关键词: 棉铃虫, 中肠, cDNA文库, 表达序列标签（EST）, 序列分析

Abstract: Midgut is the main target for Bacillus thuringiensis (Bt) action, and a number of insect midgut proteins have been proposed as putative Bt toxin receptors. In order to study the resistance mechanism of the cotton bollworm, Helicoverpa armigera to Bt, we constructed a larval midgut cDNA library of the cotton bollworm using the Switching Mechanism at 5′ end of the RNA Transcript (SMART）technique. The total RNA of 5th instar larval midgut was extracted and the double-stranded cDNA synthesized. After the normalization treatment, cDNAs were digested and ligated into vector, and then the recombinants were transformed into competent cells. The titer was tested and the cDNA library was amplified and sequenced. The quality evaluation showed that the library had a complexity of 2×10⁶ pfu/mL, and the recombination rate was 100%. The average length of inserted cDNA fragments was over 1 000 bp, and 50% fragments were in the full-length form. A total of 1 098 expressed sequence tags (ESTs) were generated successfully after editing and trimming the vector and ambiguous sequences, and 789 unigene sequences were identified, including 132 contigs and 657 singlets. The assembled 789 ESTs were analyzed with Blast in NT, NR and SWISSPORT database of NCBI. The Blast analysis showed that 218 ESTs (27.62%) had no comparable sequences in databases, 119 ESTs (15.08%) had no definite annotations, and the rest 452 ESTs (57.29%) had high homologies with the available sequences, which had definite annotation with over 300 protein products. Through this study, a high-quality cDNA library of the larval midgut of H. armigera has been constructed, which will be a useful tool for studing gene functions in H. armigera midgut.

Key words: Helicoverpa armigera, midgut, cDNA library, expressed sequence tags, sequence analysis