCTCGGCCGAACGAACGAGACCTTAACCT-3'.

Xylulokinase (xylulokinase, XK) can catalyze the phosphorylation of xylulose to form xylulose 5-phosphate, which is one of the rate-limiting steps of xylose metabolism and is at the node of xylose metabolites entering the pentose phosphate pathway (PPP) Location. Although Saccharomyces cerevisiae has a downstream enzyme system of xylulose metabolism, its low activity limits the flow of xylose metabolism to the pentose phosphate pathway (PPP), resulting in the metabolic engineering of Saccharomyces cerevisiae. Low, the production of xylitol, a by-product of fermentation, is high, and the yield of ethanol is still low.

The endogenous overexpression of xylulokinase gene XKS1 in Saccharomyces cerevisiae can accelerate the xylose metabolic flux and improve the xylose utilization rate and ethanol yield of Saccharomyces cerevisiae. The method of the present invention constructs a multi-copy integrated expression vector carrying xylulokinase gene XKS1 and is suitable for industrial strains of Saccharomyces cerevisiae, and the vector can integrate a high copy of overexpressed xylulokinase gene XKS1 into the chromosome of Saccharomyces cerevisiae On the genome, the stable overexpression of xylulokinase XKS1 has been realized, the xylose metabolic flow of Saccharomyces cerevisiae using xylose to produce ethanol has been dredged, the utilization rate of xylose has been improved, and the by-product xylitol production has been reduced. The plasmid constructed by the method of the present invention is transferred into Saccharomyces cerevisiae, and the comparative fermentation test proves that the ethanol yield of Saccharomyces cerevisiae transferred to the plasmid constructed by the method of the present invention is more than 30% higher than the ethanol yield of Saccharomyces cerevisiae after metabolic engineering transformation, indicating that After the plasmid constructed by the method of the invention is transferred into Saccharomyces cerevisiae, the restriction on the xylose conversion ethanol metabolic pathway can be eliminated, and the activity of the downstream enzyme system can be improved.

Detailed ways

Specific embodiment 1: The construction method of the plasmid expressing the xylulokinase gene in this embodiment is carried out according to the following steps: 1. Cloning the KanR gene in the plasmid pKT0150; 2. Cloning the XKS1 gene of Saccharomyces cerevisiae; 3. Cloning the ADH1 terminator in the plasmid pKT0150 Fragment; 4. Clone the 2.2kbr DNA fragment in Saccharomyces cerevisiae; 5. Recover and purify the gene fragments prepared in steps 1 to 4, and then connect them to the pGEMTEasy vector at 16°C for 10-12 hours to obtain pGMT-KanR and pGMT-XKS1 , pGMT-ADH1T and pGMT-rDNA; 6. Construct plasmid pR, and introduce the gene KanR with resistance marker with Saccharomyces cerevisiae integration vector p406ADH1 as the backbone; 7. Construct plasmid pRK, add XKS1 gene to plasmid pR; 8. Construct plasmid pRKT , adding the ADH1 terminator fragment to the plasmid pRK;

9. Add the 2.2kb rDNA fragment to the plasmid pRKT to obtain a plasmid expressing the xylulokinase gene;

The upstream primer sequence amplified in step 1 is 5'-TTC CATATG TCTGTTTAGCTTGCCTC-3', the downstream primer sequence is 5'-CTT GACGTC TATCATCGATGAATTCGA-3'; the upstream primer sequence amplified in step 2 is 5'-CGG ACTAGT CTCAATCTTCAGCAAGCGAC- 3', the sequence of the downstream primer is 5'-CGC GTCGAC AACGGGGAACAAAATGATG-3'; the sequence of the upstream primer for amplification in step 3 is 5'-CGC GTCGAC ATTTGTTACTGCTGCTGGTATT-3', and the sequence of the downstream primer is 5'-TCT CGGCCG CCCTGTTATTCCCTAGCGG-3'; In Step 4, the sequence of the upstream primer for amplification is 5'-TTC CATATG GGAACCTCTAATCATTCGCT-3', and the sequence of the downstream primer is 5'-TCT CGGCCG AACGAACGAGACCTTAACCT-3'.

