| The alkaloids of mulberry leaves have a toxic effect on non-mulberry feeding insect,but have no effect on mulberry-feeding insects such as the economic insect silkworm Bombyx mori and the important mulberry pest Diaphania pyloalis Walker.It is speculated that B.mori and D.pyloalis may use the β-fructofuranosidase(β-FFase)expressed in larval intestine to avoid the toxic effects of mulberry sugar-mimic alkaloids.Our previous studies have shown that Bm Suc1(β-FFase gene of B.mori)has an important function on regulating sucrose metabolism in the intestine of the silkworm larvae.However,the recombinant protein of the β-FFase(Dp SUC1a)identified in the larva of D.pyloalis showed not activity in vitro.Recently,more and more reports proved that intestinal microorganisms have important effects on insect growth,development,reproduction and other aspects,such as helping the host to resist pathogenic bacteria and exogenous toxic substances.Moreover,studies have shown that insect β-FFase have high homology with bacterial β-FFases.It is possible that insects can obtain β-FFase gene via horizontal gene transfer from bacteria.However,it is unknown whether there is functional differentiation of β-FFase between B.mori and D.pyloalis and whether their intestinal bacteria are related to the sucrose metabolism of the larvae.In this study,we first compared and analyzed the diversity of intestinal bacteria between the silkworm(p50 strain)and D.pyloalis and between the wild-type silkworm(Nistari strain)and the transgenic RNAi strain(RNAi-Bm Suc1).Then,sucrose-specific selection medium containing alkaloids(DNJ)was used to screen the bacteria producing β-FFase from the larvae of silkworms(p50 strain)and D.pyloalis,and their physiological and biochemical identification and comparison were conducted.The followings are the main results:1)The 16 S r DNA sequencing results showed that the intestinal microorganism distribution of silkworm and D.pyloalis larvae was significantly different.The diversity of silkworm is higher than that of D.pyloalis.The gut diversity of the RNAi-Bm Suc1 silkworm is lower than that of the wild type,suggesting that interfering with the expression of Bm Suc1 had an effect on the gut bacteria of the silkworm.2)From the level of Phylum,Proteobacteria and Firmicutes are the dominant bacteria of wild-type silkworm and D.pyloalis,respectively.At the genus level,the dominant bacteria in the wild-type silkworm are Methylobacterium(Proteobacteria),while that in D.pyloalis and RNAi-Bm Suc1 silkworm are Enterococcus and Tyzzerella,respectively(both belonging to Firmicutes).3)By the sucrose-specific selection medium,two bacterial strains were screened from B.mori,and 9 strains were screened from D.pyloalis.Through gram staining and physiological and biochemical experiments,they belonged to Proteobacteria and Firmicutes,respectively.The result of DNS enzyme activity assay showed that all the cultured bacterial crude proteins displayed β-FFase activity.4)Cloning of β-FFase encoding genes from 6 intestinal bacteria strains with relatively higher enzyme activity,full-length open reading frames of total 10 β-FFase genes were amplified.This indicated that the intestinal tract of silkworm and D.pyloalis larvae have bacteria containing β-FFase genes,and most bacteria contain more than one β-FFase coding gene.In conclusion,the dominant bacteria between the silkworm and D.pyloalis are different maybe due to their hosts’ differentiaed developmental system and immune system.But the change of the dominant bacteria of the silkworm intestine after interfering Bm Suc1 indicated that the host can cope with defects in sucrose metabolism by regulating the community structure of its intestinal bacteria.Cloning and prokaryotic expression of β-FFase gene from D.pyloalis intestinal bacteria Klebsiella variicola provides a theoretical basis for the subsequent study of its involvement in host sucrose metabolism. |
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