【作者】 林辉锋；

【导师】 林文雄；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 利用水稻化感作用开发生态安全的稻田生物除草技术是当今农业生态学上的研究热点之一。水稻化感作用特性属于数量性状,受外界环境调控,已有研究表明逆境胁迫下水稻化感作用潜力增强。研究者已有从水稻体出发揭示了其生理与分子生物学机理,但对逆境下化感水稻土壤微生物生态学特性的研究相对较少。在抗逆应答特性上,水稻是干旱胁迫敏感型作物,为此,本文主要利用环境微生物系统生物学的研究思路,运用T-RFLP(末端限制性片段长度多样性)、DGGE(变性梯度凝胶电泳)、土壤蛋白质组学三种技术,以强化感水稻品种PI312777和弱化感水稻品种Lemont为材料,分析旱直播模式和育秧移栽模式下强弱化感水稻根际土壤微生态特性,探讨旱作水稻化感作用增强及其在较强化感作用生育期的特异或高峰度表达的菌群,同时在环境蛋白质研究与化感微生物的研究结合上做了尝试性的工作,即利用土壤蛋白质组学研究揭示其功能蛋白。主要结果如下:(1)T-RFLP结果显示,旱直播栽培模式和育秧移栽模式下,不同叶龄期(5叶期和7叶期)的强、弱化感水稻秧苗根际土壤微生物组成存在显著差异。旱直播条件下,强化感水稻的根际土壤微生物多样性要高于弱化感水稻;随着生育期增加,强、弱化感水稻的根际微生物的多样性增加,分别从168种、129种增至424种、169种,强化感水稻的根际微生物的增幅远高于弱化感水稻(252%对比于131%);但在育秧移栽模式条件下,强化感水稻根际土壤微生物多样性要低于弱化感水稻。同时,两种栽培模式下强弱化感水稻的水稻根系微生物有17个共有菌群,占所有鉴定种属的8%,主要涉及氮循环(弗兰克氏菌属)、硫循环(外硫红螺旋菌属)、C循环(甲基单胞菌属)、芳香化合物利用(鞘脂单胞菌属)、利用葡萄糖产酸(微杆菌属、短芽孢杆菌属、孪生球菌属),表明两种水稻在分解次生代谢产物,地理化学循环等方面的根基微生态具有稳定性和保守性。(2)T-RFLP片段特异性分析显示,水稻旱直播栽培模式能较强诱导强化感水稻的根际微生物类群发生变化,其中PI7增加了30种特异微生物菌群,涉及粘细菌、产酸产气菌、以及其它菌,其中粘细菌有7个菌属,主要功能为降解大分子、纤维或死细胞,推测粘细菌能够通过破坏土壤种子库中杂草种子细胞,降解种子胚根、胚芽,从而达到抑制土壤种子库中种子萌芽作用效果。产酸产气菌属有7个,功能分析其是一类代谢最终产物为丙酸的菌属,推测其与种植化感水稻的根际微环境偏酸性有关。其他的特异菌属功能尚不明确。(3)土壤宏蛋白质组学初步分析结果表明,在旱直播模式下,强化感水稻根际土壤的功能蛋白质种类较弱化感水稻土壤更为丰富,其中强化感水稻根际土壤特异蛋白质增加9个,其中2个为信号诱导蛋白,5个为物质和能量代谢蛋白,2个为基因转录蛋白,而弱化感水稻根际土壤特异蛋白质仅为1个结构组成蛋白。由此推测化感水稻的化感物质进入土壤生态系统中能有效提高某些生物体分泌的诱导蛋白识别能力,提高运动趋向速率,进而通过物质和能量代谢的酶化学作用进行转化,形成具化感作用潜力的次生代谢产物对靶标受体进行作用。(4)DGGE检测分析显示,旱直播模式下的强化感水稻的特有条带16经系统发育树分析表明,该条带与鞘脂单胞菌属(uncultured Sphingomonas sp.)同源性较高,该菌属在T-RFLP分析中相对优势度表现差异:PI7:1.1%、PI5:2.1%、PI:2.1%,LE7:检测限以下,LE5:1.6%,LE:0.8%。同时,土壤蛋白质组学分析表明该菌属是化感水稻土壤特异功能蛋白来源微生物。该菌属在土壤中与芳香族化合物的降解有关,也是土壤病原菌之一。推测化感物质分泌出较多的芳香族化合物,鞘脂单胞菌属在化学趋化作用下富集,可影响伴生杂草的生长发育,甚至致病于伴生杂草,从而形成化感水稻的抑草作用,但其对伴生杂草的作用机制还有待进一步研究。综上所述,两种栽培条件下,强弱化感水稻土壤根际在分解次生代谢产物,地理化学循环方面的微生物具有保守性。旱直播条件能够能较强诱导强化感水稻的根际微生物生长,其中粘细菌对强化感水稻特异性强,推测化感作用与其降解大分子从而影响杂草种子胚根、胚芽的生长有关。此外,鞘脂单胞菌属在化感水稻各个生育期和栽培模式下均存在,推测与其植物致病性及释放相关功能蛋白有关。更多还原

【Abstract】 Using the trait of rice allelopathy to develop a kind of safe and environment-friendly technology to control field weeds is one of the current hot topics in agroecology research field. As a quantitative trait, the characteristic of rice allelopathy is easily induced by the external stressful environment conditions, which had been proved by many literatures. However, the research works were done to explore the mechanism in views of plant physiology and molecular biology, and less on soil microbial ecology. At the same time, in the responds of the stress-resistant, rice is sensitive to the drought. Hereby, the study focuses on the functional microbials of allelopathic potential in allelopathic rice rhizosphere by contrasting the microbial divisity in the strong allelopathic rice and weak allelopathic rice at seven leaf stage under dry directly sowing and seedling transplanting conditions from the system biology angle, and modern molecular methods polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)、terminal restrain fragment length polymorphism (T-RFLP) and metaproteomics were applicated to reveal the diversity of the rhizosphere micrbial. The main results were summarized as follows.(1)The results of T-RFLP showed that under dry directly sowing and seedling transplanting conditions, microbial diversity and structure in the different rice soils were significant different. Under dry directly sowing condition, either at the five leaf stage or seven leaf stage, the diversity of microorganisms in allelopathic rice PI312777 rhizosphere were higher than that of the non-allelopathic rice Lemont rhizosphere. And in the development of rice from 5-leaves to 7-leave the number of microbial species of PI312777 range 168 to 424, whereas Lemont range 129 to 169. The range of increase of rhizosphere microorganisms of allelopathic rice accession was greater than non-allelopathic rice accession (252% to 131%). However, the opposite results were showed under seedling transplanting condition. The range of variation of rhizosphere microorganisms of allelopathic rice accession was weaker than non-allelopathic rice accession. Meanwhile, 17 kinds of microbial floras were showed on all test sample in addition to control treatment, accounting for 8% of the total identified species, and of which, majority refer to nitrogen cycle (Frankia.), sulfur cycle (Ectothiorhodospira.), C cycle (Methylomonas.), the utilization of aromatic compounds (Sphingomonas.), the use of glucose to producing acid (Microbacterium., Brevibacillus., Gemella.). Those were indicated that it is high similarity at the decomposition of secondary metabolites, geo-chemistry cycles of micro-organisms under two cultivation conditions.