A kind of Antarctic yellow luffa moss cpd photorepair enzyme and its preparation method and application
A technology of photorepair enzyme and yellow silk, which is applied in the field of biology and can solve the problems of extremely limited protective effect and other problems
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Embodiment 1
[0045] Example 1. Acquisition of the cDNA sequence of CPD photoreparation enzyme of moss Antarctica
[0046] 1. Extraction of total RNA from moss Antarctica
[0047] The Antarctic Luffa moss (Pohlia nutans) was cultured according to conventional culture conditions. The conventional culture conditions were temperature (16±1)°C, 70% relative humidity, light intensity of 1000-2000 lux, and light cycle of 12h light / 12h dark; Trizol was used. The total RNA of Pohlia nutans was extracted by the method of Trizol, methyl chloride and isopropanol. RNA is easily degraded, so gloves should be worn during the operation to prevent RNA from being degraded by RNase in sweat. During the extraction process, all utensils must be strictly sterilized and RNase inactivated.
[0048] The specific operation steps of Trizol method to extract the total RNA of moss Antarctic Luffa are as follows:
[0049] 1. Put the tissue (leaf) into liquid nitrogen, grind it in a mortar, keep putting it in liquid n...
Embodiment 2
[0067] Example 2. Cloning of CPD photorepairase gene (PnPHR1) and construction of recombinant expression vector
[0068] 1. Cloning of CPD photorepair enzyme gene of moss Antarctica
[0069] According to the CPD photorepairase gene sequence and the principle of homologous recombination in the transcriptome data of Antarctic Luffa moss, the PCR primers were designed as follows:
[0070] The upstream primers are:
[0071] 5'-CCGCGTGGATCCCCGGAATTCCCGGGTCGAATGTACTTTGGGTCTACTACTTTCCCC-3',
[0072] Downstream primers are:
[0073] 5’-GCAGATCGTCAGTCAGTCACGATGGCGGCCGTTA GTGGTGGTGGTGGTGGTG CACCTTCCGCCCTGCCGGCTT-3',
[0074] Wherein, the underline is the introduced His tag sequence;
[0075] Using the PCR kit from Novozymes, the PCR reaction system was set as follows according to the instructions:
[0076]
[0077] The reaction program was: 94°C for 5 min; 94°C for 30 s, 54°C for 30 s, 72°C for 2 min, a total of 35 cycles; 72°C for 7 min and storage at 4°C. The PCR product wa...
Embodiment 3
[0094] Example 3. Inducible expression and isolation and purification of CPD photorepair enzyme protein from Physcomitrella antarctica
[0095] The recombinant Escherichia coli BL21(DE3) / pGEX-4T-1 / PnPHR1 was inoculated into 50 mL of LB liquid medium containing ampicillin, and cultured overnight at 37°C and 180 rpm. The next day, 5 mL of activated bacterial liquid was taken and continued to be cultured in 500 mL of medium. 600 When the temperature is 0.6-0.8, put the bacterial solution on ice for 30 minutes, add IPTG with a final concentration of 0.2 mM, culture conditions are 16 °C, induce low temperature at 100 rpm for 20 hours, and collect bacteria by centrifugation at 4 °C and 8000 rpm.
[0096] The bacteria were resuspended in 40 mL of buffer solution, and crushed in an ice-water bath at 300 W for 40 minutes. Before crushing, an appropriate amount of protease inhibitor PMSF was added to make the final concentration 0.1-1 mM. The crushed system was centrifuged at 12,000 rp...
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