农业有害微生物的绿色控制方法研究
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摘要
农业有害微生物在农业生产的各个环节会对农作物和农产品造成污染,这不仅导致农作物减产、农产品品质下降,造成巨大经济损失,而且有些病原性微生物会产生毒素,甚至引发流行性疾病。同时,农业有害微生物还会污染环境,对人类造成长期的潜在危险,影响到社会稳定乃至国民经济的持续发展。目前,我国防治农业有害微生物的方法多样,但在实际应用中均存在有不同的缺陷,如杀灭不完全,尤其是对一些产芽孢的烈性致病细菌的控制非常困难；大多数化学方法处理难以避免存在环境污染问题等。
论文提出利用绿色技术控制农业有害微生物,如可利用高级氧化技术,对农业生产原料、废弃物和排泄物等进行灭菌,也可对农业生产环境和农产品储藏室进行消毒。同时,可通过培育和推广作物抗病品种,在农作物收获前就控制病原菌,从而低成本高效益地预防、消除或减小农业有害微生物造成的危害。
论文的研究内容主要包括以下两部分：
第一部分,农业有害微生物的外源性羟基自由基控制研究
高级氧化技术(AOT)可通过反应产生极具氧化性的羟基自由基,它几乎能和所有的生物大分子、有机物和无机物发生不同类型的化学反应,直至彻底转化为二氧化碳和水等无害物质。论文采用强电场电离放电法制备高浓度羟基自由基,以大肠杆菌、枯草芽孢杆菌和芽孢为模型,研究外源性羟基自由基对农业有害微生物的生物学效应及作用机理,具体内容如下：
1、外源性羟基对G-细菌的杀灭效应及作用机制
研究以大肠杆菌为模型,采用不同浓度的外源性羟基自由基处理大肠杆菌,发现羟基浓度超过0.4mg/L时即能导致其全部死亡,这表明羟基浓度对G-细菌有高效的杀灭作用。对生物大分子含量及完整性的分析表明,羟基能进入细胞内损伤DNA、RNA和蛋白质。对胞内电解质渗漏率和膜脂氧化程度的分析表明,羟基能直接对细胞膜造成重大损伤。扫描电镜和透射电镜观察发现,当用0.2mg/L羟基溶液处理时,细菌形态扭曲,细胞壁与细胞膜之间出现明显的空隙；当用1.0mg/L羟基溶液处理时,细菌保持杆状,但细胞壁上出现不规则褶皱。羟基处理还使胞内的拟核变得模糊,甚至消失。综合上述结果,外源性羟基自由基不仅能损伤细胞壁、细胞膜等外部结构,也能损伤胞内结构和胞内生物大分子,从而导致G-细菌死亡
2、外源性羟基对G+细菌及芽孢的杀灭效应及作用机制。
致死效应研究表明,羟基也能有效杀灭枯草芽孢杆菌及芽孢,但完全杀灭它们所需的浓度分别为0.8mg/L和1.0mg/L,远高于杀灭大肠杆菌所需的浓度(0.4mg/L),这可能是由于枯草芽孢杆菌及芽孢的外壁比大肠杆菌厚造成的。电镜结果显示,不同浓度的羟基处理枯草芽孢杆菌时,菌体基本保持杆状,但其表面会出现褶皱,用0.2mg/L羟基溶液处理时,仅少数菌体表面出现褶皱,当羟基浓度提高至1.0mg/L时,所有菌体表面出现大量规则的竖条形纹路,超薄切片观察显示,胞内的拟核变模糊。对芽孢的扫描电镜观察表明,0.4mg/L羟基溶液不会引起芽孢表面结构的变化,而当羟基浓度提高到1.0mf/L时,芽孢体积大为缩小不足正常芽孢的1/9,并在芽孢全周布满凸起物。透射电镜结果显示,受损芽孢的外部结构呈解体状态,而芽孢内部呈深色,表明其致密度很高。由此推测,G+细菌的细胞壁和芽孢的芽孢壁等都是外源性羟基自由基的攻击靶标。
3、利用绿色荧光蛋白监测外源性羟基对大肠杆菌的杀灭效果。
研究采用基因工程技术制备了重组绿色荧光蛋白(EGFP),并构建了能组成型表达EGFP的工程菌E.coli-EGFP。离体研究表明,羟基溶液能有效淬灭重组EGFP的绿色荧光,当羟基浓度为0.2mg/L时,绿色荧光强度可降至48.25%,当羟基浓度增加到0.4mg/L以上时,绿色荧光基本消失。菌体研究表明,当羟基浓度为0.3mg/L时,工程菌的荧光强度可减至33.22%,当羟基浓度超过0.8mg/L时,荧光信号基本消失。结合工程菌的死亡率数据发现,用0.2-1.0mg/羟基溶液处理工程菌时,相对荧光强度与工程菌死亡率存在相关性,符合曲线方程：y=0.0009x2-0.0276x+0.0525(R2=0.9923)。该结果表明,借助工程菌胞内EGFP的荧光强度,可监测羟基溶液杀灭大肠杆菌是否彻底。
第二部分,农作物病原微生物的基因控制研究
培育和推广抗病品种是防治作物病害最经济、安全和有效的途径,其关键在于发掘和利用新的抗性资源。小麦白粉病是小麦三大病害之一,严重威胁着小麦生产。小麦野生近缘物种簇毛麦所携带的抗白粉病基因Pm21是目前最优秀的小麦白粉病抗源,但对其遗传基础尚缺乏足够的认识。论文借助比较基因组学高效开发携带Pm21基因的6VS缺失片段FL0.45-0.58及附近的CISP标记,构建高密度比较基因组图谱,进而采用RGA克隆法获得Pm21候选基因。研究的主要内容如下：
1、簇毛麦抗白粉病基因Pm21的精细定位
