小白菜AMT1;2基因克隆、组织表达特性检测及功能验证

Molecular cloning,tissue expression specificity detection and functional analysis of AMT1;2 from Chinese cabbage Pak-Choi

  • 摘要: 【目的】克隆小白菜铵转运蛋白基因(BcAMT1;2),检测其组织表达特异性并验证其功能,为深入探究BcAMT1;2基因在小白菜NH4+吸收和转运过程中的作用机制提供理论参考。【方法】以小白菜品种上海青为材料,采用同源克隆方法克隆BcAMT1;2基因,通过对其编码蛋白进行生物信息学分析,采用实时荧光定量PCR检测BcAMT1;2基因组织表达模式,并在拟南芥中超表达该基因以验证其功能。【结果】克隆的BcAMT1;2基因cDNA序列全长为1539bp,编码512个氨基酸残基,蛋白分子量为54.90 kD,理论等电点(pI)为8.03,无信号肽,有9个跨膜结构域,定位于细胞膜上,为稳定的两性蛋白。BcAMT1;2蛋白归属于AMT1亚家族,具有AMT1的特征结构域(DFAGSGVVHMVGGIAGLWGALIEGPR),与拟南芥AtAMT1;2蛋白的氨基酸序列相似性最高,为91.21%。BcAMT1;2基因在叶片中的表达量显著高于其他组织(P<0.05,下同),根和叶柄次之,而在茎中几乎不表达。在0.25 mmol/L NH4+处理下,超表达BcAMT1;2基因的3个拟南芥株系的生物量(地上部鲜重和地下部鲜重)、主根长度和植株内NH4+-N含量较野生型均显著增加,而在20 mmol/L甲基胺(MeA)下,超表达BcAMT1;2基因的3个株系的株鲜重较野生型显著降低,且表现出植株叶片黄化、根系生长受抑等毒害效应。【结论】BcAMT1;2基因具有明显的组织表达特异性,可调控NH4+及其类似物甲基胺的吸收和转运,表明BcAMT1;2蛋白在小白菜对NH4+的吸收和转运过程中发挥着重要作用。

     

    Abstract: 【Objective】 In this paper, an ammonium transporter gene BcAMT1;2 was cloned from Chinese cabbage Pak-Choi, its bioinformatics and tissue expression characteristics were detected, and its function was analyzed, which provided a the oretical basis for further studying the mechanism of BcAMT1;2 gene in NH4+ absorption and transportation in Chinese cabbagePak-Choi.【Method】 Homologous cloning method was applied to clone BcAMT1;2 gene from Chinese cabbage Pak-Choi variety Shanghaiqing, then its biological information was carried out on its encoded protein. The expression of BcAMT1;2 gene was detected by real-time fluorescence quantitative PCR in different tissues of Chinese cabbage Pak-Choi, and the function of BcAMT1;2 was verified by overexpression BcAMT1;2 in Arabidopsis thaliana.【Result】 The open reading frame(ORF) of BcAMT1;2 gene was1539 bp, encoding 512 amino acids residues, with molecular weights of 54.90 kD, and theoretical isoelectric points(pI) of 8.03. In addition, BcAMT1;2 protein had no signal peptide, but had 9 transmembrane domains. Stability prediction indicated that BcAMT1;2 was stable amphipathic protein, and the subcellular localization prediction showed BcAMT1;2 might locate in the plasmalemma. Conserved domain analysis suggested that BcAMT1;2 included the motif(DFAGSGVVHMVGGIAGLWGALIEGPR), which was the signature motif of AMT1 subfamily. Moreover, amino acid sequence alignment showed that BcAMT1;2 protein had the highest homology with At-AMT1;2(91.21%) in A. thaliana, so BcAMT1;2 was one member of AMT1 subfamily. The expression level of BcAMT1;2 in leaf was significantly higher than in other tissues in Pak-Choi(P<0.05, the same below), followed by the ones of root and petiole, but almost no expression in stem. Under the treatment of 0.25 mmol/L NH4+, the biomass(the fresh weight of root and shoot), the length of primary root, and NH4+-N content of the whole plants were significantly increased in three lines of overexpressing BcAMT1;2, compared with the ones of wild type. At 20 mmol/L methylamine(MeA), the biomass of three lines of overexpressing BcAMT1;2 was significantly lower than that of wild type, with yellow leaves, short roots, and other symptoms of ammonia toxicity.【Conclusion】 BcAMT1;2 gene has obvious tissue specificity and can regulate the absorption and transportation of NH4+ and its analog methylamine, indicating that BcAMT1;2 protein may play an important role in the absorption and transport of NH4+ in Pak-Choi.

     

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