Identification And Characterization Of The DHODH In Silkworm,Bombyx Mori

Pyrimidine nucleotides are abundant molecules with essential functions in a multitude of biochenmical processes.They are the building blocks for the direct synthesis of DNA and RNA that function in information storage and retrieval within the cell,but they were also directely involved in carbon metabolism providing energy-rich precursors for a lot of synthetic reaction such as UDP-glucose as a precursor in sucrose or cell wall biosynthesis.De novo pyrimidine biosynthesis,which is also referred to as the "orotate pathway",is one of the most important methoblism pathways.Dihydroorotic acid dehydrogenase(DHODH),as the fourth enzyme of the pyrimidine,can oxidize dihydroorotate to orotate.The main results are as follow:1.Identification and cloning of BmDHODH in silkworm,Bombyx mori.BmDHODH was identified through the silkworm genome database(SilkDB).Complete cDNA was obtained using PCR amplification and rapid-amplification of cDNA ends(RACE),and the result was confirmed through amplification of the complete ORF.To analyze DHODH sequences between silkworm and other species,a phylogenetic tree was constructed which showed that DHODH was conserved from invertebrates and vertebrates.Similar to other species,BmDHODH sequence contained a DHOD-2-like domain and a transmembrane domain.No signal peptide sequence was shown,which meant it belonged to a non-secretory protein.2.The expression profile of BmDHODH in silkworm,Bombyx moriWe analyzed the expressed-sequence tag(ETS)database from silkworm at stage of 3-day-old 5th instar larvae,and found that BmDHODH was highly expressed in silkworm,including testis,ovary,head,and silk gland.Next,the expression profile of BmDHODH in different developmental stage was analyzed.The result showed that BmDHODH was commonly expressed throughout the whole developmenttal stages.The BmDHODH expression level in pre-molting period was higher than other stages.3.Down-regulation of BmDHODH by DHODH specific inhibitor leflunomideAs a low molecular weight compound,leflunomide can specifically inhibit DHODH activity.This would cause de novo pyrimidine nucleotide synthesis reduction[9-12].After leflunomide treatment,cell growth and proliferation were dramatically inhibited.At the same time,it also induced cell cycle arrest at G2 phase,with a significant decrease of cyclin B and cdc2,which are the determinant of G2/M transition in the cell cycle[13,14].4.Knockdown of BmDHODH by dsRN A interferenceTo further investigate the function of BmDHODH in silkworm.dsRNA interference was employed to knockdown BmDHODH expression.After dsRNA interference,cell growth and proliferation were dramatically inhibited.At the same time,it also induced cell cycle arrest at G2 phase,with a significant decrease of cyclin B and cdc2,which are the determinant of G2/M transition in the cell cycle.