种植转胱硫醚-γ-合酶基因大豆对其根际真菌、固氮细菌和氨氧化细菌的影响

发布时间：2018-10-05 18:15

【摘要】：转基因作物自1996年商业化种植以来,其种植面积持续增加,对全球人口供养,粮食安全,可持续性和气候变化做出了巨大贡献。然而,由此带来的生态风险和环境安全也引起了广泛关注,尤其是对土壤生态环境的影响。植物和土壤的相互关系对于生态系统的功能稳定和植物健康是至关重要的。转基因植物的种植可能会影响土壤酶活性以及微生物群落组成,同时,可能会对微生物参与的生化进程产生影响。本研究以转拟南芥胱硫醚-Y-高蛋氨酸大豆ZD91和它的受体自贡冬豆ZD为材料,研究转高蛋氨酸大豆对其根际主要酶活性、微生物群落多样性、氮循环相关细菌数量和群落结构的影响。转高蛋氨酸大豆ZD91及其受体大豆ZD由中国农业科学院作物科学研究所提供,连续两年播种于四川省南充市农业科学院试验基地(30°48'N,106°04'E),转高蛋氨酸大豆ZD91及其受体大豆ZD分别设置四个重复。在苗期,花期,鼓粒期,成熟期四个生长时期进行采样。采用五点取样法采集转基因大豆和受体大豆根际土壤用于环境安全评价研究。主要研究结果如下:1、土壤酶活性测定主要测定了其根际土壤中蔗糖酶和碱性磷酸酶的活性。分别采用3,5-二硝基水杨酸比色法和磷酸苯二钠比色法进行测定。研究结果表明,两年中转高蛋氨酸大豆ZD91及其受体大豆ZD的土壤蔗糖酶和碱性磷酸酶的活性在四个生长时期均无显著性差异。生育期间存在无明显规律的差异,这种差异与采样时土壤温度和湿度有关。2、真菌群落多样性研究采用BiologFF微平板法进行。实验结果表明,转高蛋氨酸大豆及其受体大豆碳源利用活性,多样性指数,不同类碳源利用率均没有显著性差异。因此,转基因高蛋氨酸大豆ZD91对真菌群落功能没有影响。3、对氮循环相关微生物数量变化的研究选取固氮细菌和氨氧化细菌的相应功能基因nifH和amoA-AOB,运用实时荧光定量PCR的方法进行定量。研究结果表明,转高蛋氛酸大豆及其受体大豆固氮细菌的nifH在四个生育时期均无显著性差异,氨氧化细菌的amoA-AOB仅在鼓粒期有显著性差异,并且随着大豆的生长,这种差异逐渐减小,最终消失。而且,ANOVA分析表明,影响氮循环相关基因丰富度的最重要因素是植物的生育时期,而不是栽培品种。4、对氮循环相关的固氮细菌和氨氧化细菌群落结构的研究采用克隆文库的方法。结果表明,转高蛋氨酸大豆及其受体大豆的固氮细菌和氨氧化细菌群落结构没有显著性差异,优势菌群相同。并且,多样性指数和主成分分析(PCA)结果表明转基因对土壤中氮循环相关细菌群落组成没有产生显著性影响。同时,发现大部分氮循环相关细菌属于未培养细菌,使得进一步研究转基因大豆ZD91根际与固氮作用和氨化作用相关细菌变得更加重要。通过以上的研究初步说明转拟南芥胱硫醚-Y-高蛋氨酸大豆ZD91对其根际土壤生态环境无显著性影响,初步认为其种植是安全可靠的。该研究的开展不仅为转高蛋氨酸大豆的环境安全评价提高基础数据,更为今后评价转基因植物安全性提供了有效的技术参考。
[Abstract]:Since the commercialization of GM crops in 1996, its planting area has continued to increase, making a significant contribution to global population support, food security, sustainability and climate change. However, the ecological risk and environmental safety brought about by this cause have also attracted wide attention, especially on the soil ecological environment. The interrelationship between plants and soils is critical to the functional stability and plant health of ecosystems. The planting of transgenic plants may affect the activity of soil enzymes and the composition of microbial communities, and may also have an impact on the biochemical processes involved in the participation of microorganisms. In this study, the effects of high methionine soybean on the main enzyme activity, microbial community diversity, nitrogen cycle-related bacteria number and community structure were studied. The high-methionine soybean cultivar 991 and its receptor soybean protein were provided by the Institute of Science and Technology of China Academy of Agricultural Sciences, which was sown in the experimental base of the Agricultural Sciences Academy of Sichuan Province (30 掳 48 'N, 106 掳 04' E) for two consecutive years, and four replicates were set up for the high methionine soybean cultivar 91 and its receptor soy salad oil. sampling was carried out at four growth stages of seedling stage, flowering period, drum grain period and mature period. A five-point sampling method was used to collect transgenic soybean and receptor soybean root-seed soil for environmental safety evaluation. The main results were as follows: 1. The activity of sucrose and alkaline phosphatase in soil was determined by soil enzyme activity assay. The colorimetric method of 3,5-dinitrosalicylic acid (3,5-dinitrosalicylic acid) and sodium phenodisulfate were used respectively. The results showed that there was no significant difference in the activity of sucrose and alkaline phosphatase in the soil between the two-year transit high-methionine soybean cultivar 91 and its receptor soybeans. There was no obvious difference in the growth period, which was related to soil temperature and humidity at the time of sampling. The results showed that the activity, diversity index and utilization rate of carbon sources in soybean and soybean were not significantly different. Therefore, there was no effect on the function of the fungal community by the transgenic high-methionine soybean cultivar 91. 3. The relative functional genes nifH and amoA-AOB of nitrogen-fixing bacteria and ammonia-oxidizing bacteria were selected for the study of the change of the number of microorganisms related to nitrogen cycling, and the quantitative PCR was carried out by real-time fluorescence quantitative PCR. The results showed that there was no significant difference between the NifH of soybean and its receptor soybean nitrogen-fixing bacteria, and the amoA-AOB of ammonia-oxidizing bacteria only had significant difference in the period of growth, and as soybean grew, the difference gradually decreased and eventually disappeared. Moreover, ANOVA analysis indicated that the most important factor affecting the richness of nitrogen cycle-related gene was the reproductive period of plants, rather than the first one. 4. The research on the structure of nitrogen-fixing bacteria and ammonia-oxidizing bacteria community related to nitrogen cycling was the method of cloning library. The results showed that there was no significant difference in the structure of nitrogen-fixing bacteria and ammonia-oxidizing bacteria in high-methionine soybean and its receptor soybean, and the dominant bacteria group was the same. Moreover, the diversity index and principal component analysis (PCA) showed that there was no significant influence on the composition of the bacterial communities in soil with nitrogen circulation. At the same time, it was found that most of the nitrogen-cycle-related bacteria belonged to the uncultured bacteria, which made it more important to further study the relationship between the action of rice and the bacteria associated with ammonification. Through the above research, it was preliminarily indicated that there was no significant influence on the ecological environment of the root-seed soil of Arabidopsis thaliana disulfide-Y-high methionine soybean cultivar 91, and it was preliminarily considered that its planting was safe and reliable. The development of the study not only improves the basic data for environmental safety evaluation of high-methionine soybean, but also provides an effective technical reference for evaluating the safety of transgenic plants in the future.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S565.1;S154.3

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