Banana polyphenol oxidase gene, recombinant protein, and preparation method thereof
A polyphenol oxidase and recombinant protein technology, applied in the field of bioengineering, can solve the problems of cumbersome separation and purification steps, variability and inactivation, and restricted application, and achieve the effect of fast operation, simple technology and low cost
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Embodiment 1
[0042] Example 1 Extraction and amplification of banana pulp total RNA
[0043] 1.1. Extraction of total RNA from banana pulp
[0044] The specific operation process of lithium chloride precipitation method: the extraction buffer contains 2% CTAB, 100mM Tris-boric acid (pH9.0), 1.4MNaCl, 20mMEDT (pH8.0), 2% PVP-K30, add β-mercapto before use Ethanol to a final concentration of 2%, preheat it to 60°C; grind the pulp into powder with liquid nitrogen, add 1ml of extraction buffer per 0.2g of pulp, vortex and mix, place at 60°C for 30min, every 5-10min Invert and mix once; add 0.5M CaCl2 to the extraction mixture to make the molar concentration 100mM, and place at 25°C for 20min; cool to room temperature, extract with an equal volume of chloroform:isoamyl alcohol (24:1), and mix Uniformly, let stand for layering, centrifuge at room temperature 12000rpm for 5min; take the supernatant and add an equal volume of chloroform: isoamyl alcohol (24:1) for extraction, mix well, let stand ...
Embodiment 2
[0045] Example 2 Electronic cloning and RT-PCR verification of banana polyphenol oxidase gene
[0046] 2.1, PCR amplification of banana PPO gene
[0047] Using banana pulp total RNA as a template, the first strand of cDNA was synthesized. Then use the first strand of cDNA as a template and use the designed specific primers (upstream primer FP1: 5'-tcgatcc tgttctcggcttc-3' (SQE NO.4); RP1: 5'-cgatggtgcggctttttttcc-3' (SQE NO.5) ) for PCR amplification, the amplified product was detected by 1% agarose gel electrophoresis, and 2 bands of 2100bp and 1050bp were obtained, and the 2100bp band was brighter than the 1050 band fragment ( figure 1 ). The 2100bp band is consistent with the expected fragment size.
[0048] 2.2 Cloning and identification of PCR product of banana PPO gene
[0049] The 2100bp fragment was recovered by gel cutting, purified and recovered by TIANGEN ordinary agarose DNA recovery kit (DP209), and a single band ( figure 2 ). The gel-recovered PCR product...
Embodiment 3
[0053] Embodiment 3 Construction of prokaryotic expression vector pQE80L-PPO
[0054] 3.1. Banana PPO gene amplification
[0055] According to the banana PPO gene sequence that embodiment 2 obtains, design the primer with restriction site, wherein, upstream primer: FPQE25 '-CGG ggtaccCCG atgactgca aat gccaagctcgac-3'(K pn I)(SQE NO.6); downstream primer: RPQE5'-CCCAagcttGGtcaatttgggaaatcgat-3'(Hind III)(SQE NO.7). Use this pair of primers to carry out PCR amplification, wherein the PCR amplification reaction conditions are 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 10s, 30 cycles; 72°C for 5min, and obtain 1600bp BXPPO2 by PCR amplification Fragments (with Kpn I and Hind III restriction sites), such as Figure 5 shown.
[0056] Among them, the 25ul reaction system is: 2×ES Master Mix 12.5ul, upstream primer (10uM) 1ul, downstream primer (10uM) 1ul, plasmid 0.5ul, RNase-Free Water 10ul.
[0057] 3.2. Construction of recombinant plasmid pQE80L-PPO
[0058] The ampl...
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