Silkworm Insulin Signal Transduction Pathway Upstream Gene Cloning And Expression Patterns

Insulin,as a multiple-function hormone,plays an important role in the regulation of protein,lipid and carbohydrate metabolism.Insulin is also essential to modulate cell growth and proliferation and to ensure normal body growth and development.During the last decade,many progresses have been made in understanding how insulin signaling pathway regulates cell size and cell number,including gene identification,elucidating the physiological function and phosphorylation regulation of key molecules involved in the pathway.Most of the insulin signaling pathway was explored in the model animals such as the worm(Caenorhabditis elegans),the fruitfly(Drosophila melanogaster),and the mouse(Mus musculus).The insulin signaling pathway is comparatively conserved during the evolution in the animal kingdom.The silkworm(Bombyx mori) is a new model insect in the studies of genetics and developmental biology.Except for Drosophila melanogaster,Bombyx mori is the best model for insect research. The silkworm has several advantages in the studies of insect physiology and developmental biology, for example:a suitable body size,a relatively short lifespan,a strong reproductive ability and conveniences for maintenance,dissection,culture,bioassay and other experimental operations.The homologue of insulin in B.mori is bombyxin.Although the draft map of silkworm genome has been completed,studies on the molecular mechanism of bombyxin and insulin signaling pathway in B. mori is very rare.Fatbody is the center for nutrient metabolism and energy storage in insects.The insulin signaling might be important to regulate its physiological function in a short-term and rapid manner and its organ development in a long-term and slow manner.To further understand how insulin signaling regulates fatbody function and development in B. mori,several conclusions might be drawn below based on the experiments conducted in this master thesis:1.First,the amino acid sequences in the upstream genes of the insulin signaling pathway(IRS, PI3K110,PI3K60,PTEN,PDK and Akt) in B.mori have very high similarity to those of D. melanogaster,Aedes Aegypti and Tribolium castaneum,and comparatively low similarity to those of human.The protein domains of PI3K60 and PTEN are the same in insects,but are slightly different from those of human.Akt and PDK are the mostly conserved genes in this pathway.2.Second,compared with the control gene rp49,the absolute expression levels of the upstream genes of the insulin signaling(InR,IRS,PI3K110,PI3K60,PTEN,PDK and Akt) are low in all tissues,including brain,nerve cord,testis and ovary,malpighian tube,midgut,silk gland,and fat body.These genes have the highest expression levels in brain and fat body.1) Except for Akt, expression levels of the other genes are comparatively high in fat body;2) Akt is highly expressed in brain,and other genes are also comparatively highly expressed in this tissue;3) All genes have the lowest expression levels in the silk gland.3.Third,from day 1 of the third instar to day 2 of the adults,the expression levels of InR,IRS, PTEN and PI3K110 in the fat body reach a peak at the end of each larval stage.In contrast, before pupariation,the expression levels of Akt,PDK and PI3K60 in the fat body are always low and have no significant variation.4.The expression levels of InR,IRS and PDK increase dramatically after 24 hours of starvation on day 1 of the fifth instar,PI3K110,PI3K60 and Akt increase～1-fold,but PTEN has no significant change.However,on day 5 of the fifth instar,the expression levels of all upstream genes in the insulin signaling pathway do not change dramatically after 24 hours of starvation.It indicates that the expression levels of those genes in the fat body are much more sensitive to starvation on day 1 than day 5 of fifth instar.