4.3. Cloning and Sequencing Analysis

Specific primers were designed and synthesized by Sangon Biotech Company (Shanghai, China) based on the nucleotide sequence of TrufOBP4. The sense primer was 5-TGTGACGCTTTACACTAA-3, and the antisense primer was 5-AGTCGTCTAATCAGGAAA-3. PCR studies were performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Jiangsu, China). The amplification of the TrufOBP4 cDNA fragment was performed on a Veriti Thermal Cycler (AB, CA, USA) with the following PCR procedure: 94 °C for 5 min; 30 cycles at 94 °C for 30 s, at 60 °C for 30 s, and at 72 °C for 30 s; and 72 °C for 10 min. The colonies were sequenced by Sangon Biotech Company (Shanghai, China). The cDNA sequence and deduced amino acid sequence of TrufOBP4 were analyzed using DNAMAN (Lynnon Biosoft, CA, USA). The putative N-terminal signal peptides were predicted using the SignalP V4.1 program (https://services.healthtech.dtu.dk/service.php?SignalP-4.1 (accessed on 10 January 2021)). The chemical and physical properties, secondary structure and hydrophobicity scales of TrufOBP4 were predicted using the online program tools ProtParam (https://web.expasy.org/protparam/ (accessed on 10 January 2021)), SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/ (accessed on 10 January 2021)), ProtScale (https://web.expasy.org/protscale/ (accessed on 15 January 2021)) and ESPript3.0 software [28]. The larger the hydrophobic value, the stronger the hydrophobicity of this amino acid [40]. ClustalW and MEGA 5.2 were employed for multiple alignments and the phylogenetic tree construction of TrufOBP4 with similar OBPs of other insect species using the neighbor-joining method with a No. of differences model and a pairwise deletion of gaps.