Analysis On Bacterial Diversity In The Larval Intestine Of Hepialus Gonggaensis

Dongchongxiacao are traditional chinese medicinal materials, which have important medical and health care functions. But the natural resources are scarcity, because of the narrow distribution and the slow growth of life, high mortality rate of host larvae, deterioration of environment, as well as people's excessive digging. The intestinal microbial flora play an important action in larvae's development, nutrient and resistant to the diseases. In this paper, intestinal microflora diversity and predominant group of H. gonggaensis larval gut were systematically analyzed by traditional and molecular methods, It was expected to find out the key factors infected the host of Dongchongxiacao'development and antiadversity in larvae breeding, provide theoretical and technical support for the scale artificial process of the Dongchongxiacao.The main results were as follows:①The intestinal microbial diversity in the larval gut of Hepialus gonggaensis was studied by isolating the clone of bacteria, and identified by morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis method. There were 8 genera of bacteria were identified from 11 isolated bacterial populations. The dominant bacteria belonged to enterobacter in the intestine.②The molecular method of PCR-DGGE (denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V3 region gene was used to the microflora analysis. By 16S rRNA V3 region gene DGGE method analysis, eleven distinct bands were obtained from 16S rDNA amplificons. The bands were purified and sequenced. The sequences aligned with GenBank database and showed that they were belonged to 8 different genera of bacteria. Phylogenetic analysis showed that the sequences of bacteria belonged to the Proteobacteria and Firmicutes. The most dominant bacteria group was Carnobacterium in the gut and Bacillus followed by it. The different patterns were observed in different instars larvae guts from DGGE profiles, which might be caused by their different physiological development stages.③8 genera were obtained from intestine of H. gonggaensis by traditional culturing method and 16S rDNA analysis method respectively. But the two groups were not exactly same, and the dominant group was different also.④Equalized strategy was first used in microflora diversity analysis and an 16S rRNA library was established from intestinal microbiota of H. gonggaensis larvae by duplex-specific nuclease. The total DNA was purified from the larval gut of H. gonggaensis and 16S rDNA gene sequences were amplified using the bacterial universal primers and cloned. A total of 100 clones were analyzed by restriction fragment length polymorphism (RFLP), and 86 unique RFLP patterns were obtained with three restriction enzyme (HaeⅢ, MspⅠand RsaⅠ). Of these patterns, 47 clones were selected randomly for sequencing of their 16S rRNA genes. The sequences analysis showed that these 47 clones belonged to 13 families bacteria and some uncultured bacteria. High frequency intestinal microflora belonged to Carnobacterium sp. and Spiroplasma sp., and accouted for 13% and 12% of the liabrary respectively.⑤Compare the diversity of the different librarys constructed by the equalized and normal 16S rRNA library with the culturable bacterial isolated from content of intestine of H. gonggaensis larvae, the much higher bacterial diversity showed in equalized library. But there were also differences in the reported community compositions, and more phylogenetic groups were detected by an equalized 16S rDNA library. This suggested that a combination of molecular and traditional culturing methods can be used to analyze and monitor the diversity of intestinal microflora effectively, and that will give us more information of microorganism diversity.