Step 5 of this embodiment uses the DNA Gel Extraction Kit (Gel Extraction Mini Kit) produced by Shanghai Huashun Biological Engineering Co., Ltd. to recover. The four sets of ligation systems in Step 5 are all 10 μL, and they all consist of 1 μL of purified gene fragments, 1 μL of pGMT-easy, 1 μL of 10×T4 DNA ligation buffer, 1 μL of LT4 DNA ligase and the rest of double-distilled water.

The ligation products pGMT-KanR, pGMT-XKS1, pGMT-ADH1T and pGMT-rDNA obtained in Step 5 of this embodiment can be transformed into E.coli DH5α for preservation and amplification.

The recognition site of Nde I (underlined part) is added in the upstream primer of step 1 of the present embodiment, the recognition site of AatII (underlined part) is added in the downstream primer of step 1; the recognition site of Spe I is added in the upstream primer of step 2 (underlined part), the recognition site of Sal I (underlined part) is added in the downstream primer of step 2; the recognition site of Sal I (underlined part) is added in the upstream primer of step 3, and Eag I is added in the downstream primer of step 3 The recognition site of Nde I (underlined part) is added in the step 4 upstream primer, and the recognition site of EagI is added in the step 4 downstream primer (underlined part). The 2.2 kb rDNA fragment cloned in Step 4 of this embodiment is used to construct a plasmid homologous recombination site, which contains a HpaI single restriction site for linear transformation of the plasmid.

The plasmid pKT0150 of this embodiment was purchased from Addgene Company, and the genomic DNA of Saccharomyces cerevisiae was obtained by using the fungal genomic DNA extraction kit of Tianjingsha Company. The medicines, reagents, enzymes, competent cells and plasmids used in this embodiment are all readily available, and the concentration is the concentration marked on the product if there is no special requirement.

For steps 5 to 9 of this embodiment, refer to the operation manual of the kit.

The plasmids obtained in steps 5 to 9 of this embodiment can be transformed into E.coliDH5α for storage and amplification after PCR verification.

Embodiment 2: The difference between this embodiment and Embodiment 1 is that the resistance marker in step 6 is G418 (geneticin). Other steps and parameters are the same as those in Embodiment 1.

Specific embodiment three: The difference between this embodiment and specific embodiment one is: in step one, the PCR reaction system is 10 μL, consisting of 1 μL 10×PCR buffer, 0.8 μL of dNTP with a concentration of 2.5 mmol/L, and 1 μL with a concentration of 1 pmol/L The composition of μL upstream primer, 1 μL downstream primer with a concentration of 1 pmol/μL, 0.8 μL MgCl ₂ with a concentration of 25 mmol/L, 2 μL plasmid pKT0150, 0.2 μL Taq enzyme with a concentration of 5 U/mL and the rest of double distilled water; PCR reaction Conditions: Pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 45°C for 1min, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 7min. Other steps and parameters are the same as those in Embodiment 1.

The 10×PCR buffer used in this embodiment does not contain Mg ²⁺ .

Embodiment 4: The difference between this embodiment and Embodiment 1 is that the PCR reaction system in step 2 is 10 μL, consisting of 1 μL of 10×PCR buffer, 0.8 μL of dNTP with a concentration of 2.5 mmol/L, and 1 μL of dNTP with a concentration of 1 pmol/μL The composition of the upstream primer, 1μL downstream primer with a concentration of 1pmol/μL, 0.8μL MgCl ₂ with a concentration of 25mmol/L, 2μL Saccharomyces cerevisiae genomic DNA, 0.2μL Taq enzyme with a concentration of 5U/mL and the rest of double distilled water; PCR Reaction conditions: pre-denaturation at 94°C for 5min, denaturation at 94°C for 1min, annealing at 55°C for 1min, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 10min. Other steps and parameters are the same as those in Embodiment 1.

