摘要
为构建长角血蜱雄蜱抑制消减杂交cDNA文库,获得长角血蜱雄性成蜱在半饱血状态下的特异表达基因,用抑制消减杂交方法构建以pGEM-T Easy为载体的长角血蜱半饱血雄蜱全蜱cDNA文库,测序并进行生物信息学分析。以新合成的长角血蜱饥饿和半饱血雄蜱cDNA第一链为模板,用RT-PCR方法对从文库中挑选的5个表达序列标签(EST)进行鉴定。初步获得18个EST,BlastX分析显示,所得cDNA编码的功能性蛋白主要包括信号传导、组织形态形成、转录调节和糖类代谢等相关蛋白。RT-PCR鉴定结果显示,随机挑选的5个新EST中有3个在半饱血状态下差异表达。结果表明,成功地构建了长角血蜱雄性全蜱抑制消减杂交文库,获得了有潜在生物学功能的EST。
To construct a suppression subtractive hybridization（SSH） cDNA library of male Haemaphysalis longicornis and to screen the differentially expressed gene in the partially-engorged condition,the cDNA library of partially-engorged male H.longicornis was constructed in the clone vector of pGEM-T Easy by SSH.The sequencing and bioinformatics analyses were conducted afterwards.The quality of the library was evaluated by RT-PCR using the first strand of partially-engorged male H.longicornis cDNA newly synthesized as the template.Eighteen expressed sequence tags（ESTs） were obtained from the SSH cDNA library and BlastX analysis revealed that the putative proteins encoded by these ESTs had similarity to proteins reported before,with most of which were functional proteins,such as signal transduction,tissue morphogenesis,transcriptional regulation and carbohydrate metabolism proteins.These proteins are valuable to the ticks to adapt the changes of physiology after feeding.The result of RT-PCR revealed that 3 of 5 ESTs from the library were differentially expressed in the partially-engorged condition.The results suggested that the SSH cDNA library of male H.longicornis was constructed successfully,and some potential functional ESTs were obtained.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第8期659-664,共6页
Chinese Veterinary Science
基金
国家"十一五"高技术研究发展计划(863)项目(2006AA10A207)
国家基础平台项目(2005DKA21205-3)
关键词
长角血蜱
抑制消减杂交(SSH)
CDNA文库
Haemaphysalis longicornis
suppression subtractive hybridization（SSH）
cDNA library
