砷代草丁膦的合成分子机制及其对水稻土壤细菌群落结构的影响

Synthesis molecular mechanism of arsinothricin and its impact on bacterial community structure of rice soil

摘要:

目的 进一步探讨arsL、arsM基因在砷代草丁膦(arsinothricin, AST)合成中的作用，以及AST对土壤细菌群落结构的影响。方法 以俄克拉何马伯克霍尔德氏菌(Burkholderia oklahomensis) NCTC 13388为研究对象，通过PCR扩增获得该菌株的BoarsL和BoarsM基因，分别构建重组质粒pET21b-BoarsL和pET28a-BoarsM。将重组质粒转化至大肠杆菌表达菌Rosetta(DE3)感受态细胞中。采用高通量测序技术分析不同浓度AST处理对土壤细菌群落组成及多样性的影响。结果 十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)分析显示，重组菌株表达出相对分子质量分别为47.79 kDa和41.50 kDa的目的蛋白，证实BoArsL和BoArsM蛋白成功表达。单独表达BoarsL基因的细胞产生AST-OH和少量的AST，而单独表达BoarsM基因的细胞仅产生少量的二甲基砷酸。统计分析表明，不同浓度AST处理对土壤细菌群落的α多样性产生了显著影响(P<0.05)，具体表现为Chao1指数和Shannon指数均存在显著差异，低浓度处理组土壤细菌多样性和丰富度高于对照组，高浓度处理组则显著降低了土壤细菌的多样性和丰富度。进一步分析发现，不同浓度AST处理组在属水平上的细菌群落组成也呈现显著差异(P<0.05)，高浓度AST显著富集了伯克霍尔德氏菌属-卡瓦列罗菌属-副伯克霍尔德氏菌属(Burkholderia-Caballeronia-Paraburkholderia)细菌，同时对梭状芽孢杆菌属(Clostridium_sensu_stricto)、沉积物杆菌属(Sedimentibacter)等细菌则表现出显著的抑制作用。结论 B. oklahomensis NCTC 13388菌株的BoarsL基因是AST生物合成所必需的。高浓度AST显著干扰了土壤细菌群落结构。

Abstract:

Objective To further investigate the role of arsL and arsM genes in the synthesis of arsinothricin (AST) and the effects of AST on the community structure of soil bacteria.Methods Using Burkholderia oklahomensis NCTC 13388 as the research object, we obtained its BoarsL and BoarsM genes via PCR amplification, constructed recombinant plasmids pET21b-BoarsL and pET28a-BoarsM, and transformed them into the competent cells of Escherichia coli expression strain Rosetta(DE3). In addition, we employed high-throughput sequencing technology to analyze the effects of different concentrations of AST treatment on the composition and diversity of soil bacterial communities.Results Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected target proteins with relative molecular weights of 47.79 kDa and 41.50 kDa in recombinant strains, indicating successful expression of BoArsL and BoArsM. Cells expressing only the BoarsL gene produced AST-OH and a small amount of AST, while cells expressing only the BoarsM gene produced only a small amount of dimethylarsinic acid. Additionally, statistical analysis indicated that AST treatment at different concentrations had a significant impact on the alpha diversity of soil bacterial communities (P<0.05), as evidenced by significant differences in both the Chao1 and Shannon indices. The low-concentration treatment group had higher soil bacteria diversity and richness than the control group, whereas the high-concentration treatment caused statistically significant declines in both diversity and species richness. Further analysis revealed that bacterial community composition at the genus level also exhibited significant differences among the AST treatment groups of different concentrations (P<0.05), and high concentrations of AST significantly enriched bacteria of the genus Burkholderia-Caballeronia-Paraburkholderia but significantly inhibited bacteria of the genera Clostridium_sensu_stricto and Sedimentibacter.Conclusion The BoarsL gene of B. oklahomensis NCTC 13388 is essential for the biosynthesis of AST. High concentrations of AST significantly affect the structure of soil bacterial communities.