The 10×PCR buffer used in this embodiment does not contain Mg ²⁺ .

Embodiment 5: The difference between this embodiment and Embodiment 1 is that the PCR reaction system in step 3 is 10 μL, consisting of 1 μL 10×PCR buffer, 0.8 μL of dNTP with a concentration of 2.5 mmol/L, and 1 μL with a concentration of 1 pmol/L. The composition of μL upstream primer, 1 μL downstream primer with a concentration of 1 pmol/μL, 0.8 μL MgCl ₂ with a concentration of 25 mmol/L, 2 μL plasmid pKT0150, 0.2 μL Taq enzyme with a concentration of 5 U/mL and the rest of double distilled water; PCR reaction Conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 50°C for 1min, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 7min. Other steps and parameters are the same as those in Embodiment 1.

The 10×PCR buffer used in this embodiment does not contain Mg ²⁺ .

Embodiment 6: The difference between this embodiment and Embodiment 1 is that the PCR reaction system in step 4 is 10 μL, consisting of 1 μL 10×PCR buffer, 0.8 μL of dNTP with a concentration of 2.5 mmol/L, and 1 μL with a concentration of 1 pmol/L. μL of upstream primers, 1 μL of downstream primers with a concentration of 1 pmol/μL, 0.8 μL of MgCl ₂ with a concentration of 25 mmol/L, 2 μL of Saccharomyces cerevisiae genomic DNA, 0.2 μL of Taq enzyme with a concentration of 5 U/mL and the rest of double distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 53°C for 1min, extension at 72°C for 2.5min, a total of 30 cycles, and extension at 72°C for 7min. Other steps and parameters are the same as those in Embodiment 1.

The 10×PCR buffer used in this embodiment does not contain Mg ²⁺ .

Embodiment 7: The difference between this embodiment and Embodiment 1 is: in step 6, p406ADH1 and pGMT-KanR are double-digested with restriction endonucleases Nde I and Aat II, and then T4DNA ligase is used to carry out double digestion. Ligated to obtain plasmid pR. Other steps and parameters are the same as those in Embodiment 1.

Step 6 of this embodiment is performed by referring to the operation manual of the kit.

Embodiment 8: The difference between this embodiment and Embodiment 1 is: in step 7, use restriction endonuclease Spe I and Sal I to carry out double digestion of plasmid pR and pGMT-XKS1 respectively, and then use T4DNA ligase Ligation was performed to obtain plasmid pRK. Other steps and parameters are the same as those in Embodiment 1.

Step 7 of this embodiment is performed by referring to the operation manual of the kit.

Specific embodiment nine: the difference between this embodiment and specific embodiment one is: in step eight, plasmid pRK and pGMT-ADH1T are double-enzymatically digested with restriction endonuclease Sal I and Eag I, and then T4DNA ligase is used Ligation was performed to obtain plasmid pRKT. Other steps and parameters are the same as those in Embodiment 1.

In Step 8 of this embodiment, refer to the operation manual of the kit for operation.

Embodiment 10: The difference between this embodiment and Embodiment 1 is that in step 9, plasmids pRKT and pGMT-rDNA are double-digested with restriction endonucleases Nde I and Eag I, and then T4DNA ligase is used Ligation is performed to obtain a plasmid expressing the xylulokinase gene. Other steps and parameters are the same as those in Embodiment 1.

Step 9 of this embodiment is performed by referring to the operation manual of the kit.

