用简并性引物RT-PCR技术检测大鼠舌味蕾细胞谷氨酸受体表达

THE STUDY ON GluRs EXPRESSION IN RAT TASTE BUDS BY RT PCR USING DEGENERATE PRIMERS

为快速、有效地用ＰＣＲ检测不同组织，根据大鼠脑中谷氨酸受体家族ＤＮＡ序列，在高同源区设计出对谷氨酸家族有专一性的简并性引物。用此引物通过ＰＣＲ扩增来自大脑的多种谷氨酸受体，而味蕾细胞ＰＣＲ产物克隆后的ＤＮＡ序列，仅与脑内代谢型谷氨酸受体四型（ｍＧｌｕＲ４）相一致；在缺少味蕾的舌上皮组织则无此种ＰＣＲ产物。以含味蕾细胞的ＰＣＲ产物为模板，合成同位素标记的ＲＮＡ探针，用ＲＮＡ酶保护法进一步检测ｍＧｌｕＲ４在不同组织中的表达，发现含味蕾的舌上皮组织中有与阳性对照相似的ＲＮＡ片段，而在不含味蕾的舌上皮组织ＲＮＡ中无此片段。因此，推测ｍＧｌｕＲ４可能是谷氨酸味觉的化学感受器。

The expression of GluRs in rat taste buds was determined by using RT PCR and RNase protection which indicate that mGluR4 is uniquely associated with rat taste buds.According to DNA sequence of GluRs family in brain,degenerate primers were selected from high homology regions,specific for the family of GluRs.These primers were used to detect different tissues by RT PCR and readily amplified GluRs from brain mRAN.DNA sequence of PCR product which was cloned from taste bud enriched lingual tissue was shown to be the same as mGluR4 in the brain.No such PCR product was found in lingual epithelium lacking taste buds.RNase protection assay,although less sensitive,provides a reliable,quantitative estimate of mRNA expression in tissues.In order to do this,radiolabeled RNA probes were synthesized from the subcloned PCR product from taste buds and the hybridization was performed with poly(A)RNA from positive control tissue and taste buds.Similar protected fragments of the expected size were observed.In contrast,no protected fragments could be visible when RNA from epithelium devoid of taste buds was hybridized to the mGluR4 probe.The results indicate that mGluR4 might be a chemosensory receptor responsible,in part,for the taste of monosodium glutamate.