Gene Cloning, Gene Expression And Physiological Function Of Laccase1and Laccase2from The Insect Ostrinia Furnacalis

Laccase belongs to the multicopper oxidase family. Insect laccase catalyzes the oxidation of catechols to form quinines, which then cross-links with nucleophilic side chains of amino acids and may also polymerize to cause the dehydration of cuticle. Laccase strengthens and stabilizes the cuticle during molting, playing an important role in insect growth and development. Therefore, laccase attracts people’s attention because of its potential as pesticide target. However, the expression pattern in all tissues and development stages is less understood. In this thesis, the laccase-1and laccase-2genes from Ostrinia furnacalis were cloned, and their expression pattern and physiological function were studied as well. The main contents of this study include:(1) The cDNA sequence of OfLacl from O. furnacalis was cloned by using PCR and RACE. The full length cDNA of OfLacl is3065bp, containing an ORF of2403bp encoding800amino acid residues with a predicted molecular weight of90.6kDa and an isoeletric point of5.34. According to the alignment with other insect laccases, the OfLacl showed higher identity (more than70%) to both BmMCOl from B.mori and MsLacl from M.sexta, but lower identity (less than50%) to DmMCOl from D.melanogaster, AgMCOl from A.gambiae and TcLacl from T.castaneum. The gene transcriptional level of OfLacl in different tissues was analyzed using semi-quantitative PCR. The transcriptional level of OfLacl at different developmental stages of O. furnacalis and its response to ecdysone (20E) treatment were investigated by using real-time PCR. The results showed that OfLacl was mainly expressed in midgut and Malpighian tubule, less expressed in epidermis and trachea, and rarely expressed in fat body, testis and silk gland. As far as the expression levels at different developmental stages were concerned, OfLacl was highly expressed in adult, infering it might play an important role in adult. The expression of OfLacl was increased at24h after20-hydroxyecdysone(20E) treatment, indicating that the expression of OfLacl was under the regulation of20E.(2) The cDNA sequence of OfLac2from O. furnacalis was cloned by using PCR and RACE. The full length cDNA oiOfLad is3405bp, containing an ORF of2283bp encoding760amino acid residues with a predicted molecular weight of84.0kDa and an isoeletric point of6.43. According to the alignment with other insect laccases, the OfLac2showed higher identity to BmLac2(94%) from B.mori and MsLac2(92%) from M.sexta, but lower identity to DmLac2(76%) from D.melanogaster, AgMCO2(77%) from A.gambiae and TcLac2(78%) from T.castaneum. The gene transcriptional level of OfLac2in different tissues was detected by using semi-quantitative PCR. The transcriptional level of OfLac2at different developmental stages of O. furnacalis and its response to20E treatment were analyzed by using real-time PCR. The results indicated that OfLac2was mainly expressed in midgut and silk gland, less expressed in epidermis and trachea, and nearly undetectable in testis, Malpighian tubule and fat body. As far as the expression levels at different developmental stages were concerned, OfLac2was mainly expressed at prepupa stage, and the expression of OJLac2was slightly up regulated in the last day of each instar, speculating that OfLac2may have a key role in cuticle tanning after ecdysis. The expression of OJLac2was up-regulated at12h after20E treatment, indicating that the expression of OfLac2was under the regulation of20E. The RNAi experiment was performed to investigate the biological function of OfLacl during insect development. The result showed that the injection of dsOJLac2did not disturb the process of larval-pupal metamorphoses, only caused a small amount of larvae with head unable to detach from the body and abdominal epidermis unable to tan normally.(3) To obtain amount of the laccase, the ORF of OfLac2without the signal peptide was cloned into the yeast expression vector pPIC9between the restriction sites SndB I and Not I with a C-terminal six-histidine tag in frame, and then were transformed into the Pichia pastoris host strain GS115. The recombinant expression of OfLac2was not successful.