Cloning Of Novel Herbicide-degradation Genes From Soil Metagenomic Libraries

With advantages of high catalysis efficiency, low toxicity and low cost, 2,4-dichlorophenoxyacetic acid (2,4-D) and glyphosate both have become the most widely used herbicides all over the world. But the two herbicides would also cause some damages to crops during the process of killing weeds. These factors had limited the two herbicides'large-scale promotion and application until the transgenic crop era, that scientists discovered herbicide resistant genes and transformed them genetically into crops, have come. Since the inhibition mechanism of 2,4-D on plant is still unclear, currently 2,4-D-resistance of transgenic crops were obtained by overproduction of 2,4-D degrading enzyme,2,4-D a-ketoglutarate dioxygenase (TfdA) which is reported few. On the other hand, the commercialized glyphosate-resistant transgenic crops were obtained by overproduction of bacterial EPSPs from Agrobacterium sp. strain CP4 (namely CP4 EPSPs). However, glyphosate uptaken by CP4 EPSPs-transgrenic crops cannot be degraded and may accumulate in plants, which generated potential negative effects to plants and human. Studies have shown that the introduction of glyphosate degradation mechanism can solve this problem. Therefore, it is necessary to explore new 2,4-D and glyphosate-degradation enzymes.In this study, two glyphosate-degradation genes were identified from the glyphosate polluted soil metagenomic library, there were glyphosate oxidoreductase gene and glycine oxidase gene, designated as goxA and goA, respectively. There are two amino acids difference in the amino acid sequence of the encoding products of goxA with Monsanto patented gox. The encoding produt of goA was only a 88% homology with GO from Bacillus subtilis strain 168. A chimeric glyphosate oxidoreductase gene was obtained using overlapping extension PCR, designated as chgox, whose product exhibited 79% homology with Monsanto patented GOX. The two coding product of goxA and chgox exhibited the similar activity toward all the assayed substrates.Two novel 2,4-D-degradation genes,2,4-dichlorophenol hydroxylase gene (tfdB) and catechol 1,2-dioxygenase gene (tfdC), were identified from chloropenols polluted soil metagenomic library by screening catechol 1,2-dioxygenase activity, designated as tfdB-JLU and tfdC-JLU, respectively. The encoding product of tfdB-JLU, was only 48% homology with TfdB from Cupriavidus necator JMP134 (pJP4). The deduced amino acid sequence of tfdC-JLU exhibits 59% homology with the TfdC from Comamonas testosteroni S44. The purified TfdB-JLU exhibits a wider substrate spectrum and a higher relative activity towards many chlorophenols than 2,4-dichlorophenol, the preferred substrate of other known TfdBs.The three novel glyphosate-degradation genes identified in this study could become potential candidate genes for the development of glyphosate-tolerant transgenic plants without glyphosate residues. Furthermore, the identification of tfdB-JLU and tfdC-JLU in this work indicate that it is possible to obtain the 2,4-D metabolic genes by constructing metagenomic library and activity screening method. This work provides an experimental basis for the ultimate identification of new tfdA.