A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice.

Shu-yuan Yi,Zi-duo Liu,Ying Cui,Fei Zhou,Yong-jun Lin,Yan Zhao

doi:10.1038/srep19104

Abstract

As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system.

Highlights

Glyphosate tolerance: RXHXE and NXTR (X represents non-conserved amino acids), in both of which the positive charge of the side chain of Arg hinders the binding of glyphosate[17]
The results show that it can be used as an effective marker for direct selection and the generated transgenic rice lines were conferred high glyphosate tolerance
After screening about 5,000 colonies from the library, one positive colony was found to possess the ability to grow on M9 agar containing 100 mM glyphosate, indicating that the recombinant plasmid in the colony might contain a gene involved in glyphosate tolerance

Summary

Introduction

Glyphosate tolerance: RXHXE and NXTR (X represents non-conserved amino acids), in both of which the positive charge of the side chain of Arg hinders the binding of glyphosate[17]. Glyphosate insensitivity can be achieved in class I aroA enzymes through site-directed mutagenesis[23,24,25] or natural selection[26,27,28]. The double mutated aroA (TIPS) has been successfully used to produce the first commercial transgenic glyphosate-tolerant maize (GA21). A novel aroA enzyme from a glyphosate-tolerant strain Janibacter sp Sp is characterized as a new class I aroA enzyme. The results show that it can be used as an effective marker for direct selection and the generated transgenic rice lines were conferred high glyphosate tolerance (up to 3360 g/ha glyphosate). Our study indicates that the naturally occurring novel class I aroAJ. Our study indicates that the naturally occurring novel class I aroAJ. sp gene has promising potential for the development and breeding of transgenic glyphosate-tolerant crops

Methods

Results

Conclusion