【作者】 吕豪豪；

【导师】 陈英旭； 吴伟祥；

【作者基本信息】 浙江大学 ， 微生物学， 2010， 博士

【摘要】 转基因植物是运用人类智慧和技能在遵循生物学原理的基础上创制出的符合人类意愿的新型植物品种。它的种植满足了人类对粮食产量、经济效益和食物安全的需求。2009年11月,中国首次通过转Bt基因水稻的审批,Bt大米在不久的将来即会进入商品化生产阶段。当前,我国常年水稻种植面积约4.5亿亩,转Bt基因水稻一旦商品化生产,无疑将给我国带来巨大的经济和环境效益。然而,由于转基因植物通常携带外源基因,表达外源物质,且外源基因的人工插入可能改变植物原来的基因组序列,可能会影响植物的生理生化特性和物理化学结构；改变根分泌物的组成从而影响田间土壤生态；大面积转基因作物的秸秆还田也可能给农田生态系统带来很大的环境安全压力。本论文针对当前转Bt基因水稻农田生态安全风险中关注的主要问题,以转Bt基因水稻“克螟稻”和亲本水稻“秀水11”为试材,通过田间试验,系统研究比较转Bt基因与三唑磷农药施用对水稻根际土壤微生物生态的影响；转Bt基因对水稻成熟体植株物理结构与化学组成的影响；转Bt基因对水稻成熟体残茬降解以及土壤降解微生物的影响。研究结果为即将到来的Bt水稻大规模释放的农田土壤生态安全风险性评价提供重要依据。具体研究结果如下：1.转Bt基因及三唑磷施用对水稻根际微生物的影响通过田间试验,采用PCR-T-RFLP-AMMI方法对比水稻转Bt基因与螟虫常用杀虫剂三唑磷施用对水稻根际土壤细菌和真菌种群结构多样性的影响,结果发现转Bt基因与亲本及亲本喷药处理之间丛枝菌根(AM)真菌孢子浓度、球囊霉素(Glomalin)蛋白浓度未见显著性差异,表明转Bt基因未对AM真菌群落丰富度产生显著性的影响；此外,转Bt基因与三唑磷并未显著影响水稻根际的总细菌和真菌群落多样性组成。环境因素对水稻根际微生物影响显著,AM真菌及glomalin蛋白的丰度随着时间季节的变化呈现较大的波动。环境因素的变化如气温的季节变化、湿度的变化对水稻根际的总细菌和真菌的群落组成影响较大。2.转Bt基因对成熟期水稻秸秆与根物理结构与化学组分的影响通过田间试验培育的转Bt基因克螟稻及亲本水稻的成熟期秸秆与根,采用元素分析、傅立叶红外光谱(FTIR)、木质素和纤维素含量分析和扫描电镜(SEM)观察,对比转Bt基因克螟稻和亲本水稻成熟期秸秆物理结构与化学成分的差异。结果发现：转Bt基因与亲本水稻秸秆与根之间C%、N%、C：N、H%以及灰分均存在较大差异,水稻秸秆的C%、C：N和H%显著高于根,而N%和灰分含量则显著低于水稻根。但除水稻秸秆灰分含量外,转Bt基因与亲本水稻秸秆与根的元素组成均无显著性的差异；化学结构组成、傅立叶红外光谱(FITR)及表面和横切水稻组织扫描电镜(SEM)分析发现,水稻秸秆与根之间木质素和纤维素含量均显著高于根,SEM显示秸秆与根之间的物理结构存在一定的差异；但转Bt基因与亲本水稻亲本之间木质素和纤维素含量、表面基团并无显著性差异,转Bt基因与亲本水稻物理结构均呈现典型的粳稻的结构。3.转Bt基因对成熟期秸秆与根降解动力学的影响通过田间培育克螟稻及原亲本成熟期秸秆与根,在不同耕作方式(油菜-水稻、双季稻)下,采用litterbag技术将成熟期克螟稻与亲本稻秸杆与根还田,以明确克螟稻与亲本秸杆与根在田间土壤中的降解规律,结果发现：地下放置方式与双季稻模式下转Bt水稻根降解可能存在一定的差异,双季稻模式转Bt基因水稻根降解在地下放置31、68和137 d处理下显著性高于亲本稻；两种耕作模式下水稻秸秆降解过程存在较大的相似性,转Bt基因水稻与亲本水稻秸秆降解均未出现持续的显著性差异,耕作模式对水稻秸秆的降解影响较小；不同耕作模式下转Bt基因水稻与亲本稻根降解存在较大差异。双季稻模式水稻根降解前期出现平台期而油菜-水稻耕作模式下油菜种植有利与前期水稻根的降解。4.转Bt基因对成熟期秸秆与根降解微生物群落的影响采用双季稻或油菜-水稻耕作模式通过田间试验研究了转Bt基因地成熟期水稻秸秆与根降解微生物群落的影响,结果发现：基于降解动力学的结果,我们也发现在双季稻耕作模式地下放置方式下,还田31 d、68 d和137 d的转Bt基因水稻与亲本稻根降解真菌群落结构均存在一定的差异。提取前20个相对荧光强度的T-RFs,进行对比分析也表明,在地下10 cm放置处理,转Bt基因与亲本根降解真菌T-RFs在三个时期均存在一定的差异,而细菌和地表放置处理则无差异,真菌DGGE分析也表明这种差异可能由担子菌门和子囊菌门真菌种群结构发生变化所致；相比较与时间和放置方式因素,两种耕作模式转Bt基因对水稻秸秆与根的降解整个降解周期影响较不明显,方差分析也证明转Bt基因效应在整个降解周期模型中并不显著；降解微生物菌群落均在降解时间上存在较大差异,并在一定程度上表现出不同埋放方式之间的差异,表明转Bt基因水稻与亲本稻秸秆与根的降解微生物群落受降解时间的推移影响最大；两种耕作方式下微生物群落结构沿降解时间演替规律存在一定的差异,主要表现为双季稻模式下微生物群落结构分化主要在后期出现,而油菜-水稻耕作模式下主要是前期。综上所述,转Bt基因对水稻根际土壤微生物生态、成熟期植株体结构与组成、成熟期水稻秸秆的降解及降解微生物的影响并不显著。虽然如此,我们发现转Bt基因对成熟期水稻根降解前期以及降解真菌群落组成存在一定的影响。更多还原

