Cloning And Evolutionary Analysis Of Members Of CYP6 Gene Family From Philotrypesis Spp.

Cytochrome P450 monooxygenases, distributing widely in bacteria, fungi, plants and animals, play important roles in the detoxification of exogenous compounds and endogenous compounds. All cytochrome P450s (P450s) make up of an ancient gene superfamily, with each cytochrome P450 designated CYP followed by a family, subfamily and isoform number which may share a common ancestor. CYP6 gene family, a gene family of the P450 supergene family, is specific to the insect and most insect CYP6s are involved in metabolic detoxification of host plant allelochemicals toxicants, insecticide. Every P450 gene has some conserved regions and variable regions. The conserved regions, suffering from purifying selection, are so essential for the function. However, the variable regions, subjected to adaptive evolution and neutral random drift, contribute to the divergence of the P450s. The possibility that the P450s performing such tasks could be broadening or changing substrate specificity in accordance to environmental changes suggests that they could be under adaptive evolution. In fact, evidence for adaptive evolution in encoding sequence is restricted to the phylogenetically unstable P450 genes employed the software PAML 3.14 by Thomas.Gene duplication leading to a new gene functions and the divergence of gene family is a common evolutionary event at the molecular level. An emerging trend in the study of the evolution of multi-gene families is observation of rapid, adaptive evolution driven by positive selection following gene duplication. The non-synonymous substitution to synonymous (silent) substitution rate ratio (ω=dN/dS) provides a sensitive measure for evaluating selective pressure for the protein-coding genes, withω<1,ω=1, andω>1, representing negative selection, neutral evolution, and positive Darwinian selection (adaptive evolution), respectively. Specially, Site-specific model is vigorous to evaluate selective pressure on given genes.In the fig/fig wasp symbiosis, both food and habitats of fig wasps are provided by figs; and fig pollination is only dependent on fig wasps. The obligate mutualism is an excellent model for the study of such evolutionary topics as adaptation, evolution of virulence, sympatric speciation, sex ratio, etc. Besides highly species-specific fig pollinators, figs also host some non-pollinating fig wasps in the enclosed fig cavity. Fig wasps have adaptive evolution on their morphological characters and lifestyles due to their long-time hosted life in the syconia of figs.Cytochrome P450 proteins play a critical role in plant–insect interactions for insects. The metabolic detoxification of host plant allelochemicals is the main mechanism to adapt the host plants for herbivorous insects. So far, there is no paper reported about the CYP genes from fig wasps. Our study focused on amplifying the genes and analyzing their characteristics. In this context, we aim to discuss the following two questions: (1) With the special habitat, is the expansion of CYP6 gene of Philotrypesis the result of gene duplication? (2) What are differences of natural selection pressure on the encoding regions? Furthermore, which is the evolutionary pattern of the Philotrypesis CYP6 sequences: adaptive evolution, neutral evolution or purifying evolution?Nine DNA fragments sequences encoding cytochrome P450 were amplified from Philotrypesis spp.: CYP6CK1,CYP6AQ2,CYP6AQ3,CYP6AS20,CYP6AS21,CYP6AS22,CYP6AS23,CYP6AS24,CYP6AS25. These sequences include helix I and heme-binding regions which are important functional regions for all P450 genes. We run MEGA4.0 and PAML4.0 to constructed phylogenetic trees and test the selection pressure on the encoding region of the members from CYP6AS subfamily. The results revealed that the expansion of CYP6AS, CYP6AQ and CYP6CK subfamilies may be the result of gene duplication. It is very likely that serial gene duplication events had happened before the separation of the Philotrypesis sp., Philotrypesis pilosa, Philotrypesis sp.1, Philotrypesis sp. 4 with the 100% bootstrap value of the branch in the phylogenetic tree. The CYP6AS subfamily members from fig wasps are suffering from purifying selection and the encoding regions present high conservative tested by the branch-site model which based on the maximum likelihood method.