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昆虫的化学感受系统在其生存和繁殖过程中起着极其重要的作用[1-3]。在过去10多年的时间里,研究者对昆虫触角嗅觉信号传导的分子机制研究有了突出的进步。在昆虫嗅觉识别时,气味分子从触角感器孔渗入,然后被气味结合蛋白(odorant binding proteins,OBPs)或化学感受蛋白(chemosensory proteins,CSPs)识别和转移,最后激活位于嗅觉感觉神经元(olfactory sensory neurons,OSNs)树突膜上的嗅觉受体(odorant receptors,ORs)或离子型受体(ionotropic receptors,IRs),产生电位,指导昆虫做出相应的行为反应[4-8]。此外,还有一些蛋白如感觉神经元膜蛋白(sensory neuron membrane proteins,SNMPs)在昆虫气味识别过程中也扮演着至关重要的作用[5]。
昆虫SNMPs也是一种膜蛋白,与脊椎动物CD36家族为同源基因,具有2个跨膜区域,其功能主要是识别和转运亲脂性气味分子如脂肪酸和脂类化合物等[9-14]。昆虫第一个SNMP基因在多音天蚕(Antheraea polyphemus Cramer)中被鉴定,并命名为ApolSNMP1[9]。随后在烟草天蛾(Manduca sexta L.)中发现SNMP的第二个亚类型,即命名为MsexSNMP2[10, 15]。紧接着SNMP的同源基因在鳞翅目Lepidoptera[10, 15-16]、双翅目Diptera[17]、鞘翅目Coleoptera[18]、直翅目Orthoptera[13]和膜翅目Hymenoptera[19]等昆虫中都有发现。一直以来被认为SNMP基因家族就2个成员,即SNMP1和SNMP2。最近昆虫SNMP家族的第三个成员SNMP3在鳞翅目中被鉴定[20-21],但认为该基因的主要功能与昆虫的免疫反应有关,这还需要进一步的去证明。
红脊长蝽(Tropidothorax elegans Distant)属半翅目(Hemiptera)长蝽科(Lygaeidae),主要为害刺槐(Robinia pseudoacacia L.)、辣椒(Capsicum annuum L.)、葫芦(Lagenaria siceraria Molina)、油菜(Brassica napus L.)、大白菜(Brassica pekinensis Lour.)和小麦(Triticum aestivum L.)等多种植物,食性较杂。关于红脊长蝽的嗅觉基因研究较少,本研究通过前期红脊长蝽触角转录组测序结果[22],鉴定了红脊长蝽的2个SNMP基因,即TeleSNMP1和TeleSNMP2,并通过荧光定量PCR技术对红脊长蝽TeleSNMP1和TeleSNMP2在不同组织中的表达情况进行分析,为进一步探索红脊长蝽SNMPs的化学通讯功能奠定基础。
红脊长蝽感觉神经元膜蛋白基因克隆及组织表达谱分析
Cloning and Expression Pattern of Sensory Neuron Membrane Protein Genes of Tropidothorax elegans
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摘要:
目的 感觉神经元膜蛋白(SNMP)是至关重要的次要嗅觉蛋白,能够作为信息素识别的标记,而红脊长蝽(Tropidothorax elegans)作为一个重要的农林害虫,这方面的研究还很少。 方法 利用PCR方法对红脊长蝽SNMP基因进行克隆,并通过荧光定量PCR对该基因的表达情况进行分析。 结果 首次克隆和鉴定了红脊长蝽的2个SNMPs基因,并命名为TeleSNMP1和TeleSNMP2。序列分析表明,TeleSNMP1编码区开放阅读框长1 497 bp,编码498 aa;而TeleSNMP2编码区开放阅读框长1 686 bp,编码561 aa,这2个基因的等电点分别为8.26和7.05。同源性分析发现,同目昆虫同类SNMP序列一致性较高,不同目昆虫不同类SNMP序列一致性较低。TeleSNMP1和TeleSNMP2之间的序列一致性极低。进化树结果也表现同目昆虫同类SNMP基因进化关系最近。定量PCR结果显示,TeleSNMP1主要在雌雄触角中表达,而TeleSNMP2在非触角组织中也有表达。 结论 该结果为今后对红脊长蝽SNMPs基因功能的研究提供了有用的参考。 Abstract:Objective To study the sensory neuron membrane proteins (SNMPs) in Tropidothorax elegans. Method The SNMP genes were cloned by PCR, and the expression of these genes were analyzed by fluorescence quantitative PCR. Result Two SNMPs genes of T. elegans, named as TeleSNMP1 and TeleSNMP2, were cloned and identified for the first time by RT-PCR. Sequence analysis showed that the TeleSNMP1 encoding area was 1 497 bp in length, encoding 498 aa. The TeleSNMP2 encoding area was 1 686 bp in length, encoding 561 aa. The isoelectric points of these two genes were 8.26 and 7.05, respectively. Homology analysis showed that the consistency of SNMPs genes in insects of the same order was high, while the consistency of different order was low, and the sequence consistency between TeleSNMP1 and TeleSNMP2 was extremely low. The evolutionary tree results also showed that the evolutionary relationship of SNMPs genes in insects of the same order was rather close. Quantitative real-time PCR showed that TeleSNMP1 was antennae-biased, whereas TeleSNMP2 was expressed in female and male antennae but was also expressed in nonantennal tissues. Conclusion The results provide a useful reference for future studies on the function of the T. elegans SNMPs gene. -
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