Gene Expression Patterns And Functions Of The Insect Chitin Synthases From Ostrinia Furnacalis

Chitin, an important structural component of insect exoskeleton, is widely distributed in the integument, trachea, head capsule, foregut, hind gut, crops, gland as well as peritrophic matrix. Insect chitin synthase is the essential enzyme that catalyzes the polymerization of N-acetylglucosamine to form chitin to meet the needs of insect molting. As chitin is not found in mammals and plants, insect chitin synthase has attracted much research interest because of its potential as specific target for designing eco-friendly pesticides.Insect chitin synthase includes two classes, class A (CHSA) and class B (CHSB). CHSA are expressed during various developmental stages and in different tissues. It was found to contain alternative splicing exons, which encode two isoenzymes that function mainly in the integument and trachea. CHSB is mainly expressed in the midgut during the feeding stages of insect. It contains no alternative splicing exons. Since the structure of chitin varies in different tissues, chitin biosynthesis with tissue specificity is still unknown.This dissertation focused on study of chitin synthases from the destructive pest Ostrinia furnacalis. It examined the possibility of alternative splicing exons in OfCHSA and the expression patterns and functions of the alternative spliced exons during insect development. It also examined the heterogeneous expression of OfCHSB and the subcellular localization of the recombinant OfCHSB. The major findings obtained this work are as follow:1) Both cDNA (GenBank ID:EU376026) and genomic DNA sequences (GenBank ID: EU563936) of OfCHSA were obtained. The deduced protein sequence indicated that OfCHSA contains nine highly conserved motifs that are speculated to contribute differently to the catalytic processes. A novel alternative splicing exon2was found in the genomic DNA sequence of OfCHSA besides the alternative splicing exon19. This alternative splicing exon was also found in Bombyx mori, but not in other insect species, indicating it may only exist in lepidopteran insects. The spliced exons of alternative exon2contain independent promoters and response differently to20E treatment;2) Real-time PCR showed that OfCHSA was expressed in newly laid eggs and during larval-larval molting, larval-pupal transition as well as pupal-adult metamorphosis. Its expression pattern showed tissue specificity toward integument. The phenotype resulting from the knockdown of OfCHSA by RNAi showed that OfCHSA was essential for larval-larval molting and larval-pupal transition. Injection of the5th instar larvae with20E led to an up-regulation of OfCHSA expression.3) Real-time PCR and RNAi phenotypes showed that the expression patterns and the functions of the four alternatively spliced exons (OfCHSA-2a, OfCHSA-2b,OfCHSA-19a and OfCHSA-19b) during the development of insect were different, suggesting that these exons may have different physiological roles during insect development;4) RT-PCR results showed that four exon combinations can be produced through alternative splicing, and these were2a-19a,2a-19b,2b-19a and2b-19b. These exon combinations were demonstrated to play different roles during insect development. Among all the alternatively spliced exon combinations,2a-related combinations were the dominating transcript of OfCHSA, while2b-related combinations were more sensitive to the hormone20E treatment than the2a-related combinations, though normally its expression level was kept in very low level.5) The cDNA (GenBank ID:DQ294306) and genomic sequences (GenBank ID: EU258740) of OfCHSB were obtained. The core promoter region of OfCHSB contains several binding sites for regulatory elements involved in ecdysone regulation pathway. Real-time PCR showed that OfCHSB was mainly expressed during the feeding stages in a tissue-specific manner towards the midgut;6) Expression of OfCHSB in the yeast Saccharomyces cerevisiae showed that OfCHSB and its truncated versions were mostly localized in ER. Expression of OfCHSB in the insect midgut cells CF203/2.5showed that OfCHSB and its truncated forms were co-localized with Golgi marker, indicating that the enzyme was transported through Golgi apparatus.