| Protozoa is a special and independent kingdom of microorganism in theprocessing of the evolution from prokaryote to eukaryotic cell. As aunicellular eukaryote, protozoa have many unique characters, such asdeviation from the universal genetic code, individual chromosomes andnuclear duality. It is perfect material and model biology for the research ofmolecular cell biology. Euplotes octocarinatus is a representational strain ofHypotrichida Euplotidae. It contains two types of nuclei in each cell, one isgenerative micronucleus, a typical diploid, meiotic germ-line nucleus, andanother one is vegetative macronucleus, a highly specialized expressionorganelle that provides all the transcripts for cell function.Hypotrichids ciliate macronucleus chromosome was named "genesize chromosome", which carrying the gene coding sequence flankedby short non-translated regions and bounded by telomeres (C4)4(A4)3.Macronucleus chromosome is furthest simple chromosome ineukaryotic cell. This character is benificial to investigating toconstruction and function of the chromosomes, and also toconstructing artificial chromosomes in ciliate and analyzing functionof gene.Firstly, we cloned a newβ2-tubulin gene from the macronuclear andmicronuclear geneome of E. octocarinatus (DQ864704), respectively.Compared withβ1-tubulin gene, the coding regions of bothβ-tubulingenes have an identical length of 1335 bp (99.1％homology). Thoughsequences of non-coding regions of two genes differ from each other,both of them contain a TATAA box in 5' non-coding regions andaboundant in AT. Secondly, artificial chromosome EUAMC of ciliatewas constructed usingβ2-tubulin non-translated regions of E.octocarinatus. The artificial chromosome was composed ofβ2-tubulin non-translated regions, telomere, and plasmid elements of pEGFP-N1 (GFPgene, multiple cloning site and Kanr, etc). Expression of GFP gene isregulated under the 5' non-code regions ofβ2-tubulin. For high expression ofGFP gene in E. octocarinatus cells, some codons in GFP gene wereoptimized by using point mutation to fit for the codon usage of ciliates.Plasmid pBTub-tel2, containing artificial chromosome, were transfectedinto Euplotes cells with the help of liposome. Interval observations under thefluorescent microscop were performed with excition waves of 470—490 nm.The results displayed that GFP genes were expressed effectively in E.octocarinatus, and GFP was distributed unifimly around macronuclearand diffused to the entire cell gradually. The chromosomes can be kept inthe macronucleus for 90h.
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