Migratory locust chemoreception protein and its coding gene and the expression method of the protein
A technology of chemical sensing and coding gene, applied in the field of chemical sensing protein of locust and its coding gene and expression of the protein, can solve the problems of providing high-abundance protein and the like, and achieve a low cost, simple expression method and high expression amount. Effect
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Embodiment 1
[0029] Example 1. Obtaining the gene encoding migratory locust chemosensory protein
[0030] Based on the known N-terminal and C-terminal sequences of CSPsg4 (accession no.AF070964) and OS-D2 (accessionno.AJ251076) and OS-D5 (accession no.AJ251079) in the known desert locust, design the following primers:
[0031] CSPsg4(accession no.AF070964): Forward primer 5’-GAR GAR AAR TAY ACN AC-3’
[0032] Reverse primer 5’-YTG RTG NAG YTC YTT YTC-3’;
[0033] OS-D2(accession no.AJ251076): Forward primer 5’-GCN GCN GCN TAY ACN AC-3’
[0034] Reverse primer 5’-NGC NCT NAG NAG NAG YTC NGG-3’;
[0035] OS-D5(accession no.AJ251079): Forward primer 5’-GCN GCN GCN TAY ACN AC-3’
[0036] Reverse primer 5'-NGC NCT NAC YCT YTT NAG-3'.
[0037] Where R is A or G, Y is T or C, N is A or T or C or G,
[0038] Using Locusta migratoria antennae as the material, the total RNA was extracted with Trizol, and the integrit...
Embodiment 2
[0039] Example 2. Expression of migratory locust chemosensory protein
[0040] 1. Construction of recombinant expression vector and screening of engineering bacteria
[0041] Using the above pGEM-T-LmigCSP as a template, the primers are:
[0042] 5’ end: 5’-TACATATG GCT GCT GCG TAC ACC ACC-3’
[0043] 3'end: 5'-AATTGAATTC TTA TTA CGC GGA GAC CCT CTT GA-3' for PCR amplification to obtain the PCR product of the LmigCSP gene. Combine the obtained PCR product and the expression vector pET-5b ( figure 2 , Purchased from Novagen, Darmstadt, Germany) were digested with NdeI and EcoRI respectively, and then ligated at room temperature for 10 hours. The resulting ligation product was subjected to heat shock transformation. The specific method is as follows: add 1 μL ligation product to 100 μL E. coli BL21 (DE3 ) In competent cells, ice bath for 30min, heat shock in 42℃ water bath for 90s, and immediately put on ice for 5min. Add 400μL of LB medium and incubate at 37°C for 60min. Spread LB / ...
Embodiment 3
[0052] Example 3. Functional verification of migratory locust chemosensory protein
[0053] 1. Fluorescence detection and binding experiment
[0054] The fluorescence emission spectrum was recorded with Jasco FP-750 fluorescence chromatograph at 25°C. The container used is a quartz cup with a width of 1 cm and a wall thickness of 5 nm. The LmigCSP protein expressed in Example 2 was dissolved in 50 mM Tris-HCl with a pH of 7.4 to a final concentration of 5 μmol / L. The endogenous ligand oleoamide extracted from the wing was dissolved in a 1 mM methanol solution, and the concentration was increased from 2 μM to 20 μM. Excite 1 μM of the LmigCSP expressed in Example 2 at 295 nm, and measure the fluorescence intensity of tryptophan in the LmigCSP expressed in Example 2 at the emission spectrum of 300-360 nm. The fluorescence quenching of tryptophan was measured when the concentration of oleoamide was 4-20 μM. Fluorescence detection results such as Figure 5 Shown. The results show that...
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