Clone And Analysis Of Phenoloxidase 1(PPO1) Intron Sepuence In Diamondback Moth Plutella Xylostella

Phenoloxidase(PO, EC 1.14.18.1) is a structure complexed copper enzyme, it widely exists in animals, plants, insects, microorganism and human. It plays important roles in insect physiological development and endogenous immune, such as insect melanism and wound healing, etc. The Open Reading Frame(ORF) of PxPPO1 gene and its expression in different larvae and time were determined in diamondback moth in our group’s previous studies. In this paper, the whole PxPPO1 gene was cloned and analysed using the chromosome walking methods. The location and number of introns or exons were analysed according to the cloned sequence, and the introns’ AT% content, joints and base size were also calculated. All the information provided here would shed lights on the gene structure and functions in the future. The main content as follows:1. The primers were designed according to the comparison of homology of lepidopterous insect in GenBank. The conserved genomic DNA was used as template to amplify fragment within introns, and the fragment was sequenced by NCBI alignment. Based on the above fragment to design the downstream primers, another fargement was obtained. The 5’- end fragment was cloned based on the three upstream specific primers using chromosome walking method. Through several steps, stitch all those obtained fragments together as one complete sequence of PxPPO1 gene of 7202 bp of Plutella xylostella.2. The gene sequence analysis was carried out through online software EMBOSS Needle and NCBI BLAST program, the results showed that there are 12 exons, 11 introns and 407 bp 5ˊ end non-coding regions in this gene. The introns sequences were 4745 bp, which composed by 60.9% A+T and 39.1% G+C.3. Two introns, I3 and I9, were selected, using DNAMAN software, to predict the secondary structure, 5 ’splicing sites, 3’ end splicing site and branch site. The results showed that the two introns belong to the rules of eukaryotic cells mRNA splicing. All the joint sequence of introns and exons were compared, and the results indicated that the 3’ end of 8th intron is AC, the others introns’ 3’ end are GT-AG, that belong to the GT- AG regularity in eukaryotic gene splicing.4. CpG island was predicted by EMBOSS CpGplot software, the result showed that there is nocpgislandintheintrons.thepotentialtranscriptionfactorbindingsitesofgeneintronswereanalysedusingtfsearchsoftware,theresultsindicatedthatthereisonecaatboxinthe2 nd,4th,5thand7 thintron,thereisonetataboxinthe2nd,3rd,6thintron,thereistwotataboxinthe7 thintron.repeatmaskersoftwarepredicatedthegeneduplicationsequence,andtheresultsshowedthattherearetwosimplerepeatswith47 bpinthegene,whichare0.62%ofthegenesequcence.