Functional Research Of ?-fructofuranosidase In Glycometabolism Of The Silkworm Bombyx Mori

As a model lepidopteran insect,the silkworm Bombyx mori is an important silk-spinning lepidopteran insect which has been reared on mulberry leaves for thousands of years.Also,the silkworm-mulberry interact system is the classical model in the research of insects feeding.Mulberry leaves contain high concentration of sugar-mimic alkaloids,which are harmful to many insects but not to B.mori.The sugar-mimic alkaloids are strong inhibitors to a-glucosidase(EC 3.2.1.20),however,they do not affect the activity of?-fructofuranosidase(?-FFase,EC 3.2.1.26).BmSuc1encodes a midgut-and silkgland-specific expressed?-FFase of B.mori that was firstly found and cloned from an animal.BmSUC1 was considered to act as an essential midgut sucrase for B.mori larvae to against alkaloid in the mulberry leaves,while the function of BmSUC1 in the silkgland is clearly unknown so far.In order to verify the function of BmSuc1 in glycometabolism mechanism in midgut and silkgland,in this study,we established shRNA-mediated transgenic RNAi strain RNAi-BmSuc1,the followings are the main results:1.We firstly investigated sucrose hydrolysis activity in the larval midgut of B.mori within different a-glucosidase and?-FFase inhibitors and found that sucrase activity mainly stemmed from?-FFase,not?-glucosidase.2.After the shRNA screening in BmN cell line,we microinjected the best RNAi system into silkworm eggs and established RNAi-BmSuc1 transgenic strain.The results showed that after knocking down BmSuc1,the silkworm appeared the abnormal phenotypes,just like delayed larval development,reduced in body size,body weight and reduced in fat body.3.The activity investigation of several disaccharidases alone in the midgut demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut.The research about sugar distribution,total sugar and glycogen in the midgut,hemolymph and fat body demonstrated that after knocking down BmSuc1,the glucose and maltose in midgut and the glycogen and trehalose in fat body were all clearly decreased.We conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut,and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.4.We performed the analysis about BmSuc1 m RNA transcription,protein expression and sucrase activity during 5^th instar in anterior and middle parts of silk gland.We found that the BmSuc1 expression pattern was very similar to silk gland development pattern.So that we concluded BmSuc1 regulated the development of silkworm silk gland.Following,through the detection of sucrose hydrolysis activity,we found that not only BmSUC1 had the sucrase activity,it still had the activity of transfructosylase.The results about the variation of silk protein in silk gland after knocking down BmSuc1 implied that the BmSUC1 would contribute to the glycosylation of sericin.5.Last,the transcriptome analysis in midgut and silk gland indicated that,after knocking down BmSuc1,the glycometabolism genes and pathway were variation prominently,which further indicated that BmSuc1 is essential to the glycometabolism in silkworm.Moreover,we established the CRISPR/Cas9 system against BmSuc1 to better understanding the function of BmSuc1 in the biosynthesis process of silk proteins.