摘要
以构建的中国水仙不同花期的抑制差减杂交文库(SSH)为基础,采用RACE及RT-PCR技术,分离克隆到1个水通道蛋白基因NtPIP1。此基因包含有一个867 bp完整阅读框架(ORF),编码288个氨基酸,分子量为30.68 kD,理论等电点为8.97。用生物软件对NtPIP1蛋白分析表明:NtPIP1含有6个跨膜区,由5个环相连,其中环A、C和E在细胞膜外,环B、D和蛋白的N、C末端都位于细胞膜内,N末端55个氨基酸,C末端9个氨基酸,具有MIP家族典型的保守氨基酸序列HINPAVTFG和2个NPA保守肽段。三级结构预测表明和菠菜(2b5fA)水通道蛋白结构相似。聚类分析表明:该蛋白与其他12个物种的PIP1类水通道蛋白同源性在91%以上,属于质膜水通道蛋白PIP1类。半定量RT-PCR分析结果表明:NtPIP1基因在盛花期中国水仙雌蕊中的表达量最高。
Based on constructed different florescence suppression subtractive hybridization （SSH） of Narcissus tazetta var. chinensis, an encoding transmembrane aquaporin （AQP） gene NtPIP1 was separated and cloning by RACE and RT PCR. The NtPlPl eDNA was 867bp, encoding a protein of 288 amino acids with a predicted molecular mass of 30.68kD and theoretical isoelectric point of 8.97. The NtPIP1 exhibited a typical structure with six transmembrane domains which linked by five loops, loop A, C and E were at outside of membrane, loop B, D, N terminal and C terminal were at inside of membrane. N terminal has 55 amino acids and C terminal has 9 amino acids. The NtPlPl has the consensus sequence HINPAVTFG of MIP family and two highly conserved peptides AsnPro Ala （NPA） . The protein 3D structure was predicted by homology comparative modeling in Swiss Model, the result from Swiss-Model showed that the 3D structure was highly similitude with Spinacia oleracea （2b5fA） . The NtPlPl amino acids showed above 91% identity to other 12 plant species PIP1 subfamily, so NtPIP1 should be a plasma membrane intrinsic protein of PIP1 subfamily. Semi-quantitative RT-PCR revealed that NtPIP1 was expressed highest in pistil of Narcissus tazetta var. chinensis in bloom stage.
出处
《福建农业学报》
CAS
2011年第3期371-376,共6页
Fujian Journal of Agricultural Sciences
基金
福建省农业科学院科技创新重点项目(2007YCXZ02)
关键词
中国水仙
水通道蛋白
基因克隆
蛋白质结构
Narcissus tazetta vat. chinensis
aquaporin
gene cloning
protein structure prediction
