【作者】 刘微；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2009， 博士

【摘要】 近年来，随着转Bt基因植物的大量环境释放，转基因作物的生态安全问题越来越受到人们的关注与重视。尤其外源性基因(包括目的基因、标记基因和启动子等)的插入打乱了原有的基因网络，可能引起转基因作物的生理生化过程或光合产物代谢途径的改变，并由此导致以根系分泌物为底物和能源的微生物种群结构和活性变化，从而对农田土壤质量造成负面影响。本研究针对当前转Bt基因植物农田生态安全风险评价中关注的主要问题，以转Bt基因水稻“克螟稻”和亲本水稻“秀水11”为试材，通过(13)~C稳定态同位素脉冲标记技术，阐明了转Bt基因克螟稻及其亲本各器官(根、茎、叶)光合产物瞬时分配的差异以及水稻转Bt基因对土壤有机碳固定和微生物种群结构多样性的影响。在此基础上通过田间小区试验，对比杀虫剂施用和转Bt基因水稻种植对水稻根际微生物群落结构多样性的影响。研究结果为“克螟稻”等转Bt基因水稻大规模释放的农田土壤生态安全风险性评价提供了重要依据，并为同类研究提供了可借鉴的研究方法。具体研究结果如下：以克螟稻和亲本稻秀水11为研究对象，在温室条件下探讨了水稻不同生育期旗叶光合作用及其相关酶活性的变化。发现转Bt基因促进水稻拔节期和抽穗期旗叶光合速率和叶绿素含量显著升高。成熟期转Bt基因水稻与亲本水稻旗叶各生理指标无显著性差异。表明外源Bt基因的插入会引发水稻生长过程中光合生理特性的显著性变化，这种变化对农田土壤生态风险性影响有待进一步研究。基于转Bt基因对水稻光合特性的显著影响，应用(13)~C稳定态同位素脉冲标记技术分析了水稻转Bt基因对植物生长过程光合产物固定、分配及其根际活性微生物种群结构多样性的影响。结果表明，随着水稻生长，植物地上部分固定(13)~C的含量逐渐升高，而地下部分和土壤微生物中固定的(13)~C含量呈缓慢降低；苗期、拔节期和抽穗期转Bt基因克螟稻与亲本水稻叶片中(13)~C含量呈现显著性差异(P＜0．05)，然而在水稻生长的成熟期未观察到两者之间的显著性差异；苗期、拔节期和抽穗期，转Bt基因水稻根际革兰氏阳性菌(G~+)代表性脂肪酸含量显著低于其亲本水稻(P＜0．05)，革兰氏阴性菌(G~-)代表性脂肪酸含量则明显高于其亲本水稻(P＜0．05)，但在成熟期，两种水稻根际之间G~+、G~-以及磷脂脂肪酸总含量未呈现显著性差异(P＞0．05)；此外，在水稻生长的整个过程中，转Bt基因对水稻根际土壤真菌和放线菌代表性磷脂脂肪酸含量无显著性影响(P＞0．05)。总体而言，两种水稻瞬间光合产物优先进入根际革兰氏阳性菌(16：0)，真菌(cl8：2ω6)和革兰氏阴性菌(cl8：1)中。结果表明，crylAb基因的插入对苗期、拔节期和抽穗期水稻光合产物的分配以及根际细菌种群结构具有显著性影响，但这种影响并不持续到成熟期。以(13)~C标记的转Bt基因和亲本稻秸秆为试材，在温室条件下，通过秸秆还土试验探讨水稻转Bt基因对土壤有机碳固定效应的影响。结果发现，秸秆添加可显著提高淹水土壤甲烷和二氧化碳的排放通量。水稻根际微环境对秸秆的降解具有显著性的促进作用，秸秆降解过程中根际土壤有机碳(SOC)、溶解性有机碳(DOC)、微生物量碳(MBC)含量以及甲烷和二氧化碳排放速率均显著高于非根际土壤；与亲本水稻秸秆降解相比，水稻转Bt基因对根际和非根际区域秸秆的降解、土壤有机碳、溶解性有机碳和微生物量碳含量以及甲烷和二氧化碳的排放通量无显著性影响。变性梯度凝胶电泳(DGGE)图谱及其主成份(PCA)分析表明，水稻秸秆的添加对淹水土壤和水稻根际土壤产甲烷古菌的种群结构多样性具有一定的影响，但水稻转Bt基因则对淹水土壤和水稻根际产甲烷古菌没有显著性影响(P＞0．05)。通过田间试验，对比分析了水稻转Bt基因和杀虫剂施用对稻田土壤微生物学活性和根际细菌种群多样性的影响。结果发现水稻生长过程中根际土壤生物学活性发生显著性变化(P＜0．05)，但转Bt crylAb基因和三唑磷杀虫剂的施用均未对水稻根际土壤磷酸酶活性、脱氢酶活性、厌氧呼吸强度和产甲烷活性造成显著性影响(P＞0．05)。而水稻不同生长时期对水稻根际土壤微生物学活性有显著性影响(P＜0．05)。PCR-DGGE分子指纹图谱分析表明，三唑磷杀虫剂施用对拔节期和抽穗期水稻根际土壤细菌群落结构多样性具有显著性影响(P＜0．05)，而水稻转Bt基因对水稻生长过程中根际土壤细菌群落结构多样性没有显著性影响(P＞0．05)。由此可以推断，转Bt基因抗虫水稻对农田土壤细菌群落多样性影响风险较小或无风险。更多还原

【Abstract】 Use of transgenic crops is increasing at a rapid rate in worldwide. Field andlaboratory studies of transgenic Bt crops have been carried out to assess their risks toenvironment. The insertion of exogenous gene （including the target gene, the markergene and promoter） disrupted the existing network of plants, which may alter thephysiological and biochemical pathway or the process of metabolism and may resultin potential risks to non-target organism or beneficial microorganisms, and direct orindirect effect on soil health and agricultural ecosystem. In this thesis, stable carbonisotope pulse labeling had been developed for the research on variation ofphotosynthates distribution between transgenic Bt rice and its parental variety. It iscleared that transgenic Bt rice has some temporary effect on photosynthatesdistribution in rice plant. Combined with the ¹³C-PLFA technology, the soil organiccarbon sequestration and microbial community structure dynamics in rhizosphere soilwere studied. Based on these findings, seasonal effects of transgenic rice and theinsecticide triazophos on soil enzyme activities and microbial communities werecompared under field conditions. These results provided strong evidence on riskassessment of transgenic Bt rice application and provided technologies for the samekind of studies.The greenhouse experiment was carried out to investigate the effects oftransgenic Bt gene on photosynthesis and enzyme activities of rice flag leaves. Theresult indicated that there were significant and temporary differences betweentransgenic Bt rice and non-transgenic parental rice on photosynthesis and chlorophyllcontent of flag leaves. There was no significant difference between transgenic Bt andnon-transgenic parental rice of the same index at maturing stage. It was suggested theincorporation of crylAb gene resulted in some changes in rice plant. More works needto be done under to assess the risk in field condition.Based on the study of physiological differences between transgenic Bt andnon-transgenic parental rice, the temporal effects of gene transformation on carbon partitioning in rice plant and rhizosphere microbial communities were investigatedunder greenhouse conditions using the ¹³C pulse-chase labeling method andphospholipid fatty acid （PLFA） analysis. It was found that the ¹³C contents