Isolation, Identification Of Intestinal Aerobic Bacteria From The Larva Of The Chinese Oak Silkworm, Antheraea Pernyi And Molecular Cloning, Expression Analysis Of α-amylase Of A. Pernyi

The Chinese oak silkworm (Antheraea pernyi), originated from China, is a kind of valuable agricultural economic insect which has a long history of development and distinct advantages of comprehensive utilization. Digesting and absorping foodstuff for A. pernyi plays an very significant role in improving tussah cocoon yield and the quality. The intestinal bacteria and enzyme gene related to the digestive of A. pernyi were researched. Eggs and different instars of A. pernyi larvae were selected as materials to isolate aerobic bacteria of A. pernyi with traditional culture identification methods and the molecular biology technology. And foll length of A. pernyi a-amylase gene cDNA was cloned. Semi-quantitative RT-PCR and Real-time PCR technology were used to detect the expression profile in A. pernyi, which was induced by Nosema pernyi. The results were as follows:1. A strain of Bacillus was separated from the eggs of A pernyi which indicated that the intestinal bacteria of A. pernyi may be passed in the form of vertical transmission.2. The intestinal bacteria of A. pernyi larvae was rich including Bacillus, Acinetobacter, Enterobacte, Staphylococcus, Streptomyces and Sporosarcina. The structures of the bacteria community in different ages had certain differences, but also had continuity.3. A kind of cellulase-producing bacteria was isolated from the intestinal of A. pernyi. Fermentation conditions for the cellulase-producing were optimized. The optimized fermentation conditions were as follows:fermentation temperature 30℃, inoculation quantity 4%.4. Sequences of a-amylase gene of A. pernyi was cloned which including an 1 794 bp open reading frame (ORF) encoded 597 amino acids. Amino acid sequence alignment with several kinds of a-amylase showed that a-amylase of A. pernyi shared significant similarity to other a-amylase of insect, with the highest identity (86%) to Danaus plexippus. a-amylase of A. pernyi was highly expressed in intestine of the 5th instar larvae and in the induced treatment groups, the expressions of a-amylase gene in intestine was increased first, and then decreased. And the highest levels can be obtained at 21 hours. These results suggested that a-amylase of A. pernyi was involved in the digestive system and immune system of A. pernyi.