高盐咸味和酸味味觉受体的分离和功能鉴定

Taste is one of human's five special senses. Normal or abnormal taste function can directly affects a person's ability to like or dislike certain food. Overconsumption or unbalanced ingestion or disliking of food will engender many health problems. Thus,thorough investigations into the taste system can lead to novel treatment or cure of these new century's diseases. In the past few years,many progresses have been made in the molecular biology of taste. Receptors for bitter, sweet,umami（savory）substances and low concentration of sodium chloride (NaCl) have been identified, and many of their downstream signaling components have also been revealed. However,the receptors for high concentrations of salt and sour stimuli have not yet been identified.Lack of the knowledge on these two important receptors appears to hinder the further progress of the taste research. The major participant of this project has many years of experience in the molecular biology of taste, having established a powerful single taste bud cell transcriptome amplification and comparative analysis method, and identified many key taste signaling molecules, including the G protein subunits Gbeta1, Gbeta3, Ggamma13, the transient receptor potential ion channel TrpM5,and the Sarco/endoplasmic reticulum Ca2+-ATPases(Serca2 & 3),and other ion channels and cell type markers. In this study, the research group will take advantage of and further develop this single cell transcriptome approach by incorporating the functional characterization of taste bud cells using calcium imaging, and the newly developed next-generation deep sequencing of the transcriptomes. Based on calcium responses of taste bud cells to given taste compoumds, cells responding to both high concentrations of NaCl as well as a mixture of bitter compounds will be determined and collected whereas cells responsive only to the bitter compounds not to the salt will also be collected as control cells. In addition, cells responsive to i)both high concentration of NaCl and sour stimulus (10 mM citrate acid pH 6);ii)sour stimulus only will be collected while excitable neuron-like type III taste bud cells that are unresponsive to either high concentrations of NaCl or the sour stimulu but can be depolarized by 50 mM KCl are collected as control cells.Transcriptomes of these single cells of the above five groups will be amplified using antisense RNA(aRNA)method, and sequenced and bioinformatically analyzed. Genes that are selectively expressed in the receptor cells but not in their corresponding control cellls will be further analyzed,of which those encoding transmembrance proteins or proteins involved in the salt or sour metabolisms will be selected as candidate genes.The roles of these candidate genes in high salt and sour tastes will be validated in vitro and in vivo. Identification of these taste receptors can have a profound impact on the biology of taste and may enable us to devise novel approachs to the new health problems.

味觉是人类的特殊感觉系统之一. 味觉功能是否正常直接影响到人对食物的喜好或厌恶. 而过份进食或偏食或厌食会带来各种健康问题. 对味觉系统的深入研究能为这些新世纪疾病提供新的治疗方案. 最近几年,苦味, 甜味和鲜味及低盐咸味的受体和信号转导途径已经基本清楚, 但是高盐咸味及酸味的受体还没确定. 项目组主要成员长期从事味觉分子生物学的研究, 建立了一套味蕾单细胞转录本扩增和分析的方法,发现了多个味觉信号传导的关键分子.在本项目中,项目组成员将进一步利用这一成功经验, 结合胞内钙成像技术, 鉴别对高盐咸味和酸味有反应的受体味蕾细胞和相应的对照细胞, 扩增这些细胞的转录本, 用深度测序技术和生物信息分析方法来找出在受体细胞中表达而在对照细胞中不表达的候选受体基因, 再在体内和体外表达系统中确定这些候选受体的功能, 最后鉴定高盐咸味及酸味的受体基因. 这一成果将极大地推进味觉生物学和食品科学的研究.

为确定参与咸味和酸味感知的基因，从小鼠的舌头上皮上把味蕾分离下来，分散成单细胞，进行钙成像分析；把对咸味和酸味分子有响应的受体细胞收集起来，与其它味蕾细胞一道进行RNAseq分析。用生物信息学方法分析RNAseq数据发现对高盐和酸有响应的细胞是III型味蕾细胞，这些细胞在基因表达上与鲜味受体细胞的更相似，而与甜味受体细胞的基因差别更大。基因表达比较分析确定了多个可能参与高盐和酸味味觉的候选基因，包括ENaC上皮钠离子通道。RT-PCR结果表明ENaC亚基在味蕾中的表达，且有多种剪接变异体。基因组分析表明人有4个ENaC亚基：α，β，γ和δ，而小鼠只有3个：α，β和γ，小鼠的δ基因“Scnn1d”是一个假基因。把人的编码ENaCδ亚基的基因SCNN1D注射到小鼠的受精卵，得到的转基因小鼠在味蕾中表达人的ENaCδ，但这转基因对其它味觉基因的表达没有影响。对鼓索神经的电生理记录显示，与野生型小鼠相比，转基因小鼠的咸味不易受amiloride抑制，与人的咸味更接近，即转基因小鼠有了人的咸味味觉。此外，对于另一个候选基因色氨酸羟化酶2（TPH2）研究表明，味蕾中的神经递质5-HT可能是III型味蕾细胞吸收血液中的5-羟基色氨酸（5-HTP）后合成的，这一成果为解释咸味、酸味味觉可能受血液中的激素等影响提供了分子基础。