摘要
避开传统的分离培养过程,采用现代分子生物学方法探讨了杀虫剂啶虫脒污染条件下旱地土壤微生物种群多样性。通过对不同培养时间、不同浓度啶虫脒污染下旱地土壤微生物进行DGGE基因多样性的分子指纹图谱分析,发现随着培养时间不同,各处理之间的土壤微生物基因多样性出现了一定的差异。但在整个试验过程中,正常田间使用浓度(0.5mg kg-1干土)的啶虫脒对土壤微生物群落的影响不明显,DGGE图谱条带与对照没有明显差异,土壤微生物基因多样性没有明显下降,这说明在旱地中使用正常田间浓度的啶虫脒不会对微生物群落造成较大的影响,高浓度啶虫脒对土壤微生物群落基因多样性有一定的影响,但是影响时间不长。在培养第五周时,浓度为5 mg kg-1干土的土样出现了特异性条带,为对照所没有,其他处理浓度染色暗淡。经序列比对分析,与来自土壤的Uncultured bacterium具有100%的相似率,可能为不可培养或未培养过的细菌种。
Acetamiprid is an insecticide used on cotton, leafy vegetables, fruit trees, and other horticultural crops to control sucking insects. Acetamiprid has been classified as an unlikely human carcinogen and it has relatively low acute or chronic toxicity in mammals. As an insecticide, considerable part of applied acetamiprid would enter actually soil. However, it＇s possible effects on the soil bacterial community have not be well known update. We examined bacterial community composition in an acetamipridpolluted upland soil in China using PCR-DGGE method which can detect variation of microbial gene diversity in environments. Soils to which acetamiprid was applied at the normal field dose （0.5 mg kg^- 1 dry soil）, at higher dose （5mg kg^- 1 , 25 mg kg^- 1 , 50 mg kg^- 1 ） or not applied were put in pots, respectively, incubated at 28℃ , and sampled weekly after incubation for PCRDGGE analysis. Soil sample DNA was extracted using the SDS-high strength salt method and purified by CsCl2. The V3 region of 16S rDNA was amplified using the universal primers （BSF8/20 and BSR1541/20; F338GC and R518）. Amplified DNA fragments were separated by parallel denaturing gradient gel electrophoresis （DGGE）. The DGGE results indicated that there were differences in bacterial community diversity in upland soils treated with different concentrations of acetamiprid. There was no significant difference in DGGE patterns between 0.5 mg kg^-1 dry soil of acetamiprid and the control soil during the whole incubation time. This suggested that acetamiprid applied at the normal field dose would be unlikely to pose a threat to soil bacterial communities. However, changes were observed in DGGE fingerprints derived from soil after one week receiving the high concentrations of acetamiprid, such as 5, 25, and 50 mg kg^- 1 dry soil. DNA in application-responsive bands from the acetamiprid treatments was recovered and amplified using the universal primers （F338 and R518）. PCR products were recovered and cloned into pGEM-T Easy （Promega） and two clones were obtained. The two clones were sequenced using the automated Model 377 or 3730 DNA sequencing system （Applied Biosystems）. The two cloned sequences had very high similarities （100%） to many uncultured bacteria reported previously in database of NCBI.
出处
《生态学报》
CAS
CSCD
北大核心
2006年第9期3074-3080,共7页
Acta Ecologica Sinica
基金
国家自然科学基金资助项目(30370048)~~
