| Background:Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. In recent years, Cryptococcosis that causes death in AIDs patients becomes a big problem to the world.The Cryptococcus neoformans Genome Project is a collaboration among scientists at two centers: the Stanford Genome Technology Center (SGTC) and the Institute for Genomic Research (TIGR). Starting in March 2000,and they finished last year, which provides the basis of gene function analysis. Meanwhile, they are just a start point, many work should be done on the strong virulence factors helping the pathogen escapeand survive from the host immune reaction.To research an unknown gene, the classic protocol is gene replacements that leads to "knock out" of the gene. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. It is a time-consuming and very difficult work to complete. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. It is a simple and effective tool instead of the traditional technology which leads to a revolution. So when we design the research protocol, we introduce a long mRNA sequence which is a part of the same as the target gene, and it could cause the PTGS (post transcription gene silence) like touching the trigger. In our research, we focus on the CNLAC1 gene encoding a laccase, which has previously been cloned and characterized, but in this field a lot of things remains unknown to us. To analyse the presence of the Cryptococcus neoformans virulence factor, we have to construct a cnlacl gene "knock out" strain using RNAi technology.Objective:Part 1: Reconstruct the plasmids Pcnlacl-tel, Pcnlacl-S-tel , Pcnlacl-A-tel.Part 2: To prove the condition of the transformation in Cryptococcus neoformans ,conclude a better transformation system.Part 3: To form a new system that could research the function of yeast gene more effectively.Part 4: study of main virulence factor such as laccase when we get the transformed Cryptococcus neoformans.Method:Part 1: We reconstruct the plasmid Pcap59i-tel which was provided by professor Tamara. L. Doering to get the RNA interference plasmid Pcnlacl-tel. Extracting the total genomic gene of Cryptococcus neoformans , we design 4 primers containing Bgl â…¡and Nde â… site on each side. So we get four 500bp PCR products of open reading frame of CNLAC1 gene:PBN, PNB, PB, PN , and reconstrct PN and PB between promoter ACTin and Terminator GAL7 with opposite station. In the middle of them, we insert a GFP gene of 250bp as a spacer. As a result, The constrct inverted repeats of 500 bp of coding sequence of the gene separated by a spacer segment of green fluorescent protein (GFP) sequence, and the transcription product should be double-stranded RNA(dsRNA) that has 500bp the same as the gene product.Part 2: we also reconstrct the Pcnlacl-S-tel using PNB, and Pcnlacl-A-telwith PBN.Part 3: JEC21 cells were transformed by electroporation using linearized DNA with selection of transformants on minimal plates lacking uracil.Results:1. we successfully get the correct reconstructs as design : Pcnlacl-tel, Pcnlacl-S-tel, Pcnlacl-A-tel, proved by gene sequencing.2. we have tested many conditions of the transformation in Cryptococcus neoformans , and the positive control succeed.3. we failed in all of the transformation experiments.Conclusion:1. the plasmid and the host strain JEC21 are reliable and correct.2. The efficency of the transformation by electroporation is still not satisfied.3. The positive control confirms the electroporation is a better protocol in Cryptococcus neoformans.4. Are there some system risk factors in the research?...
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