in leavesof transgenic （Bt） and non-transgenic （Ck） rice were significantly different at theseedling, booting and heading stages. There were no detectable differences of ¹³Ccontent in rice leaves at the maturing stage of rice development. Although asignificantly lower amount of Gram-positive bacterial PLFAs and a higher amount ofGram-negative bacterial PLFAs were observed in transgenic Bt rice rhizosphere ascompared with non-transgenic parental rice at the seedling, booting and headingstages of rice development, there were no significant differences in the amount ofindividual ¹³C-PLFA between transgenic Bt and non-transgenic parental ricerhizosphere soil at any growing stage. There were no significant differences in theamount of fungal and actinomycete related actinomycete related PLFAs betweentransgenic Bt and non-transgenic parental rice rhizosphere soil. These findingsindicated that the insertion of crylAb gene into rice had temporary effect on thephotosynthate distribution in rice or the microbial community composition in itsrhizosphere, however, it did not last until the maturing stage of rice development.A flooded soil experiment was conducted in which ¹³C-enriched rice straw（transgenic and non-transgenic parental rice）was added to evaluate the effects oftransgenic Bt rice and straw application on the soil C pool and greenhouse gas （CH₄and CO₂） dynamics in the root zone （soil surrounding rice roots） and the non-rootzone （soil outside the root zone）. The result showed that rice straw additionsignificantly increased the methane and carbon dioxide emissions in flooding soil.Meanwhile, the rhizosphere microenvironment stimulated straw decompositionsignificantly. The presence of root exudates increased the soil organic carbon （SOC）,dissolve organic carbon （DOC）, microbial biomass carbon （MBC） concentration andmethane, carbon dioxide emission rate significantly, relative to the soil with strawaddition in non-root zone. The incorporation of ¹³C-label into SOC, DOC, MBC, CH₄and CO₂ was not affected by the Bt gene insert not only in root zone but in non-root zone. The succession profiles of methanogenic archaeal communities associated withrice straw decomposition in root and non-root zone were studied by polymerase chainreaction - denaturing gradient gel electrophoresis （PCR-DGGE） analysis followed by16S rDNA sequencing. Principal components analysis based on DGGE patternsindicated that straw incorporation affected the methanogenic archaeal communitiesirrespective of their location on rice straw （root and non-root zone） compared with therice plant cultivation only. However, no significant differences between transgenic Btrice straw and non-transgenic parental rice straw were observed of the PCA analysison DGGE pattem.Effects of transgenic rice expressing the Cry1Ab insecticidal protein activeagainst lepidoperan pests and the insecticide triazophos [3-（o,o-diethyl）-1-phenylthiophosphoryl-1,2,4-triazol] on soil enzyme activities and bacterial communitieswere compared under field conditions. Results showed that seasonal variations in theselected enzyme activities of transgenic, non-transgenic and non-transgenic parentalrice with triazophos application were clearly detected. However, there were nostatistically significant differences （P>0.05） in phosphatase activity, dehydrogenaseactivity, respiration or methanogenesis in rhizosphere soil between transgenic,non-transgenic and non-transgenic Bt rice with triazophos application over the ricecropping cycle. Denaturing gradient gel electrophoresis （DGGE） analyses were usedto compare rhizosphere bacterial composition among transgenic Bt rice,non-transgenic parental rice-and non-transgenic parental rice with triazophosapplication. Some occasional and inconsistent effects of the application of triazophoson the bacterial composition in the rhizosphere soil of rice plant were found at thebooting and heading stages as compared with that of transgenic or non-transgenicparental rice. There was no significant difference of the bacterial composition in therhizosphere soil of transgenic and non-transgenic parental rice. These resultssuggested KMD1 （Bt） rice expressing the crylAb gene had no measurable adverseeffect on the key microbial processes or microbial community composition inrhizophere soil.更多还原