Cloning, Sequencing And Analyses Of Alpha-Amylase Gene Of Bombyx Mandarina And Its Molecular Systematics Study

Bombyx mori is both an economically important insect and a model organism for the study of molecular genetics. The extensive study on Bombyx mori spurred the investigation of its consanguinity variety-Bombyx mandarina. As an abundant wild sericultural resource Bombyx mandarina is a treasure with much potential in research and development. It could increase the genetic diversity and be used as an important material for the study on the origin of silkworm. Up to now, extensive biological, chromosomal structure and variation, DNA polymorphism, breeding application as well as pest control studies have been conducted. Alpha-amylase is a functionally important enzyme for silkworm that feed on mulberry leaves. Although there are many studies on alpha-amylase of Bombyx mori, no reports on the alpha-amylase gene of Bombyx mandarina have been seen so far. In this investigation, the whole alpha-amylase gene of Bombyx mandarina has been cloned, sequenced and analyzed with that of the Bomyx mori. Additionally, based on the sequence of Intron I of alpha-amylase, a preliminary molecular systematics study has been performed on Bombyx mori and Bombyx mandarina.1. Sequencing of the whole alpha-amylase gene of Bombyx mandarina (Chongqing)Based on the published nucleotide sequence of alpha-amylase gene of Bombyx mori, seven pairs of primers were designed for the sectional amplification and cloning of the alpha-amylase gene of Bombyx mandarina. The combined sequence, i.e. the whole alpha-amylase gene of Bombyx mandarina, is 8903bp in length. Its AT content is 51.61%. The whole gene consists of five exons and four introns. The mean AT values for all the exons and all the introns are 49.35% and 62.23% respectively.There is an entire non-LTR retrotransposon in Intron II, which encodes a reverse transcriptase; also a segment in intron II is highly homologous with another retrotransposon.2. Sequence comparison of alpha-amylase gene of Bombyx mori and Bombyx mandarinaThe alpha-amylase gene structures of Bombyx mori and Bombyx mandarina are very similar, bothcomprised of five exons and four introns, with comparable length. The mean AT contents of exons and introns are not much different. The nucleotide sequence similarity of alpha-amylase gene of Bombyx mori and Bombyx mandarina is 90% and the putative amino acid sequence similarity is as high as 98%.In the alpha-amylase gene, the length of the non-LTR retrotransposon, located in Intron II, is equal in Bombyx mori and Bombyx mandarina, with a nucleotide sequence similarity of 98%; The length of the coded reverse transcriptase is equal in both nucleotide and amino acid sequence with a similarity of 98% also.Compared with Bombyx. mori, the exon regions of alpha-amylase gene of Bombyx mandarina have 13 transitions, 4 transversions and a 12bp insertions, while the intron regions have 362 transitions, 343 transversions, a 94bp insertions and a 56bp deletions. The whole alpha-amylase gene (including the 5'- and 3'-UTRs) of Bombyx mandarina has 380 transitions, 350 transversions, 113bp insertions and 57bp deletions.Statistics on code bias of alpha-amylase gene of Bombyx mori and Bombyx mandarina show that little difference exists between them.3. Sequence comparison of alpha-amylase gene of five insects.The nucleotide sequence similarity and the putative amino acid sequence similarity of alpha-amylase gene of Bombyx mandarina to those of Bombyx mori, Spodotera fruiperda, Ostrinia nubilalis on 25 and Drosophila subobscwa are 90%, 12%, 12%, 5% and 98%, 80%, 78%, 58% respectively. Those results indicate that Bombyx mandarina and Bombyx mori are closely related, while Bombyx mandarina and Drosophila subobscwa are relatively far related.4. Base composition comparison of codons of the alpha-amylase gene of Bombyx mori and Bombyx mandarinaIt was shown by base composition comparison of the codons that it is very similar in both Bombyx mori and Bombyx mandarina. The first base of codons clearly shows a bias for purines (59.90% for Bombyx mandarina and 5...