砧木南瓜硅转运蛋白基因CmLsi3的克隆与表达分析

Cloning and Expression Analysis of Silicon Transporter Gene CmLsi3 in Roots of Pumpkin

以用于黄瓜嫁接的砧木南瓜‘云南黑籽南瓜’(Cucurbita ficifolia,去蜡粉能力弱)和‘黄诚根2号’(C.moschata,去蜡粉能力强)为试材,采用同源克隆与RACE相结合的方法克隆到硅转运蛋白基因c DNA全长,命名为CmL si3(Gen Bank登录号分别为KM203110和KM359142)。两种砧木南瓜Cm Lsi3基因c DNA全长为1 206 bp,开放阅读框为795 bp,编码264个氨基酸,两者仅有4个位点的氨基酸不同,但与黄瓜、玉米、大麦等作物硅转运蛋白氨基酸序列同源性均在50%以上。CmL si3蛋白含有2个NPA模体、6个跨膜结构域以及1个由Gly(G)、Ser(S)、Gly(G)和Arg(R)组成的ar/R选择性过滤器,属于水通道蛋白NIPⅢ亚家族。实时荧光定量分析表明,Cm Lsi3基因在砧木南瓜根、茎、叶中均有表达,且‘云南黑籽南瓜’各器官Cm Lsi3基因的表达水平高于‘黄诚根2号’。

Two pumpkin cultivars for cucumber grafting‘Yunnan Figleaf Gourd＇（Cucurbita ficifolia）and‘Huangchenggen 2＇（C. moschata）,which have weak and strong de-blooming abilities respectively,were used in this study,and the full length c DNA of the silicon transporter gene were cloned by RT-PCR and RACE,and named CmL si3（Gen Bank accession numbers are KM203110 and KM359142）. The full-length of c DNA was 1 206 bp,and the open reading frame（ORF）was 795 bp,encoding 264 amino acids,and only four amino acids were different in‘Yunnan Figleaf Gourd＇and‘Huangchenggen 2＇,However the amino acid sequences shared at least 50% identity with those in cucumber,maize and barley. The transporter encoded by Cm Lsi3 contained typical two sparagine-proline-alanine（NPA）motifs,six transmembrane domains and a distinct ar/R（aromatic/arginine）selectivity filter composed from Gly（G）,Ser（S）,Gly（G）and Arg（R）,belonging to nodulin 26-like intrinsic membrane protein Ⅲ（NIP Ⅲ）subgroup of plant aquaporin. The expression of Cmlsi3 was investigated by using fluorescence quantitative RT-PCR and the result showed that the Cm Lsi3 gene was expressed in roots,stems and leaves of pumpkin and the expression level in organs were higher in‘Yunnan Figleaf Gourd＇than in‘Huangchenggen 2＇.