Embodiment 11: The difference between this embodiment and embodiment 1 is that the resistance marker in step 5 is G418; L of dNTP, 1 μL of upstream primer with a concentration of 1 pmol/μL, 1 μL of a downstream primer with a concentration of 1 pmol/μL, 0.8 μL of MgCl ₂ with a concentration of 25 mmol/L, 2 μL of plasmid pKT0150, 0.2 μL of Taq enzyme with a concentration of 5 U/mL, and Composition of the remaining double distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 1 minute, annealing at 45°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles, and extension at 72°C for 7 minutes; the PCR reaction system in step 2 is 10 μL , consisting of 1 μL of 10×PCR buffer, 0.8 μL of dNTP at a concentration of 2.5 mmol/L, 1 μL of an upstream primer at a concentration of 1 pmol/μL, 1 μL of a downstream primer at a concentration of 1 pmol/L, 0.8 μL of MgCl ₂ at a concentration of 25 mmol/L, and 2 μL of brewing Composition of yeast genomic DNA, 0.2 μL Taq enzyme with a concentration of 5 U/mL, and double-distilled water; PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 Cycle and extend at 72°C for 10 min; the PCR reaction system in step 3 is 10 μL, consisting of 1 μL 10×PCR buffer, 0.8 μL dNTP with a concentration of 2.5 mmol/L, 1 μL upstream primer with a concentration of 1 pmol/μL, and 1 μL with a concentration of 1 pmol/μL downstream primers, 0.8 μL of MgCl ₂ with a concentration of 25 mmol/L, 2 μL of plasmid pKT0150, 0.2 μL of Taq enzyme with a concentration of 5 U/mL, and the rest of double-distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C 1 min, annealing at 50°C for 1 min, extension at 72°C for 2 min, a total of 30 cycles, and extension at 72°C for 7 min; the PCR reaction system in step 4 is 10 μL, consisting of 1 μL 10×PCR buffer, 0.8 μL dNTP with a concentration of 2.5 mmol/L, 1 μL 1 pmol/μL upstream primer, 1 μL 1 pmol/μL downstream primer, 0.8 μL 25 mmol/L MgCl ₂ , 2 μL Saccharomyces cerevisiae genomic DNA, 0.2 μL 5 U/mL Taq enzyme and the balance weight Composition of distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 53°C for 1min, extension at 72°C for 2.5min, a total of 30 cycles, and extension at 72°C for 7min; in step 6, use restriction enzyme Nde I and Aat II double digested p406ADH1 and pGMT-KanR respectively, and then ligated them with T4 DNA ligase to obtain plasmid pR; in step 7, use restriction enzymes Spe I and Sal

1 carries out double digestion respectively to plasmid pR and pGMT-XKS 1, connects with T4DNA ligase then, obtains plasmid pRK; In step eight, carry out respectively to plasmid pRK and pGMT-ADH1T with restriction endonuclease Sal I and Eag I Double enzyme digestion, and then use T4DNA ligase to connect to obtain plasmid pRKT; in step 9, use restriction endonucleases Nde I and Eag I to double enzyme digest plasmid pRKT and pGMT-rDNA, and then use T4DNA ligase to connect , to obtain a plasmid expressing the xylulokinase gene. Other steps and parameters are the same as those in Embodiment 1.

The 10×PCR buffer used in this embodiment does not contain Mg ²⁺ .

Saccharomyces cerevisiae was streak-inoculated on YPD plates with different G418 concentrations, cultured at 30°C for 72 hours, and high-copy transformants were screened out. Then pick a single colony of activated Saccharomyces cerevisiae W5 and inoculate it in YPD liquid medium, culture it at 30°C and 200r/min for 48h, then take 100mL of the culture solution and centrifuge it at 3500r/min for 5min, discard the supernatant Then add 40mL 1×TE to wash the cells, then centrifuge at 3500r/min for 5min, discard the supernatant and add 2mL 1×LiAc/0.5×TE, leave at room temperature for 10min to make yeast competent cells.

The plasmid expressing the xylulokinase gene constructed in this embodiment that has been linearized with Hpa I, 10.7 μL fish and salmon sperm DNA, 100 μL yeast HDY-01 competent cells and 700 μL 1×LiAc/40PEG-4000/1×TE Mix and react at 30°C for 30min, then add 88μL DMSO stock solution and mix evenly, place in a water bath at 40°C for heat shock for 7min, then centrifuge at 13000r/min for 10s, discard the supernatant and add 1mL 1×TE to wash twice, 13000r/min Centrifuge for 10 seconds, discard the supernatant, add 50-100 μL 1×TE, spread on the YPD plate medium containing G418, and culture at 30°C for 48 hours to obtain the high-copy transformant W-XK.