(2)The results of specificity by T-RFLP showed that the dry condition had a strong stimulating effect to rhizosphere microorganisms of allelopathic rice accession. The unique 30 genuses were appeared in PI7. And they could be divided into three functional categories: Myxobacteria, bacteria producing acid & gas and un-detected species. Myxobacteria which seven generas had been showed could decompose polymer, fiber, and dead cells. It suggested that Myxobacteria might play an important role on rice allelopathy through its destruction of weeds seeds in soil seed bank, degradate the seed radicle, embryo that could inhibit the seed germination in soil. The producing acid bacteria and gas contained seven generas which suggested that the soil of allelopathice rice accession was acidic micro-environment.(3)The results of specificity by metaproteimics showed that the diversity of function protein appearing in allelopathic rice accession soil were greater than in non-allelopathic rice accession soil under dry directly sowing condition. There are 9 kinds of functional specific protein in allelopathic rice soil, of which 2 were signal-induced proteins, 5 were the material and energy metabolism-related proteins, 2 were gene transcriptional regulation-related proteins, while functional protein-specific of non-allelopathic rice was only one kind which was a structural protein. The result showed that, the root of allelopathic rice exudates allelochemical to the soil ecosystem,thereby the allelochemical induced the functional microbial to secret protein, which raise campaigns trend, further by some material and energy metabolic enzymes converting to allelochemical which was a sense of secondary metabolites, and the final metabolites affected the target receptor.(4)The results of DGGE showed that, under dry directly sowing condition, the band 16 appeared only in the soil of allelopathy rice accession, further the results of the phylogenetic analysis showed that it was high homology with uncultured Sphingomonas. sp., which was significant different on analysis of the dominance genus level by T-RFLP. The abundance of test sample are 1.1%(PI7)、2.1%(PI5)、2.1%(PI), non-detection in LE7, 1.6%(LE5)(<PI5), 0.8%(LE), respectively. Meanwhile, the genus of Sphingomonas. was also found that is one of the source of micro-organisms of specific functional protein of allelopathic rice accession by metaproteimics. Most kinds of Sphingomonas. were reported that not only were relating to the aromatic compounds degradation, but also were as one of the soil pathogens. It suggested that allelochemicals contain more aromatic compounds, and then the Sphingomonas. acted on chemotactic enrichment. In addition, it needed further research to prove that the Sphingomonas. was the pathogens for the allelopathic rice against weeds in soil, though it was the pathogens reported on plants.All in all, there was similarity in some sense at the decomposition of secondary metabolites, geo-chemistry cycles of micro-organisms under two cultivation conditions. However, dry directly sowing condition could induce the microbial and develop a higher diversity in the rhizosphere microorganisms of allelopathic rice accession than that of non-allelopathic rice. The Myxobacteria was specific in the soil of allelopathy rice accession. It suggested that Myxobacteria played an important role on rice allelopathy through its destruction of weeds seeds in soil seed bank, degradation of the seed radicle, embryo that inhibited the seed germination in soil. Moreover, the genus of Sphingomonas. was available in all test soils except LE7, and the abundance of Sphingomonas.was higher under the two cultivation conditions than that of non-allelopathic rice The explanation of those phenomena is for its function of plant pathogenicity and the related-function protein exudated from Sphingomonas.更多还原