【作者】 李刚；

【导师】 薛红卫；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 遗传学， 2006， 博士

【摘要】 磷脂酶D是普遍存在于细菌、真菌、植物及动物中的一类磷脂水解酶,可以水解磷脂分子内特定的酯键从而生成磷脂酸PA和自由的头部基团分子。在植物中,PLD及其产物PA参与了逆境胁迫、激素响应、囊泡运输、细胞骨架蛋白重组等多个细胞过程。为了在植物中研究磷脂酶D的生理功能,我们在拟南芥中分离得到了AtPLDζ2的全长cDNA克隆,并对其生理功能进行了初步研究。不同组织的RT-PCR以及对启动子区域驱动的GUS转基因植株进行染色分析,揭示了AtPLDζ2在多个组织中特异表达,并且该基因可以被生长素诱导表达。AtPLDζ2的缺失突变体atpldζ2、及缺失表达的转基因植株具有主根变短的表型,同时还降低了对外源生长素的敏感性、以及生长素调节的重力向性响应和高温下生长素促进的下胚轴伸长。AtPLDζ2的过量表达的转基因植株具有与atpldζ2及AtPLDζ2缺失表达的转基因植株完全相反的表型。PLD特异抑制剂1-butanol处理或AtPLDζ2的缺失表达可以降低生长素介导的DR5-GUS在根尖的分布以及早期生长素响应基因对生长素的响应。1-butanol处理植株还可以导致根部伸长区表皮细胞的膨大和生长素介导的DR5-GUS异常分布,并抑制生长素诱导的DR5-GUS在主根的伸长区大量积累以及生长素促进的侧根形成。外源添加PA或AtPLDζ2的过量表达可以促进DR5-GUS在根部维管束的积累以及生长素早期响应基因对生长素的响应,进一步阐明了AtPLDζ2及其产物PA在植物对生长素的响应过程中起到正向的调控作用。通过囊泡特异的荧光染料FM4-64对植株染色证明AtPLDζ2及其产物PA在植物细胞内影响了囊泡运输。进一步的实验证明,AtPLDζ2及其产物PA通过影响囊泡运输进而影响了生长素输出蛋白PIN1和PIN2在细胞质膜与细胞质之间的快速循环,从而介导了植物对生长素的响应过程。更多还原

【Abstract】 Phospholipase D (PLD),which hydrolyzes phospholipids to produce phosphatidic acid (PA) and a free head group, has been detected in bacteria, fungi, plants, and animals. In plant, PLD as well as its product PA play key roles in numerous cellular processes including stress responses, hormone effects, membrane vesicle trafficking and cytoskeleton rearrangements. To elucidate the physiological functions of PLD, an Arabidopsis thaliana cDNA encoding AtPLDζ2, was isolated and functional characterized.AtPLDζ2 was constitutively expressed in various tissues and induced by auxin, and phenotypic analysis on the AtPLDζ2 defected mutant, atpldζ2, and transgenic plants deficiency of AtPLDζ2 showed they were insensitive to auxin, and also have reduced root gravitropism and suppressed auxin-dependent hypocotyls elongation under high temperature (29oC); while the transgenic seedlings overexpressing AtPLDζ2 showed opposite responses, suggesting AtPLDζ2 positively mediates auxin signaling. Further studies using seedlings containing DR5-GUS construct and on the expression profiles of early auxin responsive genes indicated that AtPLDζ2, or PA, stimulated auxin responses. Observations on the internalization of membrane selective dyes FM4-64 showed the suppressed vesicle trafficking under deficiency of AtPLDζ2 or by treatment with PLD specific inhibitor 1-butanol, whereas which was obviously enhanced under PA or enhanced AtPLDζ2. 1-butanol has no effects on the localization of PIN1 and PIN2, but blocks the inhibition of BFA on PIN1 and PIN2 cycling. These results suggest that AtPLDζ2 and its product PA, stimulates auxin responses via accelerating vesicle trafficking.更多还原