Inoculate the high-copy transformant W-XK into 40 mL of gradiently diluted YEPD liquid medium (under non-selective pressure) and culture it in shake flasks for 60 generations (10 generations every 24 h), and count the number of colonies on the plate for stability every 10 generations The determination is calculated according to the formula, and the results show that the stability after 60 generations is 99%, indicating that under non-selective pressure, the foreign gene fragment integrated on the chromosome of Saccharomyces cerevisiae has good genetic stability and can be suitable for industrial production. Extensive environment. [Calculation formula of plasmid stability: Stability (%)=(number of colonies with integrated plasmid ÷ total number of bacteria)×100%]

The transformant W-XK was cultured in the selective pressure medium (YEPD medium containing G418) to the logarithmic growth phase, the bacteria were collected by centrifugation and washed twice with 100mmol/L phosphate buffer, and then dissolved in the newly prepared lysis buffer solution (100mmol/L phosphate buffer, 0.5mmol/LEDTA, 0.5mmol/L DTT, 1mmol/L PMSF, pH 7.0), the bacterial suspension was added with glass beads (0.5mm in diameter), and the maximum speed of the cell disruptor was Treat at low temperature for 30-50s, place in ice bath for 5min, crush and repeat 3-5 times, then centrifuge at 10000g for 10min, collect the supernatant, which is the crude enzyme solution for enzyme activity determination and analysis. (The determination of the protein content of the crude enzyme solution is carried out according to the operating instructions of the protein content determination kit.)

In this embodiment, the continuous monitoring method is used to measure the enzyme activity of xylulokinase, and the linear reaction period is selected to calculate the enzyme activity. The determination principle is as follows:

After the above coupling reactions reach equilibrium, the rate of conversion of the substrate D-xylulose by xylulokinase (XK) is equal to the rate of reduction of NADH in the reaction system. By measuring the rate of reduction of NADH, the rate of xylulokinase (XK) can be calculated. activity.

Definition of xylulokinase enzyme activity unit: under given experimental conditions, the amount of enzyme that catalyzes the conversion of 1 μmol NADH per minute is an enzyme activity unit (ie 1U).

formula:

6.22×10 ³ : Molar extinction coefficient of NADH (1·mol ^-1 ·cm ^-1 );

OD ₃₄₀ ¹ : the light absorption of the sample to be tested at a wavelength of 340nm at time t ₁ ;

OD ₃₄₀ ² : the light absorption of the sample to be tested at a wavelength of 340nm at time t ₂ ;

60: the number of seconds equivalent to 1min (s);

t ₁ , t ₂ : the time point (s) taken during the enzyme activity determination process;

C _protein : protein concentration of crude enzyme solution (mg/-mL);

0.1: the volume (mL) of the crude enzyme solution added in the reaction system;

10: The multiple of dilution of the crude enzyme solution.

According to the data in Table 1, the reaction system for the determination of xylulokinase activity in the crude enzyme solution was prepared, with a volume of 0.9 mL.

Table 1

The xylulokinase enzyme activity assay reaction system was equilibrated at room temperature for 15 minutes, added 0.1 mL of 10-fold diluted crude enzyme solution, vortexed and mixed, and distilled water was used as a blank control to measure OD ₃₄₀ , and use an automatic recording spectrometer A photometer records the change in _OD340 of the sample over time. Select the linear reaction period to calculate the xylulokinase activity in the crude enzyme solution. Enzyme activity measurement results show that the xylulokinase activity of the Saccharomyces cerevisiae recombinant strain W-XK transformed by the plasmid expressing the xylulokinase gene constructed by the method of this embodiment is greatly improved compared with the starting bacterial strain W5, and the enzyme activity of the starting bacterial strain W5 is 0.31U/mg, the enzyme activity of the recombinant strain W-XK is 2.8U/mg, and the enzyme activity has increased by 9 times, indicating that the plasmid vector constructed by the method of this embodiment is transformed into Saccharomyces cerevisiae, and the efficient and stable expression of the xylulokinase gene is realized .

sequence listing

<110> Heilongjiang University

<120> A method for constructing a plasmid expressing xylulokinase gene

<160>8

<210>1

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification upstream primers designed according to the nucleotide sequence of plasmid pKT0150 and the relative position of KanR gene in the plasmid.

