广谱抗真菌蛋白转基因水稻秸秆模拟还田对土壤真菌群落结构的影响

Effects of simulated straw return of transgenic rice expressing broadspectrum antifungal proteins on soil fungal community structure

为探讨广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响,本文在室温条件下进行田间秸秆还田模拟试验,设不添加秸秆（S）、添加转基因水稻‘转品1’秸秆（S-Z1）、添加转基因水稻‘转品8’秸秆（S-Z8）、添加非转基因水稻‘七丝软粘’秸秆（S-CK）4个土壤处理,采用传统的平板计数法和变性梯度凝胶电泳（denatured gradient gel electrophoresis, DGGE）技术,分析广谱抗真菌蛋白转基因水稻秸秆模拟还田过程中土壤可培养真菌数和土壤真菌群落的变化情况。平板计数结果表明,在秸秆降解的第40 d,转基因水稻秸秆处理（S-Z1、S-Z8）与非转基因水稻秸秆处理（S-CK）土壤之间的可培养真菌数差异显著,但秸秆降解中后期（50-90 d）, S-Z1、S-Z8和S-CK之间土壤可培养真菌数的差异均不显著。真菌18S rRNA的PCR-DGGE图谱显示, S-Z1、S-Z8和 S-CK 在秸秆降解过程中没有显著不同的条带出现,仅有个别条带在亮度上存在差异。DGGE 图谱条带多样性分析结果表明,在秸秆降解的个别时间段, S-Z1、S-Z8和S-CK之间在丰富度和Shannon-Wiener多样性指数上存在显著差异,而在秸秆降解的整个过程均匀度指数差异均不显著。对 DGGE 主要条带和差异性条带进行克隆测序后发现,子囊菌占最大比重,其次为担子菌、壶菌,而在转基因和非转基因土壤处理间亮度上存在差异的条带属于子囊菌。以上研究结果表明,广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响是短暂的、不持续的。

There has been a growing public concern about the impact of therapid introduction of genetically modified crops （GMCs） on soil ecosystem, especially soil microbial community. In order to verify the effects of decomposition of GMCs straws on soil fungal community structure, classical plate counting anddenatured gradient gel electrophoresis （DGGE） were used to analyze cultivable fungi amount and fungal community diversity. Straw of transgenic rice expressing broad-spectrum antifungal proteins was degraded under simulated field conditions. The treatments included soil without straw （S）, soil with straws of transgenic rice cultivar of ‘Zhuanpin-1’ （S-Z1）, soil with straws of transgenic rice cultivar of ‘Zhuanpin-8’ （S-Z8） and soil with straws of non-transgenic rice cultivar of ‘Qisiruanzhan’ straw （S-CK）. The results of traditional culture-dependent method showed significant differences in the amounts of cultivable fungi among transgenic treatments of S-Z1, S-Z8 and non-transgenic treatment of S-CK within 40 days after rice straw decomposition（P＆lt; 0.05）. However, no significant differences were noted among the above treatments in the period from 40 to 90 days after rice straw decomposition（P＆gt; 0.05）. The patterns of DGGE of 18S rRNA V1＋V2 fragments showed no apparent differences in bands between transgenic and non-transgenic treatments as the different observed bands were largely weak or transient.The positions and lightness of DGGE bands were digitally analyzed using Quantity One software and then the derived Shannon-Wiener index, evenness and abundance to evaluate fungal diversity of different treatments. The results showed significant differences in Shannon-Wiener index and abundance between transgenic and non-transgenic treatments in individual periods of rice straw decomposition such as the first 30 days （P＆lt; 0.05）. There were no significant differences in evenness between transgenic and non-transgenic treatments during the whole rice straw decomposition period （P＆gt; 0.05）. Sequence analysis of DGGE-bands showed that most of the bands belonged toAscomycota,Basidiomycota andChytridiomycota and the different bands between transgenic and non-transgenic treatments belonged toAscomycota. The study suggested transient effects of the straw decomposition of transgenic rice expressing broad-spectrum antifungal proteins, but no persistent effects on cultivable fungi count and fungal community structure.