A Study On DNA Bar Coding And The Rapid Molecular Identification And Detection Methods Of Cerambycidae

DNA bar coding is a new technique for species-level identification by using a short andstandard DNA sequence as species labels. It is an effective complement for traditional taxonomy.At present, the research and applications on DNA bar coding of Cerambycidae are still inexploration stage, so the primary task is to select candidate genes or DNA regions and thenidentify a general DNA bar coding. The gene analysis can be targeted at the complete sequencesof Cerambycidae mtDNACOI to seek the fragment building the DNA bar coding.In order tosolve the difficulties of extracting DNA from the specimen preserved by laboratory for manyyears,we also discussed the methods of extracting DNA from insect specimen. Finally, the DNAbar coding is built by COI sequences from the GenBank and the specimens of Cerambycidaepreserved in laborary. The main results are as follows:1. There is always a difficulty in the preservation of larva samples and the extraction of DNA.This research shows three ways to preserve Monochamus alternatus larva of more than one yearas experimental materials. The extraction of DNA could be accomplished by using traditionalmethods of Phenol-chloroform extraction with TaKaRa Kit and GenMagBio Kit. It can beconfirmed that the preserved effects of DNA of living longicorn larvae in a short time are the bestway by analysing and comparing the purity and quality of DNA obtained by different extractionmethods, and GenMagBio Kit is better way than other two methods in the purity and quality ofDNA.2. Specimens preserved have become new sources in genetics study. Reports on extracting DNAfrom old specimens successfully were abundant. But no matter which methods they used, theDNA content obtained was very low, the fragment was quite short and the PCR amplification wasdifficult to carry out. In this paper we had accomplished the trace extracting of insects specimenspersevered for many years with MagGenBio Kit for the first time. The analysis of gene fragmentshowed that MagGenBio Kit had a good extracting effect on the specimens which were preservedwith trace amounts of DNA appearing ruptures for many years. MagGenBio Kit meets the needof follow-up experiments completely. But it was failed to obtain a valid DNA from formalinimpregnated specimens.3.The whole gene of Cerambycidae mitochondria COI is about1545bp, the entire of whichcould not be used as gene bar coding. We don't know which fragment can reflect the genetic characters of Cerambycidae and effectively distinguish various longicorn beetles at the same time.This research analyzed the the whole sequences of mtDNA COI in24species belong to fivesubfamilies of Cerambycidae, and built phylogenisis tree of five subfamilies. It had a conclusionthat the genetic relationship of Aseminae and Lepturinae is closest, the second is Lamiinae, thethird is Cerambycinae and finally is Prioninae, yet the evolutionary relationship between differentsubfamilies needs further research. The phylogenesis relationship analyzed by using amino acidsequences could reflect the traits of species evolution more clearly, and the conclusion is morescientific.4. Comparing the results of whole sequences tree of longicorn beetles with two common-usedCOI gene fragment tree, we found that the gene located from seventy-seven to five hundredeighty bp could reflect the characteristic of mitochondria COI more exactly, so it was moresuitable to be gene barcoding for replacing whole sequences of longicorn gene.5. Under the theory of this experiment,the gene bar codings of160species of longcorn beetlesbrlonging to five subfamilies of Cerambycidae had been built, and the sequence werealignmented in Bioedit. The results showed that there didn't exist insertion, deletion or terminatorcodon in this four hundred eighty-three locus. There was an obvious polymorphism on thefragment among one hundred sixty species of longcorn beetles and which could be used fordistinguishing these kinds effectively.6. In order to be free from the restriction of longicorn beetles cyclogeny, insect stages anddamaged, this research screened out two pairs of primer, which could distinguish Tetropiumfuscum, Arhopalus foveicollis from the other fourteen kinds of longicorn beetles of Aseminae inphotograph of Gel electrophoresis. This study could pay a foundation for deeply study of ormolecular rapid identification technology.