论文对已报道的4个6VS标记进行缺失定位,将携带Pm21基因的缺失片段FL0.45-0.58定位于标记Xcinau188和CINAU91之间。对现有的12个6VS标记在二穗短柄草基因组中进行电子定位,构建低密度比较基因组图谱。选择二穗短柄草第3号染色体短臂(3BdS)上连续的200个基因作为目标区,经共线性分析后,针对其中的69个基因设计了94对CISP引物,筛选到22个6VS特异性CISP标记。缺失定位分析表明,4个CISP标记定位于缺失片段FL0.58-1.0,10个定位于FL0.45-0.58,8个定位于FL0-0.45,从而将携带Pm21基因的FL0.45-0.58精细定位于CISP
标记6VS-CISP027与6VS-CISP145之间,并构建了高密度比较基因组图谱。2、采用RGA克隆法克隆Pm21候选基因
研究以植物R基因保守的NBS结构域作为种子序列,对二穗短柄草全基因组中的R基因进行鉴定,发现全基因组中存在299个R基因,其中3BdS染色体上有30个R基因,其结构以CC-NBS-LRR和NBS-LRR为主。根据高密度比较基因组图谱,将二穗短柄草的R5-R8基因视为簇毛麦Pm21基因的同源物,针对其保守序列设计简并引物,采用RGA克隆法从簇毛麦中获得3个R基因片段。缺失定位分析表明,仅Hv-R1基因定位于缺失片段FL0.45-0.58。利用RT-PCR技术进行转录分析,发现Hv-R1基因能组成型表达,并受白粉菌诱导增强表达。利用TAIL-PCR技术获得完整的Hv-R1基因,全长为5309bp,编码区为2568bp,其中内含子为250bp。Hv-R1基因编码的蛋白质为855个氨基酸残基,与二穗短柄草R5-R7基因的编码蛋白具有很高的同源性(72%-77%)。蛋白质结构域分析表明,簇毛麦Hv-R1基因为典型的CC-NBS-LRR类R基因。启动子模序分析发现,Hv-R1基因上游除了CAAT-box和(?)ATA-box,还有W-box、MYB和MYC转录因子识别位点等多种抗病相关的启动子模序,它们可能对Hv-R1基因起重要的调控作用。研究不仅为Pm21候选基因的功能鉴定奠定了良好的基础,也为克隆孤儿作物中的目的基因提供了新的思路和方法。
研究揭示了外源性羟基自由基杀灭大肠杆菌、枯草芽孢杆菌、芽孢的生物学效应及作用机理,并构建了能用于监测羟基杀菌效果的基因工程菌,为外源性羟基自由基控制农业有害微生物提供了重要依据。本研究还采用基于比较基因组学的RGA克隆法从簇毛麦中获得了Pm21候选基因,为其功能鉴定奠定了基础,也为利用该基因培育抗病品种、控制小麦抗白粉病提供了必要的依据。
China is a large agricultural country. Development of agriculture is the basis for economic growth. Agricultural harmful microorganisms can attack crops and contaminate agricultural products in various aspects of agricultural production. The harmful microorganisms will cause severe crop failures, quality decline of agricultural production and huge economic losses. They may also produce toxins and do harm to human and animals. Furthermore, Agricultural harmful microorganisms will contaminate the environment causing potential long-term risk to human health, even affecting sustained and stable development of economy.
There are many physical or chemical methods for controlling of agricultural harmful microorganisms; however, there are different defects of the methods in application. Sometimes, physical methods could not entirely kill microbes, especially bacteria producing spores. Chemical methods usually could cause environmental pollution.
