Physical Mapping Of The Trichomonas Vaginalis Genome And Study The Condition Of Constructing BAC Genome Library

Trichomonas vaginalis is unicellular parasitical protozoa. It is a flagellated protist that causes trichomoniasis, a common but overlooked sexually transmitted human infection, with -170 million cases occurring annually worldwide. Acute infections are associated with cervical cancer, increased risk of human immunodeficiency virus 1 (HIV-1) infection. T. vaginalis is a member of the parabasilid lineage of microaerophilic eukaryotes that lack mitochondria and peroxisomes but contain unusual organelles called hydrogenosomes. However, the research about T. vaginalis is focus on cell biology, epidemiology, diagnosis methods, energy metabolism and single gene analysis etc. at present. The whole genome research of T. vaginalis aims at detecting and studying genes associated with infection, discovering the mechanism of T. vaginalis cells adhere to human epidermic cells and that of drug-fast, in order to provide molecular biology foundation of discovering new drug targets and vaccine to prevent and cure trichomoniasis. The aim of this study is to construct a physical map and study the condition of constructing BAC genome library.The genome size of T. vaginalis is larger than most other protozoa, which is about 160Mb, and at least 65% of it is repetitive. So a integrated physical map is needed in order to sequence and finish its genome. In this reaserch, we use an approach to integrate the Trichomonas vaginalis physical map by using PCR-based STS markers generated from ESTs sequences and WGS sequences to screen Fosmid DNA superpools. We designed 1353 STSs, and screened 46 superpools, 17664 clones. At last, we assembled 810 STSs and 2555 clones to 382 contigs and 102 singlets, about 21.9Mb. The longest contig contains 26 STSs and 109 clones, about 410.2 kb.Because the T. vaginalis genome is much larger than 20Mb, we couldn't give a good physical map use the low coverage Fosmid library. Then, in order to give a complete physical map, we studied the condition of constructing a BAC genome library.1. Preparation and Digestion of IncertAgarose PlugsLysis of T. vaginalis cells releases potent nuclease activity that is difficult to inhibit. The nuclease activity rapidly degrades T. vaginalis genomic DNA during parathion of plugs. 1.5% DEPC was added to the suspension buffer to prevent genomic DNA degradation.2. Determining of Endonuclease and Optimal Partial Digetion Condition BamH I ,EcoR I ,Hind III and Mbo I were used to digest the genomic DNA,and the result is that there are most Mbo I site in the genome. Then the partial digestion condition was optimized as 0.08U Mbo I /plug, 37℃, 60min.3. Determining of Optimal Transformation Condition2.1kv and 1.8kv were test to transform, and it was discovered that low voltage(1.8kv) made low transformation efficiency, but it could increase incert size.