Cloning And Sequencing Of MT CDNA Gene In Housefly Larvae

Basing on the research of housefly MT by the Insect Resource Institute of Hua Zhong Agricultural UnivorsiLy, We wont further to explore cloning cDNA sequence of housefly MT gene. In the experiment, the first instar housefly larvae' metallothionein mRNA expressing reach maximur. after induced by Cd (2mM) for 48 hours, then total RNA was extracted with guanidinium isothiocyanate, and mRNA was isolated by the PolyATtract mRNA Isolation Systems surplied by Promega company. The isolated mRNA was used as template. Two specific primers were designed and synthesized based on the conservative sequence contrasted to Drosophila and other species MT gene by computer retrieving, which were named primerl and primer2, The sequence of primer1 was: 5' CAA GTG AAT CAT CTC AGT GC3' ,and that of primer2was:5' TGT AGA GAG ACA AGA TGC AG 3' . RT-PCR technique was adoped to synthesizing the first-band of cDNA, the second-band of cDNA and expanding DNA directly only by one step. The expanding products were recovered by the PCR Fragment Recovery Kit, and then were cloned to pMD18-T vectors. Four white blots were selected randomly through anti-Amp and a -complementation, and then the recombinant plasmids were identified by Restriction analysis of EcoR I and Hind IIIand PCR expanding. Two positive clones were Sequenced, and the results showed that its nuclcotidc sequence includes an open reading segment which codes for a 45-amino acids protein and three endonuclcase sites which arc1 BgII , BamH I and BgI II, This protein was identified as metallothionein based on its characteristic described above and its similarity(85%) to the MTn gene of Drosophila: the 10 cysteine residues present occur in five pairs of Cys-X-Cys, X is Serine, Valine, Ilistidine or Lysine. This verified the conservativity of MT in the evolution. Deduced amino acid composition results showed that themolecular weight is 5456. 8Da and the content of cysteine is the highest which attain 22.2%, following are Lysine (8.0) and Serine(7.7%). However, there are aromatic amino acid residues and Histidine in the amino acid sequence such as Tyrosine(3. 3%), Phenylalanine(3.0%) and Histidine(2. 8%), which is similar tothe MT protein of Ostrea edulis while different from that of Drosophila and mammalian (no aromatic amino acid and Histidine ) .Comparison of the deduced amino acid sequence of housefly MT with other species' MT showed that its identity with Drosophila was highest, attain 65%; the different metallothioneins, within a part species of invertebrate, were 35%-41% isologous; Its identi ty wi th the MT-II of green monkey was 41% and that with the human MT-II was 35%.