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BioMed Research International
Volume 2016, Article ID 5657491, 8 pages
http://dx.doi.org/10.1155/2016/5657491
Research Article

Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis

Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China

Received 8 December 2015; Revised 8 March 2016; Accepted 16 May 2016

Academic Editor: Marta W. P. L. de Vasconcelos

Copyright © 2016 Wei Fang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between γ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of one γ-ECS from rubber tree latex (Hbγ-ECS1). According to qPCR analysis, the expression levels of Hbγ-ECS1 were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression of HbγECS1 increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow. HbγECS1 was expressed in E. coli Rosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827 U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role of Hbγ-ECS in thiols biosynthesis, which is influenced by tapping.