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 492bp,包含1个900bp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。RTPCR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 NIPS-1 gene （nodulin26-1ike 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 LotNIPS-1 was detected via real-time quantitative PCR （qRT-PCR） and RT-PCR. The results showed that： 1） LotNIPS-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 /eaves,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.