【作者】 周琳；

【导师】 张杰；

【作者基本信息】 中国农业科学院 ， 农业微生物， 2010， 博士

【摘要】 14年来,全球转基因作物的种植带来了巨大的经济、生态和社会效益。但是,转基因作物给人类带来巨大的利益的同时也存在潜在的环境安全风险。水稻和大豆是世界上最为重要的粮食作物和经济作物,我国较早开展了抗病虫转基因大豆和水稻的研究,全面准确地评价转基因水稻和大豆的环境安全性是进行商业化种植的前提保证。本文针对转基因抗虫水稻和抗真菌病害大豆对根际微生物的影响,通过常规培养计数、DGGE分析及荧光蛋白标记等手段,研究了转基因植株对土壤微生物、转基因微生物对作物及环境微生物之间的影响,并建立了以不动杆菌为指示菌的外源基因水平转移体系,主要结果如下:1、转基因抗病大豆和转基因抗虫水稻对根际微生物群落的影响:结合传统的平板计数法和变性梯度凝胶电泳分析连续两年对田间种植的转chi + rip抗病大豆G0431、G0433及其各自的亲本非转基因大豆黑农35、吉林30和温室内种植的转cry1Ac+ sck水稻MF86及其亲本非转基因水稻明恢86的根际土壤可培养细菌(包括芽胞杆菌、荧光假单胞菌)、真菌、放线菌进行平板计数和群落结构分析。结果显示,时间变化是造成根际可培养微生物数量及土壤细菌和真菌群落结构变化的主要因素,而同一时间内转基因大豆、转基因水稻和其各自亲本对照非转基因大豆和水稻根际土壤的的可培养微生物数量以及细菌和真菌群落结构之间并无显著不同。2008年,大豆根际土壤细菌群落多样性指数为5.32-5.37,群落分布均匀度指数为0.91-0.95;大豆根际土壤真菌群落多样性指数为4.78-4.91,群落分布均匀度指数为0.81-0.89;水稻根际土壤细菌群落多样性指数为5.11-5.16,群落分布均匀度指数为0.93-0.99;水稻根际土壤真菌群落多样性指数为4.78-5.14,群落分布均匀度指数为0.66-0.95;2009年,大豆根际土壤细菌群落多样性指数为5.40-5.45,群落分布均匀度指数为0.93-0.97;大豆根际土壤真菌群落多样性指数为5.21-5.26,群落分布均匀度指数为0.96-1.00;水稻根际土壤细菌群落多样性指数为5.28-5.35,群落分布均匀度指数为0.88-0.95;水稻根际土壤真菌群落多样性指数为4.99-5.39,群落分布均匀度指数为0.67-0.98。以上结果说明几丁质酶和核糖体失活蛋白双价基因导入到大豆中以及Bt杀虫蛋白基因cry1Ac和sck双价基因导入到水稻中并没有对大豆和水稻根际可培养细菌、真菌和放线菌的数量以及细菌和真菌的群落结构产生显著影响。2、转基因水稻外源基因水平转移检测体系的建立为了明确转基因水稻中外源基因是否会向土壤微生物发生转移,建立了以目前已知自然状态下接受外源DNA能力最强的不动杆菌Acinetobacter baylyi ADP1为指示菌的外源基因水平转移检测体系。Acinetobacter baylyi ADP1能够以较强的能力将外源DNA整合到自己的基因组上,以超声波破碎处理的转基因水稻MF86的基因组片段转化Acinetobacter baylyi ADP1,对5.0×106个转化子进行PCR检测,未发现含有Bt cry基因的转化子。由此推测,转基因水稻中的外源基因不会或者只可能以低于10-6的频率能够转移到土壤微生物中去。3、苏云金芽胞杆菌工程菌发酵上清液中重组质粒的水平转移检测:Bt工程菌发酵后期,细胞裂解,重组质粒随着发酵液进入环境,这些携带外源基因的质粒能否水平转移至常见的微生物体内,对环境微生物产生不良影响是Bt工程菌环境安全评价的重要方面。经PCR和大肠杆菌热激转化检测证实重组的质粒DNA随着菌体的裂解进入到发酵上清液中。以提取自静置不同时间处理的苏云金芽胞杆菌工程菌HD73(pSTK)发酵上清液中的DNA以及相应的过滤除菌的发酵上清液分别转化不动杆菌Acinetobacter baylyi ADP1、苏云金芽胞杆菌HD73、枯草芽胞杆菌Bs168以及蜡样芽胞杆菌Bc14579的感受态细胞,未得到阳性转化子。研究结果表明:即使在人为的高浓度条件下,这些残留的重组质粒和外源基因也不会或只可能以低于10-8的频率转移到其他的环境微生物中去,从这个角度再次证明Bt工程菌对环境微生物是安全的。4、HD73(pSTK-gfp)进入水稻体内研究:为了检测Bt工程菌能否从根际进入植物体内,本文用绿色荧光蛋白标记苏云金芽胞杆菌HD73,得到了gfp标记的HD73(pSTK-gfp);在激光共聚焦显微镜下观察,在细菌生长的任何时期都产生绿色荧光。接种HD73(pSTK-gfp)于水稻幼苗根际后,结合平板计数法和激光共聚焦显微镜技术,在常规水稻植株体内没有检测到经过gfp基因标记的苏云金芽胞杆菌工程菌,说明Bt工程菌在环境中的扩散能力是有限的,从这个层面证实了它在田间的使用是安全可靠的。更多还原

