Abstract:

【Aim】 To lay a foundation for exploring the mechanism of digestive physiology of Hyphantria cunea (Drury) in host transformation. 【Methods】 We first cloned a serine protease gene by screening the H. cunea cDNA library. We then analyzed the expression patterns of this gene in different developmental stages of H.cunea by qPCR. The distribution and expression patterns of this gene in different tissues of the 5th instar larvae of H.cunea were analyzed by semi-quantitative RT-PCR and qPCR, respectively, and the expression patterns in the 4th instar larvae of H.cunea feeding on leaves of different host species (Populus deltoids, Cerasus serrulata var. lannesiana, Cerasus serrulata, Camptotheca acuminata and Platanus orientalis) were detected by qPCR. 【Results】 A serine protease gene HcSP1 (GenBank accession no.: MH663425) was successfully cloned from H. cunea. The open reading frame (ORF) of HcSP1 is 882 bp in length, encoding 293 amino acids, with the predicted molecular weight (MW) of 30.5 kD and the theoretical isoelectric point (pI) of 9.86. The predicted N-terminal hydrophobic region contains signal peptide consisting of 15 amino acid residues. The protein encoded by HcSP1 contains the enzyme activity catalytic center formulated with His, Asp and Ser residues, which is a typical feature of serine proteases. HcSP1 also possesses some typical features of putative trypsin precursors, such as containing a signal peptide, activation peptide and conserved N-terminus (IVGG). The NCBI BLAST alignment revealed that the amino acid sequence identities between HcSP1 of H. cunea and other lepidopteran serine proteases are between 50% and 70%. The qPCR results indicated that the expression of HcSP1 inH. cunea in different developmental stages showed dynamic change, increasing with the larval instar. Semi-quantitative RT-PCR results showed that HcSP1 was expressed in various tissues of the 5th instar larva of H. cunea including head, salivary gland, midgut, fat body, cuticle, Malpighian tubules and hemolymph, and qPCR results further revealed that this gene had the highest expression level in the midgut. The expression level of HcSP1 was significantly higher in larvae of H. cunea feeding on C. acuminata leaves than in larvae feeding on other host plants. 【Conclusion】 In this study, we obtained a serine protease gene HcSP1 inH. cunea, and detected its developmental and tissue expression patterns, and its expression levels in the 4th instar larvae of H. cunea feeding on leaves of different host species. The results lay a foundation for exploring the mechanism of digestive physiology of H. cunea in host transformation, and also provide new insight for the development of new management tools for H. cunea.

Key words: Hyphantria cunea, serine proteases, trypsin, sequence analysis, temporal and spatial expression patterns, host plant