中文摘要：

为探究尿囊酸酶（allantoicase）基因在盘基网柄菌（Dictyostelium discoideum）发育中的作用，依据RNA干扰（RNAi）的原理和盘基网柄菌中构建RNAi载体的基本经验，扩增出尿囊酸酶基因642bp的长片段，397bp的短片段，并反向连接成含有发卡结构的寡核苷酸片段，克隆至表达载体pAct15Gal，构建了以盘基网柄菌尿囊酸酶基因为靶标的RNAi载体pAct15Gal—allCi。将此载体转染野生型KAx-3细胞，经G418筛选出阳性克隆RNAi-allc。将野生型细胞和RNAi-allc转染细胞发育20h后发现，转染细胞仅能形成类似细胞丘的小突起，不能完成完整的发育，而野生型细胞则能完成完整的发育，形成子实体。Western印迹几乎检测不到转染细胞有尿囊酸酶的免疫条带：流式细胞术检测转染细胞内尿囊酸酶的表达，发现表达率由野生型细胞的72．18％降为转染细胞的0．67％。表明本研究设计的核苷酸序列对靶标基因的表达有较明显的干扰作用，说明尿囊酸酶对盘基网柄菌细胞发育有一定的调控作用。

英文摘要：

This study was aimed to limit cell development in Dictyostelium discoideum by RNAi strategy targeting allantoicase gene （allC）. In accordance with the allC sequence in Genbank, partial cDNA sequences （642 bp, 397 bp） from Dictyostelium discoideum were amplified by RT-PCR. The gene-specific sequences （642 bp, 397 bp） of allC were linked in the antisense orientations in order to form hairpin RNA. The resulted construction was then inserted into vector pAct15Gal to obtain the RNAi expression vector pAct15Gal-allCi. Positive clones were obtained by stable transfection KAx-3 using pAct15Gal-allCi and G418 screening. Compared with untransfected KAx-3, transfected cells, arrested early stage of mound, could not complete integrative cell development. Western blot analysis showed allantoicase protein in interference sequence transfected cells was significantly down-regulated. The transfection rate was estimated by FACS analysis, indicating the allantoicase expression decreased from 72.18% to 0.67% after RNAi transfection. The results suggest that allantoicase is interfered, which is required for the development of Dictyostelium discoideum.