Molecular Cloning, Recombinant Expression Of Cytochrome Cyp4Mandcyp6Ab From Mamestra Brassicae Linnaeus

Cytochromes P450or CYP genes constitute one of the largest families of genes with representatives in virtually all living organisms, from bacteria to protists, plants, fungi, and animals. The cytochrome P450monooxygenaases constitute a large and complex superfamily of heme-thiolate proteins. In insect, P450enzymes are found in virtually all tissues, where they are involved in processes that are vital for insect growth, development, and reproduction, including ecdysteroid and juvenile hormone biosynthesis and aspects of ecdysteroid inactivation, as well as metabolism and detoxification of xenobiotics. P450enzymes also play important roles in adaptation of insects to toxic compounds in their host plants and are involved in metabolism of all commonly used insecticides. P450enzymes bind molecular oxygen and receive electrons from NADPH to introduce an oxygen atomto the substrate. Reactions catalyzed by P450s include oxidative transformations, such as monooxygenations, dehydrogenations, and roxidase-type oxidations, and non-oxidative reactions, such as dehydrase activity, nitric acid reductase activity, and isomerization. The isolation and characterization of insect P450s is a critical first step towards understanding the P450s involved in these important metabolic processes. Such studies have provided great insight into insecticide resistance, and insect development at biology and physiology.In this study, Total RNA was isolated from the fifth instar larvae of Mamestra brassicae.. The cDNA sequence of Mamestra brassicae CYP4M and CYP6AB were cloned by RT-PCR and rapid amplification of cDNA ends (RACE)The Mamestra brassicae CYP4M cDNA,1909base pairs in length, contained an open reading frame of1572base pairs, coding for a polypeptide of523amino acid residues with a predicted molecular weight of60.9kDa and p18.65. The cDNA sequence has been deposited in GenBank with accession No. JF731346. The putative protein contained the classic heme-binding sequence motif F××G×××C×G (residues461-470) conserved among all P450enzymes as well as other characteristic motifs of all cytochrome P450s. CYP4M was cloned into the expression vector pET21b and expressed in E. coli (BL21) host cell. Induced by IPTG, the protein was expressed and detected in the cells.The Mamestra brassicae CYP6AB cDNA,1966base pairs in length, contained an open reading frame of1536base pairs, coding for a polypeptide of512amino acid residues with a predicted molecular weight of58.8kDa and pi8.43. The cDNA sequence has been deposited in GenBank with accession No. JQ901385. The putative protein also contained the classic heme-binding sequence motif F××G×××C×G (residues446-455) conserved among all P450enzymes as well as other characteristic motifs of all cytochrome P450s. The CYP6AB of Mamestra brassicae was cloned into the expression vector pET21b and expressed in E.coli (BL21) host cell.