| We select the populations of field bindweed in Hebei, Beijing and Shandong Province as the research objects and studied the difference of glyphosate tolerance levels, the EPSPS gene sequences and in vitro activities, EPSPS gene expression at the transcription level treated by glyphosate of different field bindweed populations. We eluciate the mechanism of glyphosate tolerance in different field bindweed populations.The following conclusions:1. The tolerance of different field bindweed populations to glyphosate was significantly different.The results showed that GR50 of glyphosate to Hebei Yuanshi, Hebei Xushui, Shandong Zhanhu’a, Hebei Guangyang, Hebei Chengde, Beijing Fangshan, Hebei Zhengding and Beijing Haidian population were 4014.92,2834.04,2243.29,1889.31,1829.31,1789.56,1754.33 and 957.65 g a i-ha-1, respectively. The GR50 value of Hebei Yuanshi population was 4.19 times higher than that of the Beijing Haidian population.2. The accumulation level of shikimic acid was inversely proportional to the tolerance level of filed bindweed population in different places. Therefore we can compare the glyphosate tolerant differences of filed bindweed populations in different places by detect the shikimic acid accumulation level of field bindweed treated by glyphosate.3. The full length of cDNA in EPSPS gene measured from five different region field bindweed populations were all same with 1707bp and the alignment identity was 98.12%; The full-length of open reading frame in deduced amino acid sequence was 1560 bp, encoding 520 amino acids.The deduced amino acid sequence is 99.73%identical.4. EPSPS amino acid sequences of the five different field bindweed populations in two conserved regions were same; and the EPSPS in vitro activity of different populations were no difference in sensitivity to glyphosate. Compared with Arabidopsis, Japanese bindweed and other plants, in the conserved B of mature EPSP synthase, the 101 point of field bindweed EPSP synthase was a polar amino acid serine, which had polar hydroxyl group and water affinity. But in other plants it was a non-polar hydrophobic amino acid phenylalanine at the same site. The different level of field bindweed populations’tolerance to glyphosate has nothing to do with the EPSPS gene itself and its ivitro activities. The 101 amino acids of field bindweed is different with other plants. This difference may cause the EPSP synthesis changing in structure and function, which may be the reason that field bindweed naturally resistance to glyphosate.5. After glyphosate treatment, the quantity of mRNA encoded by EPSPS of two regions increased rapidly, but there were differences. The EPSPS gene expression of Hebei Yuanshi population increased obviously 1 DAT and reached the highest value at 3 DAT which was 3.17 times higher than that of control, then reduced to the same level as the control. Meanwhile, the EPEPS gene expression of Beijing Haidian population significantly increased at 2 DAT and reached its peak value at 5 DAT (2.42 times higher than that of control). Therefore, the difference of the expression time and quantity between the two field bindweed populations was the reason that the field bindweed populations were different in glyphosate tolerance.In summary, the tolerance levels to glyphosate of field bindweed in several regions in North China were investigated for the first time. The glyphosate tolerant mechanism of field bindweed was elucidated both at the enzyme level and gene level. This study has very important theoretical and practical significances to the research work of glyphosate tolerance mechanism. |
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