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| Genetic Transformation of Masson Pine (Pinus massoniana) PAP1 in Tobacco (Nicotiana tabacum) and Its Functional Analysis |
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Abstract Abstract Plant purple acid phosphatase (PAP) is crucial in response to low phosphorus stress. To further validate its function in phosphorus absorption, Pinus massoniana (PmPAP1) gene was genetically transformed into tobacco (Nicotiana tabacum) 'Xanthin' via Agrobacterium-mediated method, and 25 transgenic PmPAP1 lines were obtained through PCR detection in the current study. With semi-quantitative RT-PCR and qRT-PCR analysis, 13 transgenic PmPAP1 lines were highly over-expressed in transcriptional level of PmPAP1, among which 2 transgenic PmPAP1 lines (Line-13 and Line-10) were treated with different concentration of phosphorous for 45 d, and Semi-quantitative RT-PCR and qRT-PCR were used to detect the expression of PmPAP1 gene in transgenic plants under different phosphorous concentration. Meanwhile, the total phosphorus content, inorganic phosphorus content, phosphatase (APase) activity in roots and leaves, leaf protective enzyme activity, leaf content of malondialdehyde (MDA) as well as biomass indexes of wild type(WT) and transgenic PmPAP1 plants were measured. The results indicated that the expression of PmPAP1 were up-regulated in leaves and roots of the transgenic PmPAP1 tobacco as subjected to phosphorus deficiency in comparison with the normal condition (control), and the expression of its was bare in roots and leaves with the high phosphorus treatment. Under the condition of low phosphorus, the activities of acid phosphatase (APase) in roots and leaves of transgenic PmPAP1 tobacco were elevated 1.27 and 1.25 times respectively, compared to the WT plants, and the contents of total phosphorus and inorganic phosphorus increased by 28.32%, 45.48% in Line-10 and 36.45%, 62.31% in Line-13 respectively compared with the WT plants. Furthermore, the activities of peroxidase (POD) and superoxide dismutase (SOD) content analysis between transgenic PmPAP1 tobacco and WT plants revealed that the transgenic PmPAP1 tobacco were significantly elevated (P＜0.05) compared to those of the WT plants. The average activity of POD and SOD in transgenic PmPAP1 tobacco were 1.56 and 1.42 times of the WT plants respectively, while the results showed that the content of MDA in transgenic PmPAP1 tobacco leaves decreased significantly (P＜0.05). The MDA concent of transgenic PmPAP1 tobacco decreased 29.62% and 23.41% in line-10 and line-13 respectively compared with the WT plants. Effect of phosphorus on the growth of plants revealed that the total biomass and root biomass as well as the ratio of root to shoot of the transgenic significantly increased (P＜0.05) in transgenic PmPAP1 tobacco. Under the condition of no-phosphorus and low-phosphorus, the average root weight were 48.57% and 78.19% which were higher than that of the WT plants respectively, and the average root to shoot ratio increased by 60.28% and 53.02% compared with the WT plants respectively. Under the condition of normal phosphorus, there were no significant difference between the transgenic PmPAP1 tobacco and WT in phenotype of plants, while the transgenic PmPAP1 tobacco grew better than WT under phosphorus deficiency and demonstrated the high tolerance to low-phosphorus. There was no obvious difference in the enzyme activities, MDA concent, phosphorus concent and biomass of transgenic and WT plants under the condition of adequate phosphorus. Therefore, the over-expression of PmPAP1 might considerably enhance the tolerance to low-phosphorus stress in transgenic PmPAP1 tobacco, suggesting the involvement of this gene in phosphorus utility, particularly under low-phosphorus stress. These foundings provide basis theory for the better understanding for molecular mechanism of low-phosphorus tolerance in Pinus massoniana and creation of novel tobacco germplasm with low-phosphorus resistance.
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Received: 09 November 2016
Published: 01 June 2017
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