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| Construction of Salicylic Acid Methyltransferase Gene Over-expression Vectors and Genetic Transformation in Poplar (Populus alba × P. glandulosa) |
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Abstract When plants of annual herb are being invaded by a pathogen, the gene of salicylic acid methyltransferase (SAMT), which converts salicylic acid (SA) produced at the inoculation site into methyl salicylate (MeSA), plays an important role in plant systemic acquired resistance (SAR) and signal transduction of SA. However, the function of SAMT in perennial woody plants needs to be further verified. In this study, a pair of primers was designed according to the complete CDS of Populus trichocarpa SAMT and was used to amplify the SAMT gene of 84K popular by polymerase chain reaction. Under Gateway technology, we performed a BP recombination reaction between the attB-flanked SAMT fragment and an attP-containing donor vector pDONRTM222 with the action of BP clonase enzyme to convert the gene of interest into entry vector. Then the vector were transformed competent Escherichia coli DH5α. Plasmids, which were extracted and digested by MLU Ⅰ enzyme, and over-expression vector pMDC32 were performed an LR recombination reaction with the action of LR clonase enzyme to transform the SAMT into expression vector. Successfully over-expression vector of SAMT gene were constructed, and SAMT-overexpressing transgenic 84K popular plants were obtained by Agrobacterium-mediated genetic transformation using leaf-disk transformation method. Then transgenic lines were tested and analyzed by RT-PCR and qPCR methods. From the results, leaf-disk transformation is appropriate transformation method, with high conversion efficiency. Eccept one line of the SAMT -overexpressing genetically modified lines is lower, 57 times more than CK. Compared with control, other lines of transgenic 84K popular plants are more than 3 729 times. These results laid the foundation for the future research of the gene function and SAR in poplar and other roles, as well as provided the reference about the function of the gene in other perennial woody plants.
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Received: 30 May 2016
Published: 01 October 2016
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