谷子非特异性磷脂酶C基因SiNPC4的克隆及功能分析

Cloning and Functional Analysis of Nonspecific Phospholipase C Gene SiNPC4 in Foxtail Millet(Setaria italic)

非特异性磷脂酶C(NPC,nonspecific phospholipase C)水解磷脂酰胆碱或磷脂酰乙醇胺产生第二信使二酰甘油(DAG),NPC类基因在植物处于干旱、高盐、低磷等非生物胁迫条件下时发挥重要的作用,并参与脱落酸(ABA)、油菜素内酯(BL)等激素信号传导途径。本研究以谷子品种龙谷25为试验材料,通过序列比对,克隆到一个新的NPC类基因,命名为Si NPC4。该基因被定位在谷子第8条染色体上,编码区全长2877 bp,由3个外显子和2个内含子组成,编码512个氨基酸,蛋白分子量为56.77 k D。系统进化树分析表明该基因位于NPC基因家族第3亚族,保守结构域分析表明该蛋白含有保守的磷脂酶结构域和4个基序。亚细胞定位结果显示,Si NPC4蛋白被定位在细胞质、细胞膜、细胞核中。基因表达谱分析结果表明,该基因主要在谷子根部表达,并受干旱、盐、低温、黑暗、ABA、BL、赤霉素(GA)和茉莉酸甲酯(Me JA)等的诱导表达。将Si NPC4基因转入拟南芥中,转基因拟南芥与野生型拟南芥相比对ABA和BL激素的敏感性降低,推测该基因可能作为负调控因子参与植物对ABA和BL激素的响应过程。在干旱和高盐处理下,过表达植株与野生型植株长势没有明显差异。

Nonspecific phospholipase C（NPC） catalyzes the hydrolysis of phosphatidylcholine or phosphatidylethanolamine to generate diacylglycerol（DAG）, which is an important second messenger in cells. NPC family plays a key role in response to some stresses such as drought, high salinity and phosphorus deficiency, and is involved in some hormone signaling pathways such as abscisic acid（ABA） and brassinolide（BL）. Taking foxtail millet as material, we cloned a novel NPC gene named Si NPC4 by sequence alignment. This gene was detected to be located on chromosome 8. The full length of Si NPC4 was 2877 bp with three exons and two introns encoding 512 amino acid residues and the protein molecular weight was 56.77 k D. Phylogenetic analysis of NPC protein sequences indicated that Si NPC4 distributed to the third subfamily. The predicted protein structure of the gene contained conserved phosphoesterase domain and four motifs. The protein subcellular localization analysis revealed that Si NPC4 was localized in cytomembrane, cytoplasm, and nucleus. The gene expression profile results indicated Si NPC4 mainly expressed in root and was induced by drought, salt, cold, dark, ABA, BL, methyl jasmonate（Me JA）, and gibberellic acid（GA） treatments. Arabidopsis carrying Si NPC4 decreased the sensitivity to ABA and BL compared with WT. Thus, we deduced that Si NPC4 may act as a negative regulator in ABA and BL signaling pathways. Besides, there were no significant difference in growth between transgenic and wild type plants under drought and high salinity treatments.