Gene Cloning And Sequence Analysis Of Bt Toxin Receptor-APN And CAD In The Midgut Of The Pink Stem Borer, Sesamia Inferens (Walker)

The pink stem borer(PSB), Sesamia inferens (Walker) is one of the important rice pest in the main rice planting area in China. Since 1990, with the expansion of area of hybrid rice, as well as changes in climate and rice cultivation system, the population of S. inferens has been increasing gradually. In addition, the use of some chemical insecticides (e.g., fipronil) with high control efficacy for Chilo suppressalis, but minor or no lethal effect on S. inferens, has been an important factor contributing to the increase in population density. The occurrence of transgenic insect-resistant rice expressing Cry toxins from Bacillus thuringiensis (Bt) has provided new strategy for the control of S. inferens. However, potential resistance risk of pests to Bt crops is a major factor to influence its sustainable utilites. Therefore, it is very necessary to illuminate the resistant mechanism of insect to Bt crop and implement the strategy of resistance management. In this study, gene cloning and sequence analysis of aminopeptidase N (APN) and cadherin-like (CAD) in the midgut of S. inferens were conducted and the results would provide the scientific basis for the molecular resistant mechanism of insect to Bt crop. The main results were shown as following.1. The full length cDNAs-encoding Aminopeptidase N (APN) was cloned by Reverse transcription PCR combined with RACE techniques from the midgut of S. inferens. It was the APN3 in APN family and its GenBank accession no. was HQ636624. The full length of APN3 was 3411bp, the opening reading frame (ORF) of APN was 3021bp, encoding a protein of 1006 amino acid residues. The predicted molecular weight was 114 kDa and isoelectric points was 4.95. The contents of GC, T, C, A and G were 43.6%, 27.7%, 28.7%, 23.5% and 20.1%, respectively. Sequence analysis showed that the coding areas of 5 'end, 3' end and ORF were 1-49, 3072-3411 and 50-3070 amino acid residues, respectively. The starting anticodon of 5'end were ATG and the end terminated anticodon of 3' end were TGA. A hydrophobic signal sequence with 18 amino acids in the N-terminal region, cracking sites was located between 18 and 19 (ATA-LS). One potential N-glycosylation site (N-glycosylation site) (734NETF737) and twelve potential O-glycosylation sites (O-glycosylation site) were found in the amino sequence. The deduced protein sequence was highly similar to that of Plutella xylostella with 93% identity and other lepidopteran APN protein.2. The full length cDNAs-encoding Cadherin-like(CAD) was cloned by Reverse transcription PCR combined with RACE techniques from the midgut of S. inferens. It was named CAD and its GenBank accession no. HQ636625. The full length of CAD was 5597bp, the opening reading frame (ORF) of CAD was 5217bp, encoding a protein of 1738 amino acid residues. The predicted molecular weight was 195kDa and isoelectric points was 4.36. The contents of GC, T , C, A and G were 51.7%, 21.1%, 25.7%, 27.2%, 26%, respectively. Sequence analysis showed that the coding areas of 5 'end, 3' end and ORF were 1-184, 5402-5597 and 185-5401 amino acid residues, respectively. The starting anticodon of 5 'end were ATG and the end terminated anticodont of 3'end were TAA. A hydrophobic signal sequence with 24 amino acids in the N-terminal region, cracking sites was located between 24 and 25 (AQG-SF). Three potential N-glycosylation site(N-glycosylation site) (⁹²⁹NVSY⁹³²,¹⁰⁵⁶NLTT¹⁰⁵⁹,¹⁶⁵³NETV¹⁶⁵⁶) ,O-glycosylation site(O-glycosylation site()634,1241,1348,1738)were found in the amino sequence. The deduced protein sequence was highly similar to that of Heliothis virescens and Plutella xylostella with 99% identity and other lepidopteran cadherin-like protein.