【Abstract】 By 2009, genetically modified (GM) crops were adopted in 25 countries covering an area of more than 134 million hectares with an annual growth rate of 7%. The Chinese government implemented a＄3.5 billion GM Crops Initiative that included Bt rice to secure its food supply and to address land degradation, chronic water shortages, and a growing population that already numbers 1.3 billion. Transgenic Bt rice is not approved in any country so far, but is likely to be approved in China in the near future. However, the random insertion of a foreign gene into the plant genome may cause changes in the amount and composition of crop residues and may also affect organic matter decomposition, soil microbial community, andnutrient cycling in soil. So far, little is known about the effects of Bt rice on rihzosphere microbial community, residues composition and its decomposition process. Hennce in this thesis the effects of cry1Ab gene transformation in rice on rihzosphere and its residue decomposed microbial community were conducted under field conditions. The results were summaried as follow:1. The effect of Bt rice on rihzosphere soil microbial community compositionThere were no significant differences of arbuscular mycorrhiza fungi (AMF) spores and glomalin protein density between Bt and non-Bt rice variety indicating that Bt gene transformation have little effect on soil AMF in paddy field. Furthermore, the bacterial and fungal mommunity composition were shifted during the rice growing stage and environment condition in paddy field. The effects of Bt gene transformation on rihzosphere soil microbial community were minor.2. The effect of Bt transformation on rice residues compositionLignin, cellulose, carbon and nitrogen contents in Bt and non-Bt parental rice residues were not significantly different. However, significantly higher lignin, cellulose and carbon content, and lower nitrogen content were detected in rice straw compared with rice roots. The FITR showed no significant differences of surface functional groups between Bt and non-Bt residues. The physical histology of rice straw was quit different compared with root using scanning electron microscope (SEM). However, no differencs of physical histology were also shown in Bt and non-Bt rice residues.3. The effect of Bt rice on rice residue decomposition dynamicsFor rice straw in the litterbags, the time-course of AFMR, TN, TC and the C/N ratio showed similar trends despite a transient increase in TN at the beginning of decomposition. A significantly higher decomposition level of straw as indicated by AFMR, TN and TC under incorporated placement was observed as compared to the surface placement. However, there were no further significant differences between Bt and non-Bt treatments. Percent AFMR, TN, TC and C/N ratio of rice straws were significantly affected by time and placement factors.For rice root, there were no significant differences of AFMR TN, TC and the C/N between Bt and non-Bt treatment in there rapeseed-rice cropping system. However, concerns have to be raised in rice-rice cropping sysytem Bt roots in buried litterbags displayed significantly lower AFMR, TN and TC contents at the initial decomposition stage compared to the conventional rice. 4. The effect of Bt rice on rice residue decomposed microbial community compositionFor rapeseed-rice cropping system, AMMI analysis of two bacterial T-RFLP data sets revealed a relationship between soil microbial community and decomposition time for rice straw and roots. The MANOVA on the first two dimensions of AMMI scores for soil bacterial and fungal communities showed significant effect of decomposition time in all treatments except the second interaction principal component (IPC 2) of bacterial T-RFLP data during the root decomposition. The fungal T-RFLP fingerprints from both straw and root samples were also clearly grouped by time. An effect of litterbag placement on the microbial community associated with the residues was also detected by AMMI analysis. However, no significant effect of rice genotype was observed at any time for either bacteria or fungi associated with straw and root residues.For rice-rice cropping system, total AMMI model analysis revealed that microbial community composition in the litterbags was affected by temporal and spatial factors. Compared with the non-Bt rice residue treatment, Bt rice straw had no significant effects on the soil bacterial and fungal community composition during the study period, regardless of the litterbags being placed on the surface or buried in the soil. There were no significant differences in the bacterial community composition profiles in root decomposition between Bt transgenic and non-Bt varieties. However, significant differences in soil fungal community composition between the buried Bt and non-Bt rice roots were observed in soils sampled on days 31,68 and 137, indicating that Bt roots incorporated into paddy soil may affect soil fungal community during the initial stage of their decomposition更多还原