| Microspora are obligate intracellular parasitic protozoa without mitochondria. They are known to cause diseases in insects, fish, rabbit, silkworm and so on. It also can infect various mannals, including humans and can cause digestive and nervous clinical syndromes in HIV-infected or cyclosporine-treated people, quicken patient' s death. So the research for microspora has important function to producion, sanitation, antiepidem and environment et al N. bombycis is quarantined in egg production of silkworm because it tends to have offspring infection.The paper using Nosema bombycis as material and TriplEx2 as vector, Nosema bombycis cDNA library had been constructed. We selected randomly partial colones and sequenced, compared homogenously and analysed the gene function. 1. Construction of Nosema bombycis cDNA libraryN. bombycis was purified in a discontinuous percoll gradient. The purified spore is homogeneously refractive and without other tissue and bacteria shrebs, which can be used for the futher experiments. The total RNA of N. bombycis was extracted with Trizol kit, the agar gel electrophoresis shows the total RNA has three bands and the degree of purity can fit tor the following experiments.Using SMART IV Oligonucleotide and CDSIII/3' PCR Primer as primer, Powerscript Reverse Transcriptase as polymerase, first-strand cDNA synthesized and following dscDNA synthesized by primer extension. After proteinase K and sfi I digestion, small cDNA fragment and some rRNA removed by CHROMA SPIN-400 column. Then cDNA fraction was ligated to TriplEx2 vector and packaged,cDNA library was gottenPackaging product can be stored at -70 C for at least one year in 500ulSM, 20ulchloroform and 7%DMSO. 2. Checking of cDNA library qualityAdding some diluted phage to BM25. 8 overnight culture,then adding 0.6% top agar37 C culture in the plates. Through counting the plaques and calculating, the titer of the pliage is 1. 86 +106:Using EcoRI and HindIII digestion, the results show the inserting fragment is over 500bp 3. EST SequencingPartial clones were selected from cDNA library, after EcoRI and Hindlll digestion, 1% agar gel electrophoresis shew most of the clones had inserting fragment. We had sequenced 180 clones in the experiment.using T3 primer to sequence from 5' end, 130 useful sequence were gotten finally.The ratio of success is 72.2%. Average EST length is 552bp. 4. Analysis of sequencing result130 useful sequence were analysed in Genebank The result of sequence analysis shew 96 sequence coded ribomal RNA. Nosema bombycis had low homology with Encephalitozoon cuniculi in nucleotide level;while in amino acid level that seven EST sequence had high homology with Encephalitozoon cuniculi. The correspondence coding proteins are llistone H4, DnaJ protein(hsp70), monoubiquitin/carboxy-extension fusion protein, helicase MOT1, Syntaxin-like protein and ADP/ATP Carrier protein, at the same time, twenty-seven unknown function genes were gotten in Nosema bombycis ESTs. 5. Amplification of ADP/ATP Carrier Protein in Nosema bombycis genomeIn order to amplify the ADP/ATP Carrier Protein coding gene in Nosema bombycis genome, forward and reverse primers were synthesized according to EST sequence. And the related PCR was carried out in genome of Nosema bombycis. One band (about 400bp) was obtained. We cloned the band.After cloning and sequencing , the results show the sequence obtained from the genome of Nosema bombycis is the same as EST sequence, which verified the gene existing in Nosema bombycis genome. At the same time, through analysis the result shew that no intron was found in the gotten sequence of ADP/ATP Carrier Protein. The struction of the gene needs to be futher studied. 6. ADP/ATP Carrier Protein used for RNAi Research.RiboMAXTM Large Scale RNA Production System are used to synthesize two-strand RNA of ADP/ATP Carrier protein. When the spore was the period of SC, dsRNA was imported to the cell with DOTAP infection system. The results of counting show that RNA has about 10% difference in the...
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