Enzyme catalyzed specificity changed patchoulol synthase mutant and application thereof
A technology of patchouli alcohol and mutants, applied in the biological field, can solve the problems of few studies on enzyme engineering
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Embodiment 1
[0068] Example 1, Extraction of Patchouli total RNA, PCR amplification of target gene ADS and site-directed mutagenesis
[0069] A, the extraction of patchouli total RNA
[0070] The wild cultivated strain T002 was provided by Firmenich Fragrances (Shanghai) Co., Ltd. According to the identification and analysis of the morphology and volatile oil components of Patchouli, it can be divided into 3 ecological types (Sugimura et al., 1990). T002 is Class I (patchouli alcohol type), and its volatile oil is rich in patchouli alcohol, with a content of about 30%.
[0071] Take the patchouli material (about 100 mg) and grind it thoroughly in liquid nitrogen. Transfer to a 1.5ml centrifuge tube, add 1mL Trizol (Invitrogen, Cat. 15596-018), mix well, and stand at room temperature for 5min. Centrifuge at 12,000rpm for 10min and discard the precipitate. Add 200 μL of chloroform to the supernatant, mix well, and centrifuge at 12,000 rpm for 10 min. Take the supernatant and add 500 μL ...
Embodiment 2
[0096] Embodiment 2, vector construction and Escherichia coli transformation
[0097] A. Vector construction
[0098] KOD-plus DNA polymerase amplifies the PTSiso coding region fragment (PTSiso) and mutant fragments (PTSiso-A445S, PTSiso-A445G, PTSiso-A445C), which are digested with BamHI / SacI and ligated into the pET-32a vector (Amp r , Novagen, America), to obtain the recombinant vector.
[0099] B. Competent cell preparation
[0100] Escherichia coli DH5α or BL-21 stored at -70°C were streaked on a solid LB plate and cultured overnight at 37°C; a single colony was picked and cultured in 5 mL of liquid LB medium at 250 rpm overnight. The next day, scale up and inoculate into 500mL liquid LB medium at a ratio of 1 / 50, culture at 18-22°C until OD 600 ≈0.5 (about 5-6h), cooling on ice for 10min. Centrifuge at 2,500 g for 10 min at 4°C, resuspend the cells with 160 mL of transformation buffer, discard the supernatant, and finally resuspend the cells with 40 mL of transformat...
Embodiment 3
[0105] Embodiment 3, RT-PCR
[0106] 1 μg total RNA, Oligo(dt) 20 As primers, 10 μL reaction system was used for reverse transcription according to the requirements of the reverse transcription kit (TOYOBO, Osaka, Japan). React at 42°C for 30 minutes. After boiling water for 5 minutes, place on ice. The reverse transcription product (or diluted 10 times) can be directly used for PCR detection.
[0107] According to the known PTSiso sequence, PCR primers were synthesized, and Patchouli Pat18S (GenBank: EF529587) was used as an internal standard reference when analyzing the expression of PTSiso. The PCR conditions are as follows: the annealing temperature and extension time of the reaction are determined by the primers and the length of the amplified fragment. The general reaction conditions are: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 72°C for 30 s, amplification for 25 to 35 cycles; and incubation at 72°C f...
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