<400>1

ttccatatgt ctgtttagct tgcctc 26

<210>2

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification downstream primers designed according to the nucleotide sequence of plasmid pKT0150 and the relative position of KanR gene in the plasmid.

<400>2

cttgacgtct atcatcgatg aattcga 27

<210>3

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification upstream primers designed according to the gene sequence of Saccharomyces cerevisiae XKS1.

<400>3

cggactagtc tcaatcttca gcaagcgac 29

<210>4

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification downstream primers designed according to the gene sequence of Saccharomyces cerevisiae XKS1.

<400>4

cgcgtcgaca acggggaaca aaatgatg 28

<210>5

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification upstream primers designed according to the nucleotide sequence of plasmid pKT0150 and the relative position of the ADH1 terminator fragment in the plasmid.

<400>5

cgcgtcgaca tttgttactg ctgctggtat t 31

<210>6

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> PCR amplification downstream primers designed according to the nucleotide sequence of plasmid pKT0150 and the relative position of ADH1 terminator fragment in the plasmid.

<400>6

ctcggccgcc ctgttatccc tagcgg 26

<210>7

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223>The upstream primer for amplifying the 2.2kb rDNA fragment was designed according to the gene sequence of Saccharomyces cerevisiae rDNA.

<400>7

ttccatatgg gaacctctaa tcattcgct 29

<210>8

<211>29

<212> DNA

<213> Artificial sequence

<220>

<223> Downstream primers for amplifying a 2.2kb rDNA fragment designed according to the gene sequence of Saccharomyces cerevisiae rDNA.

<400>8

tctcggccga acgaacgaga ccttaacct 29

Claims (10)