In this study, we propose to apply environment-friendly technologies to control agricultural harmful microorganisms. We can utilize advanced oxidation technology (AOT) to sterilize the raw materials, wastes and excreta in agricultural production, or sterilize the progress rooms or storage room for agricultural products. We can also apply resistance gene for crop breeding, which contribute to controlling of plant pathogens and decreasing or avoiding environmental pollution and health risk causing by mass farm chemicals.
This paper includes the two following sections:control of harmful microorganisms using exogenous hydroxyl radicals and control of plant pathogens by disease resistance genes.
Section Ⅰ Control of Harmful Microorganisms Using Exogenous Hydroxyl Radicals
Advanced oxidation technology can produce hydroxyl radicals by reactions. Hydroxyl radical, a strong oxidant, can oxidate all kinds of organics and degraded them in to water, carbon dioxide and other harmless substances. In this paper, hydroxyl radicals were generated by generated by strong ionization discharge. As the models, Escherichia coli (Gram-negative bacteria), Bacillus subtilis (Gram-positive bacteria) and its spores were used to reveal the biological effects and mechanisms of exogenous hydroxyl radicals on agricultural harmful microorganisms. The major works are as the follows:
1. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-negative bacteria
E. coli were treated by exogenous hydroxyl radical solutions with different concentrantion. The results showed that0.4mg/L hydroxyl radical solution could entirely kill bacteria. It indicated that hydroxyl radical could efficiently kill Gram-negative bacteria. The assays on biomolecules (including DNA, RNA and protein) revealed that hydroxyl radical could enter the cell and destroy these biomolecules. The assays of cellular electrolytes leakage and MDA contents indicated that hydroxyl radical could lead to membranes damage. Cellular structures were also observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that cells were deformed and a great space appeared between the bacterial cell wall and membrane in the sample treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, bacteria kept rod-shaped; however, irregular wrinkles appeared on the surface of cells. Furthermore, the nucleoid changed vague, even disappeared in cells treated by hydroxyl radicals. The above results revealed that, exogenous hydroxyl radicals kill Gram-negative bacteria by damaging intracellular biomolecules and extracellular structures including membrane and cell wall.
2. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-positive bacteria
The study on lethal effects showed that exogenous hydroxyl radicals could also efficiently kill B. subtilis and its spores; however, the lethal concentrations were0.8mg/L and1.0mg/L, respectively, which were more higher than that (0.4mg/L) on E. coli. Under the SEM, treated B. subtilis cells were still rod-shaped. Like E. coli, wrinkles appeared on the surface of cells treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, wrinkles were more clear and regular. Under the TEM, the nucleoid become vague after treated by hydroxyl radicals. The SEM results showed that0.4mg/L hydroxyl radical solution could not lead to obvious change on the surface of spores. After treated by1.0mg/L hydroxyl radical solution, violent change was observed on the spores. Spores become small, about one ninth of the controls, and had many granules on the surface. The TEM results revealed that the surface structure of damaged spore were in disintegration and the inside of spore was showed in deep color, which indicated that the inside of spore with high-density. We proposed that the cell wall of Gram-positive bacteria and spore wall were also the major target attacked by hydroxyl radicals.
3. Tracing the lethal effect of hydroxyl radicals on E. coli by green fluorescence from EGFP.
Using genetic engineering method, recombinant enhanced green fluorescent protein (EGFP) was prepared and the engineered bacteria E. coli-EGFP strain was constructed in this study. In vitro, hydroxyl radical solution could efficiently decrease the green fluorescence intensity of purified recombinant EGFP. When treated by0.2mg/L hydroxyl radical solution, the fluorescence intensity was48.25%. When the concentration of hydroxyl radical reached to0.4mg/L, the fluorescence signal disappeared. In vivo, the fluorescence intensity deceased to33.22%after treated by0.3mg/L hydroxyl radical solution. When the concentration of hydroxyl radical reached to0.8mg/L, the fluorescence signal disappeared. Combined with the lethal effect on engineered bacteria, relative fluorescence intensity was related to mortality of engineered bacteria when the concentration range of hydroxyl radical reached was0.2to1.0mg/L. The data met the curve equation:y=0.0009x2-0.0276x+0.0525(R2=0.9923). This result indicated that the lethal effect of hydroxyl radical solution on engineered bacteria could be evaluated by fluorescence signal of EGFP.
