Abstract: 【Objective】 The objectives of this study are to clone two genes encoding OfLac1 and OfLac2 from Ostrinia furnacalis, and to predict physiological functions of these two genes based on the stage-specific and tissue-specific expression patterns of the two genes during development as well as their responses to the ecdysone (20E) treatment. 【Method】 The first fragment of OfLac1 or OfLac2 was amplified by RT-PCR using degenerated primers designed according to the conserved amino acid sequences of the other insects’ Lac1 and Lac2, respectively, and cDNAs generated from RNA isolated from O. furnacalis at pupa stage as template. The 3′ and 5′ regions of OfLac1 or OfLac2 were obtained by RACE using gene specific primers designed according to the first fragment of OfLac1 or OfLac2, respectively. Seven different tissues including epidermis, fat body, trachea, midgut, Malpighian tubule, silk gland and testes were collected from the fifth-instar day-4 larvae of O. furnacalis and the expression levels of OfLac1 and OfLac2 in these tissues were analyzed through semi-quantitative PCR. Twenty-nine samples were collected at different growth periods of O. furnacalis from egg to adult and the expression levels of OfLac1 and OfLac2 were analyzed by using real-time PCR. The fifth-instar day-2 larvae of O. furnacalis were treated with ecdysone (20E) and samples were collected at 1, 4, 8, 12 and 24 h post treatment. The expression levels of OfLac1 and OfLac2 were then analyzed by using real-time PCR. 【Result】 The full-length cDNA of OfLac1 and OfLac2 from O. furnacalis were obtained. The cDNA sequence of OfLac1 gene was 3 065 bp, containing a 5′-untranslated region (5′-UTR) of 222 bp and a 3′-untranslated region (3′-UTR) of 440 bp. It contained an ORF of 2 403 bp encoding 800 amino acid residues with a predicted molecular weight of 90.6 kD and an isoeletric point of 5.34. OfLac2 was 3 405 bp in length, containing a 5′-UTR of 162 bp and a 3′-UTR of 960 bp. The ORF of OfLac2 was 2 283 bp, encoding 760 amino acid residues with a predicted molecular weight of 84.0 kD and an isoelectric point of 6.43. OfLac1 was predicted to contain an N-terminal signal peptide with 22 amino acids in length and OfLac2 was predicted to contain an N-terminal signal peptide with 23 amino acids. The expression pattern analysis revealed that OfLac1 was mainly expressed in midgut and Malpighian tubule, and low amount of OfLac1 transcripts could also be detected in trachea and epidermis. During development, OfLac1 was mainly expressed in adult, and the expression level of OfLac1 during larval and pupal stages was low. OfLac2 was mainly expressed in midgut and silk gland, low amount of OfLac2 transcripts could also be detected in trachea and epidermis. During development, OfLac2 was mainly expressed at prepupa stage during the larval-pupal molting. The expression of OfLac2 was slightly up-regulated in the last day of each instar. The expression levels of OfLac1 and OfLac2 were significantly increased at 24 h after 20E treatment. 【Conclusion】 Two genes encoding OfLac1 and OfLac2 were cloned from O. furnacalis. The expression patterns of OfLac1 and OfLac2 were different, but they were both up-regulated by 20E. OfLac2 was related with the cuticle tanning during molting.

Key words: Ostrinia furnacalis , laccase , gene cloning , expression analysis