種植轉(zhuǎn)胱硫醚-γ-合酶基因大豆對(duì)其根際真菌、固氮細(xì)菌和氨氧化細(xì)菌的影響
發(fā)布時(shí)間：2018-10-05 18:15
【摘要】：轉(zhuǎn)基因作物自1996年商業(yè)化種植以來,其種植面積持續(xù)增加,對(duì)全球人口供養(yǎng),糧食安全,可持續(xù)性和氣候變化做出了巨大貢獻(xiàn)。然而,由此帶來的生態(tài)風(fēng)險(xiǎn)和環(huán)境安全也引起了廣泛關(guān)注,尤其是對(duì)土壤生態(tài)環(huán)境的影響。植物和土壤的相互關(guān)系對(duì)于生態(tài)系統(tǒng)的功能穩(wěn)定和植物健康是至關(guān)重要的。轉(zhuǎn)基因植物的種植可能會(huì)影響土壤酶活性以及微生物群落組成,同時(shí),可能會(huì)對(duì)微生物參與的生化進(jìn)程產(chǎn)生影響。本研究以轉(zhuǎn)擬南芥胱硫醚-Y-高蛋氨酸大豆ZD91和它的受體自貢冬豆ZD為材料,研究轉(zhuǎn)高蛋氨酸大豆對(duì)其根際主要酶活性、微生物群落多樣性、氮循環(huán)相關(guān)細(xì)菌數(shù)量和群落結(jié)構(gòu)的影響。轉(zhuǎn)高蛋氨酸大豆ZD91及其受體大豆ZD由中國(guó)農(nóng)業(yè)科學(xué)院作物科學(xué)研究所提供,連續(xù)兩年播種于四川省南充市農(nóng)業(yè)科學(xué)院試驗(yàn)基地(30°48'N,106°04'E),轉(zhuǎn)高蛋氨酸大豆ZD91及其受體大豆ZD分別設(shè)置四個(gè)重復(fù)。在苗期,花期,鼓粒期,成熟期四個(gè)生長(zhǎng)時(shí)期進(jìn)行采樣。采用五點(diǎn)取樣法采集轉(zhuǎn)基因大豆和受體大豆根際土壤用于環(huán)境安全評(píng)價(jià)研究。主要研究結(jié)果如下:1、土壤酶活性測(cè)定主要測(cè)定了其根際土壤中蔗糖酶和堿性磷酸酶的活性。分別采用3,5-二硝基水楊酸比色法和磷酸苯二鈉比色法進(jìn)行測(cè)定。研究結(jié)果表明,兩年中轉(zhuǎn)高蛋氨酸大豆ZD91及其受體大豆ZD的土壤蔗糖酶和堿性磷酸酶的活性在四個(gè)生長(zhǎng)時(shí)期均無顯著性差異。生育期間存在無明顯規(guī)律的差異,這種差異與采樣時(shí)土壤溫度和濕度有關(guān)。2、真菌群落多樣性研究采用BiologFF微平板法進(jìn)行。實(shí)驗(yàn)結(jié)果表明,轉(zhuǎn)高蛋氨酸大豆及其受體大豆碳源利用活性,多樣性指數(shù),不同類碳源利用率均沒有顯著性差異。因此,轉(zhuǎn)基因高蛋氨酸大豆ZD91對(duì)真菌群落功能沒有影響。3、對(duì)氮循環(huán)相關(guān)微生物數(shù)量變化的研究選取固氮細(xì)菌和氨氧化細(xì)菌的相應(yīng)功能基因nifH和amoA-AOB,運(yùn)用實(shí)時(shí)熒光定量PCR的方法進(jìn)行定量。研究結(jié)果表明,轉(zhuǎn)高蛋氛酸大豆及其受體大豆固氮細(xì)菌的nifH在四個(gè)生育時(shí)期均無顯著性差異,氨氧化細(xì)菌的amoA-AOB僅在鼓粒期有顯著性差異,并且隨著大豆的生長(zhǎng),這種差異逐漸減小,最終消失。而且,ANOVA分析表明,影響氮循環(huán)相關(guān)基因豐富度的最重要因素是植物的生育時(shí)期,而不是栽培品種。4、對(duì)氮循環(huán)相關(guān)的固氮細(xì)菌和氨氧化細(xì)菌群落結(jié)構(gòu)的研究采用克隆文庫(kù)的方法。結(jié)果表明,轉(zhuǎn)高蛋氨酸大豆及其受體大豆的固氮細(xì)菌和氨氧化細(xì)菌群落結(jié)構(gòu)沒有顯著性差異,優(yōu)勢(shì)菌群相同。并且,多樣性指數(shù)和主成分分析(PCA)結(jié)果表明轉(zhuǎn)基因?qū)ν寥乐械h(huán)相關(guān)細(xì)菌群落組成沒有產(chǎn)生顯著性影響。同時(shí),發(fā)現(xiàn)大部分氮循環(huán)相關(guān)細(xì)菌屬于未培養(yǎng)細(xì)菌,使得進(jìn)一步研究轉(zhuǎn)基因大豆ZD91根際與固氮作用和氨化作用相關(guān)細(xì)菌變得更加重要。通過以上的研究初步說明轉(zhuǎn)擬南芥胱硫醚-Y-高蛋氨酸大豆ZD91對(duì)其根際土壤生態(tài)環(huán)境無顯著性影響,初步認(rèn)為其種植是安全可靠的。該研究的開展不僅為轉(zhuǎn)高蛋氨酸大豆的環(huán)境安全評(píng)價(jià)提高基礎(chǔ)數(shù)據(jù),更為今后評(píng)價(jià)轉(zhuǎn)基因植物安全性提供了有效的技術(shù)參考。
[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 擄 48 'N, 106 擄 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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S565.1;S154.3
本文編號(hào)：2254379
[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 擄 48 'N, 106 擄 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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S565.1;S154.3
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