Green plant bug water-soluble trehalase, coding sequence, vector, strain and application
A technology of trehalase and chlorophyll, which is applied in the field of agricultural science, can solve problems such as the lack of recombinant trehalase, and achieves the effects of being suitable for a wide range of reaction temperatures, high enzyme activity, and efficient expression
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Embodiment 1
[0032] Example 1: Acquisition of ALTre-1 Gene
[0033] The TRIzol method was used to extract the total RNA of Lygus japonica, and the total RNA samples with good electrophoretic patterns and OD260 / OD280 values between 1.8 and 2.0 were selected and combined for mRNA purification, and the first-strand cDNA was synthesized by reverse transcription. The PCR reaction system was: 0.5 μg total RNA, 0.5μl Oligo-dT18, 2μl 5× reaction buffer, 2μl 10mM dNTP, 40U RNasin, 200U SuperscriptⅡ, add ddH 2 0 to 10 μl; PCR reaction parameters: 42°C for 30 min, 75°C for 30 min, 4°C for 5 min. Primers ALTre-1-F (5′-TACTACTGGGACTCTTATTGG-3′) and ALTre-1-R (5′-CCAGGCGTTAGGGAAGTCCC-3′) suitable for PCR amplification were designed according to the conserved sequence of known insect water-soluble trehalase genes , obtain the conserved sequence of ALTre-1 gene, the conserved sequence refers to SEQ ID NO: 3 in the sequence listing, the PCR reaction system is: 2.5 μl 10× reaction buffer, 2 μl 25mM Mg 2...
Embodiment 2
[0035] Embodiment 2: Construction of ALTre-1 gene prokaryotic expression vector
[0036] The method for constructing the prokaryotic expression vector containing the ALTre-1 gene T vector is as follows: the above sequencing is correctly connected into the pGEM-TEasy vector and pET28a are both digested with restriction endonucleases NdeI and NotI, and the large fragments are connected ( figure 2 ). The enzyme digestion system is: 1.0 μl of each of the two restriction enzymes, 2.0 μl of 10×Buffer buffer, 10.0 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 20 μl, and bathe in water at 37°C for 3h. The ligation system is: 5.0 μl of the recovered vector after enzyme digestion, 10.0 μl of the gene fragment, 1.0 μl of T4 DNA ligase, 2.0 μl of 10×Buffer, and ddH 2Make up O to 20μl, and react at 16°C for 12-16h. After the ligation product was transformed into Escherichia coli BL21(DE3), positive clones were screened by PCR. Transform the correctly ...
Embodiment 3
[0038] Example 3: Denaturation and renaturation of ALTre-1 fusion protein and purification of protein
[0039] Extraction and purification of the target fusion protein: BL21(DE3) cells containing the expression vector were cultured in large quantities after being detected by SDS-PAGE electrophoresis. Centrifuge at 4°C at 8000rpm / min to collect a sufficient amount of bacteria. Sonicate the resuspended bacteria 99 times (power 200W, sonicate for 3S, pause once for 5S, always keep the centrifuge tube on ice), centrifuge at 8000rpm / min at 4°C for 15min, and collect the supernatant and precipitate. Suspend the centrifuged pellet in a solution (1×PBS, 8M urea), incubate at room temperature for 3 hours, centrifuge at 6000rpm / min at 4°C for 15 minutes, and take the supernatant. Put the supernatant on a Ni-NTA column equilibrated with Binding buffer (1×PBS, containing 8M urea, 15mM imidazole, 300mM NaCl, 0.5%Triton X-100, pH8.0) (flow rate is 45mL / h), Collect the breakthrough fluid. ...
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