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| Transient Expression Vector Construction of AS Gene from Suspension Cells of Eriobotrya japonica and Its Subcellular Localization |
| LI Hui-Hua*, ZENG Bi-Yu, WANG Wei, CHANG Qiang, SU Ming-Hua |
| Fujian Key Laboratory of Physiology and Biochemistry for Subtropical Plant, Fujian Institute of Subtropical Botany, Xiamen 361006, China |
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Abstract Suspension culture of Eriobotrya japonica (loquat) can produce rich pentacyclic triterpenoids. Amyrine synthase (AS), a key enzyme in the triterpenoid synthesis pathway, determines the branching point of major metabolism and secondary metabolism. But the function of the AS in Eriobotrya japonica has not been reported before. In this study, using the AS overexpression plasmid pDONR207-AS as the template, the positive open reading frame of the AS gene was amplified by PCR, then ligated into the fusion expression vector pBWA(V)HS-GFP after digestion to construct the expression vector pBWA(V)HS-AS-GFP-Tnos. Next, using loquat suspension cells (homologous system with AS isolated) as receptor materials, transient expression was carried out under the mediation of Agrobacterium tumefaciens GV3101. Finally, the subcellular localization of AS protein were analyzed by imaging of co-transformed GFP under laser confocal microscopy, and the catalytic products of AS protein were analyzed by conjoint analysis of gas chromatography-mass spectrometry. Results were as follows: The subcellular localization of AS protein was in the endoplasmic reticulum in loquat. And the catalytic products of AS protein contained α-amyrin and β-amyrin, which indicated that AS of loquat belonged to the multifunctional enzyme. This study provides a reference for improvement of the synthesis of pentacyclic triterpenoid and for the further transgenic application in suspension cells of loquat.
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Received: 30 May 2019
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Corresponding Authors:
Huihua80@aliyun.com
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