Cloning And Sequencing Analysis Of Cytochrome P450 From Bemisia Tabaci In Insecticide Susceptible And Resistant Strains

Cytochrome P450-dependent monooxygenases (P450 enzymes) are a very important multienzyme complex. These monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds. It was discovered in 1958, because of its many foreign substances and endogenous metabolism, they had aroused widespread attention. The cytochrome P450 involved in insect growth, development, feeding and other processes, contibuting to insect resistance to insecticides and the formation of toxic substances on plant tolerance and improve in phytophagous insects and plants co-evolutionary process , cytochrome P450 played a significant role. At the molecular research on insect cytochrome P450, we can understand the mechnism deeply.Bemisia tabaci belonged to Homoptera of Section tabaci whitefly subfamily, originating in tropical and subtropical regions. In recent years, Bemisia tabaci is an economically important pest around the world. It had caused damage to many different crops..In this paper, the main results obtained were as follows:1. Identifying the insecticide resistance difference between the field and breeded in the interior of susceptible strains of B.tabaci in Fuzhou region.2. Mitochondrial cytochrome oxidase I (mt CO I) gene sequences were utilized as markers to identify biotype of Bemisia tabaci used in our study in Fuzhou region.The result indicated that Bemisia tabaci used in our study was of the biotype B.3.In this study, we used degenerate primers based on conserved P450 helix I and heme-binding regions to amplify P450 cDNA sequences in B. tabaci, and obtain a sequence numbered 100.Based on NO.100 sequence to design specific primers GSP,Using GSP and universal primers AUAP, and utilizing 3 'and 5'RACE ( Rapid Amplification of cDNA Ends ) techniques to clone the fragments of cytochrome P450.We obtained a 436bp fragment and a 519bp fragment, numbered 101 and 102, respectively. After these three sequences had been spliced , a fragment of 1002bp has been obtained, numbered 103, which include the stop codon TAA ,F--G---C-A, which is conserved in all cytochrome P450 and a putative"meander"-binding sequence (EXXR and PXRF) inhelix–K, so this sequence belong to P450s .After further sequence alignment , we presumed that this gene fragment belong to CYP6 families.4.We designed a pair of specific primers based on NO.103 sequence. By using the pair of specific primers, we obtained two fragments from two B. tabaci strains, resistant and susceptible to iinsecticide,numbered 107 and 108 respectively. According to107 and 108 amino acid sequence analysis , we found two amino acid residues are different .If these two amino acids on the conversion will bring the possibility of involvement in resistance have to be further verified .