家蚕胰岛素信号传导途径中上游基因的克隆与表达模式

【作者】 刘艳；

【导师】 黄原； 李胜；

【作者基本信息】 陕西师范大学 ， 动物学， 2008， 硕士

【摘要】 胰岛素作为一种具有多种生物学效应的激素,除了在人们所熟知的蛋白质、脂肪、糖原的代谢中发挥重要的调节作用外,在调控细胞生长、增殖,保证正常的生长发育等方面也有重要的作用。近10年来,胰岛素信号传导途径对细胞大小和数目调控的研究取得了突破性的进展,弄清楚了整个信号传导途径中的大多数基因,关键分子的功能及其磷酸化调控。整个胰岛素信号传导途径是在线虫、果蝇和老鼠等模式动物中发现的,是一条相对保守的信号传导途径。家蚕是新兴的遗传与发育研究的模式昆虫,是除果蝇以外最好的昆虫遗传学研究材料。利用家蚕研究昆虫生理和发育生物学可以克服以果蝇作为模式生物的一些缺点:家蚕个体大小适中,发育周期相对较短,繁殖能力较强,容易饲养,便于进行解剖、培养、生理测定等实验操作。家蚕中的胰岛素类似物是家蚕素(bombyxin);虽然家蚕基因组框架草图已经完成,但是很少有家蚕bombyxin的分子作用机制和胰岛素信号途径的相关报道。脂肪体是昆虫的营养代谢中心和能量贮存中心,胰岛素信号可能既快速地调控昆虫脂肪体的生理功能,又长期缓慢地调控其器官发育。为了进一步研究家蚕胰岛素信号传导途径对脂肪体生理功能和器官发育的分子机理,本毕业论文已得出以下结论:1家蚕胰岛素信号传导途径中上游基因IRS、PI3K110、PI3K60、PTEN、PDK和Akt同人、果蝇、伊蚊和赤拟谷盗的对应基因比对表明,它们编码的氨基酸序列和昆虫相对应的序列相似性很高,和人的序列比较相似性相对较低。其中,家蚕和其它昆虫的PI3K60和PTEN具有相同的蛋白质结构域,和人PI3K60和PTEN的蛋白质结构域有较大的差别,昆虫之间的也有较大差异:而Akt和PDK在这几个基因中最为保守。2检测家蚕中胰岛素信号通路中上游基因InR、IRS、PI3K110、PI3K60、PTEN、PDK和Akt在脑、神经节、生殖腺、马氏管、中肠、丝腺和脂肪体组织中分布及表达水平发现,相对于内参基因rp49,这些基因表达的绝对量都很低,但这些基因在脂肪体和脑中表达量相对较高。1)除了Akt外,其它几个基因均在脂肪体中有较高水平的表达;2)脑中Akt表达水平较高,其它几个基因也有较高水平的表达;3)在丝腺中所有基因的表达水平都很低。3检测从3龄1天到成虫2天,家蚕脂肪体中InR、IRS、PI3K110、PI3K60、PTEN、PDK和Akt的表达水平发现,InR、IRS、PTEN和PI3K110的表达量都分别在3龄末期、4龄末期和5龄末期出现了明显的峰值,而Akt、PDK和PI3K60在预蛹之前的表达量都非常低,没有显著的变化。4 5龄1天的家蚕在受到饥饿刺激后,脂肪体中InR、IRS和PDK都有大幅度升高,PI3K110、PI3K60和Akt的表达水平都上升了1倍,PTEN的表达量基本没有变化。5龄5天的家蚕受到饥饿刺激后,所有基因的表达水平都没有非常显著的变化。这说明5龄1天家蚕脂肪体中胰岛素信号传导途径中上游基因的表达水平对于饥饿刺激更加敏感。更多还原

【Abstract】 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.更多还原