A Hydroxylase Gene Involved in Myricetin Biosynthesis and Its Application
A hydroxylase and gene technology, applied in the field of hydroxylase MrF3'5'H gene, can solve the problem of limited research on other functions of F3'5'H gene
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Embodiment 1
[0016] Example 1: Acquisition and identification of the full length of the MrF3'5'H gene
[0017] 1. Myrica rubra tissue material
[0018] Water chestnut and Dongkui bayberry tissues (fruits, flowers, leaves) were harvested on the same day, frozen in liquid nitrogen and stored in a -80°C refrigerator. Three biological replicates were set up for each tissue sample, and each replicate had 7-8 fruits. The quality of each repeated flower is more than 500g, and each repeated 10-15 whole leaves.
[0019] 2. RNA Extraction and cDNA Synthesis
[0020] Red bayberry tissue samples were ground into powder in a liquid nitrogen environment, and total RNA was extracted by ordinary CTAB method. After passing the electrophoresis test, DNA was removed by referring to the instructions of TURBO DNAase Kit (Ambion). According to the requirements of iScript cDNA Synthesis Kit (Bio-Rad), the total RNA was extracted. 1.0 μg RNA, reverse transcribed into cDNA.
[0021] 3. Obtain the full length of...
Embodiment 2
[0025] Embodiment 2: Construction of pYES2-MrF3'5'Hs expression vector
[0026] According to the multiple cloning site sequence of pYES2 NT / C (Invitrogen) vector and the full-length gene sequence of MrF3'5'H (SEQ: NO.1), the primer sequence including BamHI and EcoRI restriction sites was designed: SEQ: NO.5 And SEQ: NO.6, the primer design includes a start codon and a stop codon, amplifies the MrF3'5'H sequence containing BamHI and EcoRI restriction sites. The PCR reaction system was 50 μL, and the components were: 1 μL Phanta high-fidelity enzyme (Vazyme), 25 μL buffer (2×), 1 μL dNTP (10 mM), 2 μL each of upstream and downstream primers (10 μM, Hua Gene), 1 μL cDNA, 18 μL H 2 O. The reaction program was: pre-denaturation at 95°C, 2min; denaturation at 95°C, 15s; annealing at 58°C, 15s; extension at 72°C, 1min, 35 cycles; thorough extension at 72°C for 5min, and storage at 4°C. Digest the pYES2 vector with BamHI (NEB) and EcoRI (NEB) respectively, use II ligase (Vazyme) l...
Embodiment 3
[0027] Example 3: Heterologous expression of MrF3'5'H in Saccharomyces cerevisiae
[0028] 1. Yeast Transformation with Recombinant Vectors
[0029] The successfully constructed pYES2-MrF3'5'H recombinant plasmid or pYES2 empty vector was transformed into Saccharomyces cerevisiae strain INVScI (Invitrogen) by the LiAC method through the yeast transformation kit (Clontech). Then it was spread on SD / -Ura culture plate and cultured at 30°C for 3 days, single colony was picked, and the recombinant plasmid or empty load was detected by PCR. Select a single colony with the correct PCR band, which is Saccharomyces cerevisiae INVScI containing the pYES2-MrF3'5'H recombinant plasmid, and store it in a -80°C refrigerator with 25% glycerol for future use.
[0030] 2. MrF3'5'H induced expression
[0031]Pick a single colony and put it in 5mL SD / -Ura+20g / L glucose culture solution, and culture it on a shaker at 30°C and 250rpm for 12h. Centrifuge at 700g for 5 minutes at room temperatur...
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