Alcohol dehydrogenase and gene and recombinase thereof
A technology of alcohol dehydrogenase and gene, applied in the direction of genetic engineering, plant gene improvement, recombinant DNA technology, etc., to achieve the effect of expanding resources
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Embodiment 1
[0039] Example 1 Design of PCR degenerate primers
[0040] According to the bacterial alcohol dehydrogenase protein sequence included in GenBank, the specific strains are shown in Table 1, after the analysis of the AlignX multiple sequence alignment program of the Vector NTI Advance 10 software package, two highly conserved amino acid sequence regions (BlockS) were found, These two conserved regions correspond to the highly conserved zinc ion binding domain and cofactor binding domain of alcohol dehydrogenase, respectively. Convert the protein sequences of these two conserved regions into degenerate nucleotide sequences, and calculate the degree of degeneracy, and design a pair of degenerate primers based on the degenerate nucleotide sequences. The sequences of the primers are shown in Table 2 and the sequence listing SEQ ID Nos. 3 and 4.
[0041] Table 1. The bacterial species of the alcohol dehydrogenase protein referred to in the design of the degenerate primers of the alc...
Embodiment 2
[0046] Example 2 Extraction of Lactobacillus kefir DSM 20587 strain genome total DNA
[0047] 1) Cultivate 300 ml of Lactobacillus kefir DSM 20587 cells (purchased from the American National Type Culture Collection, Number: 35411).
[0048] 2) Centrifuge at 3,000g for 10min, and resuspend in 5ml SET solution. Add lysozyme (final concentration 1 mg / ml) and incubate at 37°C for 1 hour.
[0049] 3) Add 1 / 10 volume of 10% (w / v) SDS and proteinase K (final concentration 0.5 mg / ml), and incubate at 55° C. for 2 hours (mix by inverting occasionally). Add 1 / 3 volume of 5M NaCl, 1 volume of chloroform, incubate at room temperature for 0.5 hours, and mix by inverting frequently.
[0050] 4) Centrifuge at 4,500 g for 15 min, and transfer the upper aqueous phase into a new tube.
[0051] 5) Add 1 volume of isopropanol, gently invert the tube and mix well, after centrifugation, the DNA precipitate is washed with 70% ethanol, dried in vacuum, and dissolved in TE solution.
Embodiment 3
[0052] The PCR amplification of the partial fragment of alcohol dehydrogenase gene of embodiment 3
[0053] The Lactobacillus kefir DSM 20587 strain genome total DNA obtained in Example 2 was used as a template, and the primers designed in Example 1 (synthesized by Invitrogen Shanghai Yingjun Biotechnology Co., Ltd.) were used to carry out PCR amplification (the amplification reaction was purchased from TaKaRa The DR001A PCR kit) reaction system of Dalian Bao Biological Engineering Co., Ltd. is shown in Table 3, and the reaction procedure is as follows:
[0054] Table 3. Composition of PCR reaction system
[0055]
[0056] PCR reaction program:
[0057] Step 1 Pre-denaturation at 95°C for 3 minutes,
[0058] Step 2 Denaturation at 95°C for 30sec,
[0059] Step 3 Anneal at 60°C for 30sec,
[0060] Step 4 Extend 30sec at 72°C,
[0061] Step 5 Repeat Step2 to Step4 for a total of 30 cycles,
[0062] Step 6 Extend at 72°C for 10 minutes,
[0063] Step 7 Keep at 4°C.
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