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植物紫色酸性磷酸酶(purple acid phosphatase,PAP)在植物响应低磷胁迫过程中起重要作用,为了验证马尾松(Pinus massoniana)紫色酸性磷酸酶(PmPAP1)基因在磷吸收上的功能,本研究通过农杆菌(Agrobacterium tumefaciens)介导法转化烟草(Nicotiana tabacum),获得25个转基因株系,RT-PCR及qRTPCR分析表明,PmPAP1在13个株系中高表达;对其中2个高表达株系(Line-10和Line-13)进行不同浓度磷处理,结果表明,缺磷可以诱导转基因植株中PmPAP1基因在根和叶中上调表达;低磷条件下,转基因植株根系及叶片酸性磷酸酶(acid phosphatase,APase)活性分别为野生型(wild type,WT)的1.27和1.25倍;转基因植株总磷与无机磷含量分别比WT提高了28.32%、45.48%(Line-10)与36.45%、62.31%(Line-13)。除此之外,缺磷条件下转基因植株过氧化物酶(peroxidase,POD)、超氧化物歧化酶(superoxide dismutase,SOD)均较WT显著提高(P<0.05),而丙二醛(malondialdehyde,MDA)含量则显著下降(P<0.05)。磷胁迫对植株生长影响表明,低磷条件下转基因植株总生物量、根系生物量及根冠比均显著提高(P<0.05)。而在正常和高磷条件下,转基因与野生型烟草的酶活性、丙二醛含量、磷含量和生物量等与均无明显差异。因此,高表达PmPAP1基因能显著提高烟草植株在低有效磷下磷的利用能力,进而增强耐低磷胁迫能力。本研究为深入了解马尾松耐低磷的分子机制与创制耐低磷烟草新种质提供了理论依据。
Plant purple acid phosphatase (PAP) plays an important role in the plant response to low-P stress. In order to verify the role of PmPAP1 in phosphorus uptake by Pinus massoniana, Twelve transgenic lines were obtained by Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. RT-PCR and qRTPCR analysis showed that PmPAP1 was highly expressed in 13 lines. -10 and Line-13). The results showed that P deficiency could induce the up-regulation of PmPAP1 gene in roots and leaves of transgenic plants. Under low phosphorus conditions, the root and leaf acid phosphatase APase activities were 1.27 and 1.25 times higher than wild type (WT) respectively. The content of TP and inorganic phosphorus in transgenic plants increased by 28.32%, 45.48% and 36.45% and 62.31% -13). In addition, peroxidase (POD) and superoxide dismutase (SOD) of transgenic plants were significantly increased compared with WT (P <0.05), while malondialdehyde MDA content decreased significantly (P <0.05). The effects of phosphorus stress on plant growth indicated that the total biomass, root biomass and root / shoot ratio of transgenic plants were significantly increased under low phosphorus (P <0.05). Under normal and high phosphorus conditions, there was no significant difference in enzyme activity, malondialdehyde content, phosphorus content and biomass of transgenic and wild type tobacco. Therefore, the high expression of PmPAP1 gene can significantly improve the ability of tobacco plants to utilize phosphorus under low available phosphorus, thereby enhancing the ability of resistant to low-phosphorus stress. This study provides a theoretical basis for understanding the molecular mechanism of low phosphorus tolerance of P. massoniana and creating new germplasm of low-phosphorus-tolerant tobacco.