Construction Of The CDNA Library Of Spodoptera Exigua And Cloning A Fragment Of The Chitin Synthase

Chitin is a major component of insect cuticle and is synthesized from cytoplasmic pools of UDP-N-acetylglucosamine (UDP-GlcNAc) by chitin synthases (CHS). Though CHS is very important in the formation of insect cuticle and peritrophic membrane, little is known of CHS maybe due to its instability. By far only seven insect CHS have been completely cloned and sequenced.A cDNA library has been proven to be a rapid and efficient approach by which to identify the novel genes and analyze the function of genes. The mRNA of fifth-instar larva of the beet armyworm (Spodoptera exigua) has been extracted by using FastTrack? 2.0 Kit for isolation of mRNA. The OD₂60/OD₂80 was 2.0. The result showed that the mRNA was pure enough and undegraded. On the basis, a cDNA library of Spodoptera exigua was constructed by using ZAP Express? cDNA Synthesis Kit and ZAP Express? cDNA Gigapack? III Gold Cloning Kit perchased from Stratagene. In order to remove out the low molecular weight cDNA fragments shorter than 400bp, size fractionation of double strand cDNA was completed by Sepharose CL-2B. Primary titer of the library was 1.1×10⁵ pfu/ml and rate of the recombination was 97%. The titer of amplified library was 5.4×10⁷pfu/ml.A 988bp fragment of the chitin synthase I was obtained from the library. A 5207bp cDNA sequence (SeCHSl) was then obtained by assembly of the overlapping fragments gained either by previous resercheres or by the author in this paper. The putative translation product shared 91% similarity to tobacco hornworm(Manduca sexta) and also showed high homology to other insect chitin synthases. By compared with other insect chitin synthases, SeCHSl was considered as the full lenth sequence of type A of the chitin synthase, with an ORF of 4698bp.