Cloning Of Ubx And Wg Genes And The Phylogenetic Relationship Of Higher Taxa In Orthoptera

The order Orthoptera belongs to Neoptera, Pterygota, insecta, including the grasshopper, cricket, mole crickets, and so on, with more than 25,000 species known in the world and 2,360 species recorded in China. The phylogeny of the Orthoptera higher taxa has not been clear, as the two suborder systematics which is accepted by the majority was questioned by the molecular data.In this dissertation, the phylogenetic relationships among suborders of Orthoptera were analyzed based on two regulatory genes (Ubx and Wg). 117bp Ubx sequences of 3 insect and 549bp Wg (incloding the gaps) sequences of 9 insect were cloned by PCR, RT-PCR and Nested PCR. Nucleotides composition, substitution saturation and genetic distance were analyzed with MEGA, and the molecular phylogenetic trees were reconstructed by the microsoft MEGA, PHYLIP, MrBayes and PAUP based on the two regulatory genes, Ubx and Wg, the outgroup sequences and the data of insecta were download from GenBank.1. The Ubx sequences are completely conserved with 13 (11.1%) sites variation in Orthoptera. Total substitutions occured at the third codon position, thus does not cause the variation in amino acid sequences. The base composition of this Ubx fragment varied among the individuals, with a strong G+C bias (92.9%) at the third codon position versus a poor A+T bias (66.6%) at the second codon position. For Amino acid composition, Aspartic acis (Asp), Glycine (Gly), Proline (Pro), Serine (Ser), Valine (Val) and Tyrosine (Tyr) were not detected, and Arginine (Arg) is the highest with average content of 17.9%.2. The Wg sequences comprised of 549 nucleotides including the gaps, and with 375 (68.3%) conserved sites in Orthoptera. A total of 103 (56.3%) substitutions occured at the third codon position, while only 33 (18.0%) at the second codon position. The base composition of this Wg fragment varied among the individuals, with a strong G+C bias (96.8%) at the third codon position versus a poor A+T bias (52.9%) at the second codon position. For Amino acid composition, 12 species of Orthoptera covering of 20 kinds of amino acid types, in which Tryptophan (Trp) is the least with an constant content of 0.56%, followed by isoleucine (Ile), 1.91%. Arginine (Arg) is the highest with average content of 11.26%, followed by Glycine (Gly), 11.12 %.3. Analysis of substitution saturation both in Ubx and Wg genes suggested that saturation of Transition (si) and Transversion (sv) had occurred between certain pairs of the taxa which may lead to higher levels of homoplasy. Genetic distance analysis based on Ubx and Wg sequences showed that the genetic distances were closer in Orthoptera, comparing with outgroup.4. Phylogeny trees reconstructed by Neighbor Joining (NJ), Maximum-parsimony (MP), Maximum-likelihood (ML) and Bayesian (BI) criteria using Ubx partical sequence do not support clear phylogeny of Orthoptera, this may be related with the completely conservation in length and amino acid sequence of Ubx fragment. On the contrary, phylogeny trees reconstructed by Neighbor Joining (NJ), Maximum-parsimony (MP), Maximum-likelihood (ML) and Bayesian (BI) criteria using Wg partical sequence highlights a consistent phylogeny of Orthoptera: the outgroup Daphnia magna separated first from Insecta taxa at the base of the tree; in Insecta, the Order Arceognatha separated first from Pterygota taxa at the base of the tree; in Pterygota, the Orthoptera was supported as a monophyletic group and was divided into two lineages with high confidence values.