1. A method for constructing a plasmid expressing a xylulokinase gene, characterized in that the method for constructing a plasmid expressing a xylulokinase gene is carried out in the following steps: one, cloning the KanR gene in the plasmid pKT0150; two, cloning Saccharomyces cerevisiae XKS1 Gene; 3. Cloning the ADH1 terminator fragment in plasmid pKT0150; 4. Cloning the 2.2kb rDNA fragment in Saccharomyces cerevisiae; 5. Recovering and purifying the gene fragments prepared in steps 1 to 4, and then combining them with pGEMT Easy vector at 16°C Connect for 10-12 hours to obtain pGMT-KanR, pGMT-XKS1, pGMT-ADH1T, and pGMT-rDNA; 6. Construct plasmid pR, and introduce the gene KanR with a resistance marker using Saccharomyces cerevisiae integration vector p406ADH1 as the backbone; 7. Construct plasmid pRK , the XKS1 gene is added to the plasmid pR; Eighth, the plasmid pRKT is constructed, and the ADH1 terminator fragment is added to the plasmid pRK; Ninth, the 2.2kb rDNA fragment is added to the plasmid pRKT to obtain a plasmid expressing the xylulokinase gene; wherein step 1 amplifies The sequence of the upstream primer is 5'-TTCCATATGTCTGTTTAGCTTGCCTC-3', the sequence of the downstream primer is 5'-CTTGACGTCTATCATCGATGAATTCGA-3'; the sequence of the upstream primer in step 2 is 5'-CGGACTAGTCTCAATCTTCAGCAAGCGAC-3', and the sequence of the downstream primer is 5'-CGCGTCGACAACGGGGAACAAAATGATG -3'; the sequence of the upstream primer for amplification in step 3 is 5'-CGCGTCGACATTTGTTACTGCTGCTGGTATT-3', the sequence of the downstream primer is 5'-CTCGGCCGCCCTGTTATTCCCTAGCGG-3'; the sequence of the upstream primer for amplification in step 4 is 5'-TTCCATATGGGAACCTCTAATCATTCGCT-3', The downstream primer sequence is 5'-TCTCGGCCGAACGAACGAGACCTTAACCT-3'. 2. The method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that the resistance marker in step 6 is G418. 3. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that in step 1, the PCR reaction system is 10 μL, and the concentration of 1 μL 10×PCR buffer and 0.8 μL is 2.5 mmol/L dNTP, 1 μL of upstream primer with a concentration of 1 pmol/μL, 1 μL of a downstream primer with a concentration of 1 pmol/μL, 0.8 μL of MgCl ₂ with a concentration of 25 mmol/L, 2 μL of plasmid pKT0150, 0.2 μL of Taq enzyme with a concentration of 5 U/mL and other Weigh the composition of distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 45°C for 1min, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 7min. 4. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that the PCR reaction system in step 2 is 10 μL, and the concentration of 1 μL 10×PCR buffer and 0.8 μL is 2.5 mmol/L dNTP, 1 μL upstream primer with a concentration of 1 pmol/μL, 1 μL downstream primer with a concentration of 1 pmol/μL, 0.8 μL MgCl ₂ with a concentration of 25 mmol/L, 2 μL Saccharomyces cerevisiae genomic DNA, and 0.2 μL Taq enzyme with a concentration of 5 U/mL and the rest of double distilled water; PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles, and extension at 72°C for 10 minutes. 5. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that the PCR reaction system in step 3 is 10 μL, and the concentration of 1 μL 10×PCR buffer and 0.8 μL is 2.5 mmol/L dNTP, 1 μL of upstream primer with a concentration of 1 pmol/μL, 1 μL of a downstream primer with a concentration of 1 pmol/μL, 0.8 μL of MgCl ₂ with a concentration of 25 mmol/L, 2 μL of plasmid pKT0150, 0.2 μL of Taq enzyme with a concentration of 5 U/mL and other Weigh the composition of distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 50°C for 1min, extension at 72°C for 2min, a total of 30 cycles, and extension at 72°C for 7min. 6. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that the PCR reaction system in step 4 is 10 μL, and the concentration of 1 μL 10×PCR buffer and 0.8 μL is 2.5 mmol/L dNTP, 1 μL upstream primer with a concentration of 1 pmol/μL, 1 μL downstream primer with a concentration of 1 pmol/μL, 0.8 μL MgCl ₂ with a concentration of 25 mmol/L, 2 μL Saccharomyces cerevisiae genomic DNA, and 0.2 μL Taq enzyme with a concentration of 5 U/mL and the rest of double distilled water; PCR reaction conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 1min, annealing at 53°C for 1min, extension at 72°C for 2.5min, a total of 30 cycles, and extension at 72°C for 7min. 7. The method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that in step 6, p406ADH1 and pGMT-KanR are double-enzymed with restriction endonucleases Nde I and Aat II, respectively. Cut and ligate with T4 DNA ligase to obtain plasmid pR. 8. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that in step 7, the plasmids pR and pGMT-XKS1 are double-treated with restriction endonucleases Spe I and Sal I, respectively. Restriction digestion, and then ligation with T4 DNA ligase to obtain plasmid pRK. 9. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that in step 8, the plasmids pRK and pGMT-ADH1T are double-treated with restriction endonucleases Sal I and Eag I, respectively. Restriction digestion, and then ligation with T4 DNA ligase to obtain plasmid pRKT. 10. A method for constructing a plasmid expressing xylulokinase gene according to claim 1, characterized in that in step 9, the plasmids pRKT and pGMT-rDNA are double-treated with restriction endonucleases Nde I and Eag I, respectively. Restriction digestion, and then ligation with T4 DNA ligase to obtain a plasmid expressing the xylulokinase gene.