Tribolium Castaneum Phosphine Resistant Strains Of Micro-satellite Polymorphism Missing Its Locus Sequence Analysis,

The Tribolium castaneum (Herbst) is a kind of economical pest which can damage grain and stored product mostly, which are widely distributed around the world. The harmful ways are including direct feeding, speed of moldy corn, pollution of manure and so on. Phosphine (PH3) is one of the best fumigant, widely recognized as the performance control stored product insects. Over the years, because of the inconsequence of scientific methods, many kinds of pests produced resistance in many areas generally. As the phosphine has incomparable superiority, it will still play a very important role in the future integrated pest management (1PM) of stored grain. In recent years, the incidence of Tribolium castaneum is increasingly wider and the damage increasingly heavy, so the problem of resistance has been more attented. Starting from the pest itself, to clarify the functions of gene can help to research new theories and technology of controling pest. Microsatellite DNA is short tandem repeats of DNA sequences, which is widely distributed in prokaryotic and eukaryotic genome. Loss of polymorphism is a special form of expression of microsatellite instability. In this study, experimental materials is including 120 castaneum adults from resistant strains and sensitive strains of Sichuan and Henan Provinces, and we get total genomic DNA using the method of phenol chloroform extraction. We also process DNA amplification and polyacrylamide gel electrophoresis on the polymorphic microsatellite marker loci DT802192, EB754173, DN648427, CB337218 and DN647734, and are cuting plastic recycling, purifying, amplifying, sequencing, making sequence analysis for polymorphic microsatellite missing DT802192 point, in order to find genes associated with resistance to phosphine. The results show that the DT802192 loss of polymorphism occurs frequently as 100%(30/30), EB754173 as 6.67% (2/30), DN648427 as 3.33%(1/30), CB337218 and DN647734 both as 0%(0/30) in the five microsatellite loci, which means that the latter two sites are found no loss of polymorphism. For loss of polymorphism and analysis of correlation with resistance to phosphine of DT802192 site, the result by X2 test. P< 0.01, shows that the locus exist the relationship of loss of polymorphism and resistant strains with statistical significance. By using bioinformatics to analyze DT802192, the sequence locates in the third chromosome of T. castaneum. The sequence with consistency of the accession number for the LOC655048 sequence is the 3 'downstream non-coding region of T. castaneum P53 gene by BLAST EST sequences of T. castaneum cDNA.The conclusion of this study as following:1. The study of T. castaneum sensitive strains and resistant strains showed that the two strains are both polymorphic in the microsatellite marker loci EB754173. DN648427. CB337218 and DN647734, but the sensitive strains in the microsatellite marker DT802192 are polymorphism and the resistant strains appears high-frequency loss of polymorphism. This indicates that phosphine resistance DT802192 may exist the anti-phosphine-related genes, and provide a good way for selecting phosphine resistance genes.2. We analysis loss of polymorphism of T. castaneum sensitive and resistant strains by selecting a polymorphic microsatellite marker loci and using polyacrylamide gel electrophoresis. Based on the different results in DT802192 sensitive and resistant strains we can confirm the two strains at the molecular level, for providing a new method of the two strains detection and detecting sites of loss of polymorphism as an important index of phosphine resistance.3. Bioinformatics analysis of ESTs sequences of T. castaneum cDNA showed that, DT802192 sequences locate the third chromosome, and the similar 3'non-coding regions of P53 gene with T. castaneum have a higher consistency. This section of non-expressed sequence may be involved in the P53 gene expression, which results in producing the resistant to the phosphine, for further studying occurrence mechanisms of T. castaneum phosphine resistance to provide effective guideline of molecular genetics.4. The microsatellite markers of PCR multiple sites and many points can detect small deletion within gene and instability of genome rapidly and effectively, of which are tested the existence by microsatellite instability, and provide a valuable method for the studying of testing T. castaneum genome.