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目的克隆中华按蚊水通道蛋白(AsAQP)基因的cDNA全长序列,分析其基因序列特征,为研究AsAQP的生物学功能提供分子基础。方法根据已报道的昆虫水通道蛋白(AQP)氨基酸序列的保守区域,采用兼并引物从中华按蚊cDNA中获取AsAQP基因片段,在此基础上利用cDNA末端快速扩增(RACE)技术克隆该基因cDNA全长序列,并用生物信息学方法对获取的序列进行分析。结果利用兼并引物从中华按蚊成蚊cDNA中分离到AsAQP基因片段,利用RACE技术克隆到该基因的全长cDNA。序列分析表明,该基因cDNA全长762 bp,编码253个氨基酸,蛋白分子量约为63.2 kD。生物信息学分析表明,AsAQP具有典型的6个跨膜区结构和2个天冬酰胺酸脯氨酸丙氨酸(NPA)结构,该结构是主要内在蛋白(MIP)家族典型的结构特征。AsAQP与致倦库蚊(Culex quinquefasciatus)AQP及埃及伊蚊(Aedes aegypti)AQP蛋白的同源性分别为76%和78%。氨基酸序列聚类分析表明,AsAQP与其他蚊种的水通道蛋白遗传距离较近。结论利用兼并引物结合RACR技术首次获得了编码AsAQP基因的cDNA全长序列,该基因属于MIP蛋白家族成员,具有典型的功能域,为进一步研究该蛋白的功能奠定了基础。
Objective To clone the cDNA sequence of AsAQP gene from Anopheles sinensis and analyze its gene sequence and provide the molecular basis for the study of biological function of AsAQP. Methods The AsAQP gene fragment was obtained from Anopheles sinensis cDNA using the conserved region of reported amino acid sequence of insect aquaporin (AQP). Based on this, the gene cDNA was cloned by rapid amplification of cDNA ends (RACE) The full-length sequence was analyzed by bioinformatics methods. Results AsAQP gene fragment was isolated from adult mosquitoes of Anopheles sinensis using annexing primers, and the full-length cDNA was cloned by RACE. Sequence analysis showed that the cDNA was 762 bp in length and encoded 253 amino acids with a molecular weight of 63.2 kD. Bioinformatics analysis showed that AsAQP has typical six transmembrane domains and two asparagine proline alanine (NPA) structures, which are typical structural features of the major intrinsic protein (MIP) family. The homologies of AsAQP to Culex quinquefasciatus AQP and Aedes aegypti AQP protein were 76% and 78%, respectively. Amino acid sequence clustering analysis showed that AsAQP had a closer genetic distance to aquaporin from other mosquito species. Conclusion The full-length cDNA of AsAQP gene was obtained by using the combination of primers and RACR. The gene belongs to MIP protein family and has typical functional domains, which lays the foundation for further study on the function of this protein.