Genetic and phenotypic characterization of a Chlamydomonas motility defected mutant

• Main Authors: Wan-Jun Chen, 陳宛君
• Other Authors: Che-Chia Tsao
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/33306930332059306551

碩士 === 國立臺南大學 === 生物科技學系碩士班 === 103 === Cilia and flagella are found in diverse eukaryotes and are responsible for cell motility and sensory functions. When cilia or flagella are defected in humans, it leads to many kinds of genetic diseases. We used unicellular Chlamydomonas reinhardtii CW15 strain, which has two equal length of flagella and the ability to swim, as the experimental model to perform insertional mutagenesis. Six mutants with defected flagella (AphVIII 3, AphVIII 5, AphVIII 15, AphVIII 21, WJ21 and Lu3) were obtained by a visual screen for motility defects. Their genomic DNA were analyzed by RESDA PCR and inverse PCR to know where the resistant drug cassette was inserted. Lu3 is a mutant strain without flagella and was further characterized in this study. Genotyping revealed that the insertion of the exogenous Aph7’’ marker caused a deletion of a 60-kb region, which contains 13 genes including flagella inner dynein arm I1 intermediate chain (IC140/IDA7) and B9 domain-containing protein 1 (B9D1). The growth of Lu3 strain showed no difference comparing to CW15 cells. TEM analysis showed that the morphology of the transition zone in Lu3 cells appeared normal, but the Lu3 cell could only extend a short axoneme beyond the transition zone. Transforming with the DNA fragment containing IDA7-B9D1 could rescue the flagella defect of Lu3 while the DNA containing only B9D1 could not. However, Lu3 cell transformed with theIDA7 DNA showed two phenotypes. One is without flagella, and the other is with short flagella and partial motility. The amounts of tubulins and intraflagellar transport complex proteins IFT46and IFT139 were decreased in Lu3, and the Lu3 cells transformed with DNA containing only IDA7 or B9D1 did not restore the amounts of IFT46 and IFT139 proteins. Since the flagella assembly depends on intraflagellar transport, it is likely that the deletion ofIDA7 and B9D1 at the same time leads to the instability of IFT complex and results in the abnormal assembly of flagella.