Construction method of plasmid expressing xylose reductase gene
A construction method and reductase technology, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problem of lack of xylose metabolism ability, inability to effectively ferment plant fiber hydrolyzate, lack of xylose reductase, etc. question
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specific Embodiment approach 1
[0006] Specific embodiment one: the construction method of the plasmid expressing the xylose reductase gene in this embodiment is carried out according to the following steps: one, clone the XYL1 gene of Candida shohata; two, clone the ADHt gene sequence in the plasmid pKT0150; three 1. Clone the KanR gene in the plasmid pKT0150; 4. Recover and purify the gene fragments prepared in steps 1 to 3, and then connect them to the pGEMT Easy vector at 16°C for 10-12 hours to obtain pGMT-XYL1, pGMT-ADHt and pGMT -KanR; 5. Construct the plasmid pADH, and introduce the ADHt gene with the Saccharomyces cerevisiae integrated plasmid p406ADH1 as the backbone; 6. Construct the plasmid pAK, and introduce the KanR gene into the plasmid pADH; 7. Introduce the XYL1 gene into the plasmid pAK to obtain the expression of xylose reduction The plasmid of the enzyme gene; wherein the upstream primer sequence amplified in step 1 is 5'-ACT TCTAGA TACATCCACAATGAGCCC-3', the downstream primer sequence is...
specific Embodiment approach 2
[0012] Specific embodiment two: the difference between this embodiment and specific embodiment one is: the PCR reaction system in step one is 10 μ L, by 1 μ L 10 × PCR buffer, 0.8 μ L concentration is the dNTP of 2.5mmol / L, 1 μ L concentration is 1pmol / L μL of upstream primer, 1 μL of downstream primer at a concentration of 1 pmol / μL, 0.8 μL of MgCl at a concentration of 25 mmol / L 2 , 2 μL of Candida shohatae 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 5min, denaturation at 94°C for 1min, annealing at 50°C for 1min, Extend at 72°C for 2min, a total of 30 cycles, and extend at 72°C for 10min. Other steps and parameters are the same as those in Embodiment 1.
[0013] The 10×PCR buffer used in this embodiment does not contain Mg 2+ .
specific Embodiment approach 3
[0014] Specific embodiment three: the difference between this embodiment and specific embodiment one is: the PCR reaction system in step two is 10 μ L, by 1 μ L 10 × PCR buffer, 0.8 μ L concentration is the dNTP of 2.5mmol / L, 1 μ L concentration is 1pmol / L μL of upstream primer, 1 μL of downstream primer at a concentration of 1 pmol / μL, 0.8 μL of MgCl at a concentration of 25 mmol / L 2 , 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 2min, denaturation at 94°C for 45s, annealing at 49°C for 30s, extension at 72°C for 45s, a total of 30 cycle, extending at 72°C for 7 min. Other steps and parameters are the same as those in Embodiment 1.
[0015] The 10×PCR buffer used in this embodiment does not contain Mg 2+ .
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