Pellerone F I, Archer S K, Behm C A, Grant W N, Lacey M J, Somerville A C
School of Biochemistry & Molecular Biology, Faculty of Science, Australian National University, ACT 0200, Canberra, Australia.
Int J Parasitol. 2003 Sep 30;33(11):1195-206. doi: 10.1016/s0020-7519(03)00173-5.
The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.
海藻糖被认为在秀丽隐杆线虫的生理学中具有重要作用，它可能在糖类运输、能量储存以及抵御环境压力方面发挥功能。在本研究中，我们以秀丽隐杆线虫为模型，研究了海藻糖代谢在秀丽隐杆线虫中的作用，同时还鉴定出了可能编码丝虫中海藻糖代谢途径相关基因的互补DNA克隆。在秀丽隐杆线虫中，两个假定的海藻糖-6-磷酸合酶（tps）基因编码催化海藻糖合成的酶，五个假定的海藻糖酶（tre）基因编码催化该糖类水解的酶。我们通过逆转录聚合酶链反应（RT-PCR）或Northern分析表明，这些基因中的每一个在秀丽隐杆线虫生命周期的所有阶段均以信使核糖核酸（mRNA）的形式表达。数据库搜索和表达序列标签克隆测序显示，至少一个tps基因和两个tre基因在马来布鲁线虫中表达，而盘尾丝虫中鉴定出一个tps基因和至少一个tre基因。我们利用RNA干扰的喂食方法在秀丽隐杆线虫中暂时敲低每个tps和tre基因的表达。半定量RT-PCR分析证实每个基因的表达都被RNA干扰沉默。单独沉默任何一个基因时，我们都未观察到明显的表型，但气相色谱分析显示，同时靶向两个tps基因时，海藻糖水平下降了90%以上。海藻糖含量的这种下降并未影响线虫的活力或发育。
