百合水通道蛋白LotNIP5-1基因的克隆及表达分析
Cloning and expression analysis of aquaporins LotNIP5-1 gene from lily

为研究NIP5-1基因（Nodulin26-like intrinsic protein）在百合生长发育方面的作用机理，利用RACE技术克隆得到百合NIP5-1基因的全长cDNA序列。运用多个软件进行生物信息学分析；采用实时荧光定量PCR（qRT-PCR）及RT-PCR技术检测其表达特性。结果表明：1）百合NIP5-1基因全长为1 492 bp，包含1个900 bp的完整开放阅读框（Open reading frame，ORF），共编码299个氨基酸；2）百合NIP5-1蛋白属于跨膜通道蛋白MIP超家族。具有6个跨膜区；在B、E环分别具有NIP5-1蛋白特有的NPS、NPV模体，具有该蛋白Ar/R filter分子筛特有的AIGR模体；与小果野芭蕉、香瓜、葡萄、黄瓜、番茄的相似性分别为86%、83%、84%、83%和82%；3）荧光定量结果显示，百合NIP5-1基因在茎中表达量最高，在花瓣中表达量较低，在叶、鳞茎、根中表达量均极低。RT-PCR半定量检测显示，其在“湿”柱头上表达量显著高于“干”柱头，这与转录组测序结果相符合。上述研究暗示，百合NIP5-1可能是硼酸通道蛋白，在促进茎生长及生殖活动中发挥作用。
To study the mechanism of NIP5-1 gene(nodulin26-like intrinsic protein)involved in the growth and development of lily,the full-length cDNA of LotNIP5-1 gene was cloned via RACE technology.Bioinformatics analysis was performed by using multiple softwares,and the expression of LotNIP5-1 was detected via real-time quantitative PCR (qRT-PCR) and RT-PCR.The results showed that:1) LotNIP5-1 gene cDNA was 1 492 bp in length containing an open reading frame of 900 bp,which encoded 299 amino acid;2) LotNIP5-1 protein belonged to transmembrane channel protein MIP super family,which had six transmembrane regions,one specific NPS motif in B loop and one NPV motif in E loop of NIP5-1,and a specific Ar/R filter's AIGR motif of NIP5-1 protein.The amino acid sequence of LotNIP5-1 was 86%,83%,84%,83%,82% identical to that of Musa acuminata,Cucumis melo,Vitis vinifera,C. sativus,Solanum lycopersicum,respectively.3) qRT-PCR results showed that the expression level of NIP5-1 was the highest in stems,lower in petals,and the lowest in leaves,bulbs and roots;RT-PCR analysis showed that the expression level of NIP5-1 in "wet" stigma was significantly higher than that in the "dry" stigma,which was consistent with the results of transcriptional sequencing.It was suggested that the LotNIP5-1 might be a boron acid channel protein,which functioned in the shoot growth and reproductive process of lily.