【Abstract】 The consistent and substantial, economic, environmental and welfare benefits were generated from biotech crops over the last fourteen years. However, at the same time, potential risk to environment originated from transgenic crops is existed. Rice and soybean are two important food and economics crops. Study of insect-resistant rice and disease-resistant soybean was developed earlier in domestic. Comprehensive and exactly environmental safety assessment of transgenic soybean and rice was important guarantee of commercial plantantion. Classical counting, DGGE and fluorescence labeling were used to investigate the effect of both transgenic crops and genetically engineered Bacillus thuringiensis strain on native microbes in the soil. Furthermore, a detection system of horizontal transfer of foreign gene was established by Acinetobacter baylyi ADP1 as indicator.1. Effects of the community structure of microbes in rhizosphere soils of transgenic soybean and rice:G0431 and G0433 were genetically modified soybean by transformation of two anti-fungus genes,“chi”(chitinase gene from bean) and“rip”(barley ribosome-inactivating protein gene) into Heinong 35 and Jilin30 natural strain, respectively, and MF86 was genetically modified rice by transformation of two insecticidal genes, cry1Ac (from Bacillus thuringiensis) and sck (modified Cowpea Trypsin Inhibitor gene) into Minghui 86 natural strain. Classical plate counting and denaturing gradient gel electrophoresis were executed to analyze the numbers of culturable microbes in rhizosphere soil and the community structure of bacteria and fungi. The results showed that there was no significant difference between transgenic crops and their host plant in the number of the culturable soil microorganism. Moreover, the results of cluster analysis of DGGE profile of bacteria and fungus indicated that the time was the main factor which affected the community structure in rhizosphere soil microbes. In the year of 2008, the Shannon’s index of the bacterial community of the soybean rhizosphere was 5.32-5.37 and the evenness index was 0.91-0.95; the Shannon’s index of the fungi community of the soybean rhizosphere was 4.78-4.91and the evenness index was 0.81-0.89; the Shannon’s index of the bacterial community of the rice rhizosphere soil was 5.11-5.16 and the evenness index was 0.93-0.99; the Shannon’s index of the fungi community of the rice rhizosphere was 4.78-5.14, and the evenness index was 0.66-0.95. In the year of 2009, the Shannon’s index of the bacterial community of the soybean rhizosphere was 5.40-5.45 and the evenness index was 0.93-0.97; the Shannon’s index of the fungi community of the soybean rhizosphere was 5.21-5.26 and the evenness index was 0.96-1.00; the Shannon’s index and evenness index of the bacteria community of the rice rhizosphere soil were 5.28-5.35 and 0.88-0.95, respectively; while the Shannon’s index and evenness index of the fungi community of the rice rhizosphere soil were 4.99-5.39 and 0.67-0.98. In conclusion, there was no remarkable difference in the rhizosphere soil between the transgenic and non-transgenic crops in the numbers of culturable bacteria (including Bacillus and fluorescent Pseudomonas), fungi and actinomycetes. All the results demonstrated that both transgenic soybean and rice tested were no significant effect to native microorganisim in the field. 2. Detection system of horizontal transfer for foreign gene using Acinetobacter baylyi ADP1 as indicator.An analytical method was established using Acinetobacter baylyi ADP1 as indicator to investigate whether the foreign gene from rice could be transferred into native microbes in soil. Acinetobacter baylyi ADP1 integrates foreign DNA into the chromosome with a high effficiency. PCR identification of 5.0×10~6 transformants obtained by transforming Acinetobacter baylyi ADP1 with sonicated genomic DNA of transgenic rice MF86, no positive transformant containing Bt cry gene fragment was found. This result indicated that Bt cry gene, one of foreign gene from transgenic rice could not be transferred into Acinetobacter baylyi ADP1 or the transformation frequency was only lower than 10-6 possibly.3. Dectection of the horizontal transfer of recombinant plasmid in fermentation supernatant of engineered Bacillus thuringiensis strain:In the anaphase on the fermentation, after cell lysis, the recombinant plasmid DNA in engineered Bacillus thuringiensis strain entered supernatant, and was confirmed by both PCR and transformation. It was an important aspect to evaluate the environmental safety of engineered Bt, whether the plasmid haboring foreign gene could transfer into familiar microbes to lead to adverse influence. There was no positive transformant after the different strains were transformed with DNA prepared from fermentation supernatant and sterile fermentation supernatant that treated with different placement time. The results indicated that the recombined plasmid harboring foreign gene from B. thuringiensis engineered strain couldn’t transfer into other environmental microbes or the transformation frequency was only lower than 10-8 possibly, and this conclusion demonstrated again that B. thuringiensis engineered strain is safe.4. Monitoring of spread of B. thuringiensis engineered strain into rice plant:In order to detect if engineered Bacillus thuringiensis can enter the interior rice through root, a gfp-labeled B. thuringiensis strain HD73 (pSTK-gfp) was constructed. Green fluorescence was observed all through the life cycle of HD73 (pSTK-gfp) under the laser scanning confocal microscope (LSCM). Classical plate counting and LSCM were used to analyze the HD73 (pSTK-gfp) after inoculating into the root of the rice (Nipponbare). There were no positive strain (HD73-pSTK-gfp) detected from inner of the rice. The results showed that the spread ability of engineered Bt was very limited. So, we could conclude that B. thuringiensis engineered strain was safe in this level.更多还原