Section Ⅱ Control of crop pathogens by disease resistance genes
Breeding and application of resistant cultivars is the most economical, safe and effective way to control crop disease. The key of this way is to discover and use new resistance resources. Wheat powdery mildew, one of the most severe diseases, is a great threat to wheat production. Haynaldia villosa, a wild relative of wheat, carres Pm21gene conferring the highest resistance to wheat powdery mildew. However, there is little known about Pm21gene. This paper attempts to develop CISP markers around6VS bin FL0.45-0.58carring Pm21gene based on comparative genomics and construct a high-density comparative genomic map. According to the map, we try to clone the Pm21candidate gene using the RGA method. The major works are as the follows:
1. Fine-localization of Pm21gene in H. villosa
In this paper, four reported6VS-specific markers were utilized to perform chromosome localization analysis by using the deletion materials. It indicated that chromosome bin FL0.45-0.58carring Pm21gene was between the marker Xcinau188 and CINAU91. The126VS markers reported could be localized to Brachypodium distachyon chromosomes. And a low-density comparative genomics map was constructed. In the target region of the short arm of B. distachyon chromosome3(3BdS), two hundred genes was selected to analyzed the colinearity between B. distachyon and rice. Among them,69genes with good colinearity were used to designed94CISP primers, and226VS-specific markers were founded. Four CISP markers were further localized on the6VS bin FL0.58-1.0, ten on FL0.45-0.58and8on FLO-0.45. So the bin FL0.45-0.58carring Pm21gene was between the CISP marker6VS-CISP027and6VS-CISP145. A high-density comaparative genomics map were further constructed.
2. Cloning of Pm21candidate gene using the RGA method.
As seed sequences, the conserved NBS domains of plant resistance (R) genes were used to perform BLAST analysis. In B. distachyon genome,299R genes were found. Among them,30R genes were on the3BdS chromosome. According to the high-density comaparative genomics map, only R5-R8gene locus corresponded to the region of6VS FLO.45-0.58in H. villosa. The conserved sequences of R5-R7were used to design degenerate primers, and three RGA fragments were PCR-amplified from H. villosa. Among them, only Hv-R1was localized on the bin FL0.45-0.58. Transcriptional analysis showed that Hv-R1constitutively expressed and the expression level was enhanced after inoculation by Blumeria graminis f. sp. tritici (Bgt). The full length sequence was obtained by TAIL-PCR. The result showed that Hv-R1gene was5309bp in length wih a250-bp intron and the coding region was2568bp in length. The predicted protein,855aa in length, had high homology (72%-77%) with R5-R7genes of B. distachyon. Protein domain analysis revealed that Hv-R1was with the classic CC-NBS-LRR domains. Promoter motif analysis indicated that CAAT-box, TATA-box, W-box, MYB and MYC transcription factor motif existed in the upstream of Hv-R1gene. These motifs might play critical roles in the expression regulation of Hv-R1gene. This study will contribute to revealing the function of Hv-R1gene and providing a new strategy for cloning of target gene in orphan crops.
These researches revealed the lethal effects and mechanisms of exogenous hydroxyl radicals on E. coli, B. subtilis and its spores, and constructed engineered bacteria with green fluorescence for fast tracing lethal effect of exogenous hydroxyl radicals. These results provided a basis for green controlling of agricultural harmful microorganism by using exogenous hydroxyl radicals. In this paper, a Pm21candidate gene, Hv-R1was obtained from H. villosa using a RGA cloning method combined with comparative genomics. This work contributed to discoving the function of the candidate R gene and made a basis for breeding applification of this gene in controlling wheat powdery mildew.
论文提出利用绿色技术控制农业有害微生物,如可利用高级氧化技术,对农业生产原料、废弃物和排泄物等进行灭菌,也可对农业生产环境和农产品储藏室进行消毒。同时,可通过培育和推广作物抗病品种,在农作物收获前就控制病原菌,从而低成本高效益地预防、消除或减小农业有害微生物造成的危害。
论文的研究内容主要包括以下两部分：
第一部分,农业有害微生物的外源性羟基自由基控制研究
高级氧化技术(AOT)可通过反应产生极具氧化性的羟基自由基,它几乎能和所有的生物大分子、有机物和无机物发生不同类型的化学反应,直至彻底转化为二氧化碳和水等无害物质。论文采用强电场电离放电法制备高浓度羟基自由基,以大肠杆菌、枯草芽孢杆菌和芽孢为模型,研究外源性羟基自由基对农业有害微生物的生物学效应及作用机理,具体内容如下：
1、外源性羟基对G-细菌的杀灭效应及作用机制
研究以大肠杆菌为模型,采用不同浓度的外源性羟基自由基处理大肠杆菌,发现羟基浓度超过0.4mg/L时即能导致其全部死亡,这表明羟基浓度对G-细菌有高效的杀灭作用。对生物大分子含量及完整性的分析表明,羟基能进入细胞内损伤DNA、RNA和蛋白质。对胞内电解质渗漏率和膜脂氧化程度的分析表明,羟基能直接对细胞膜造成重大损伤。扫描电镜和透射电镜观察发现,当用0.2mg/L羟基溶液处理时,细菌形态扭曲,细胞壁与细胞膜之间出现明显的空隙；当用1.0mg/L羟基溶液处理时,细菌保持杆状,但细胞壁上出现不规则褶皱。羟基处理还使胞内的拟核变得模糊,甚至消失。综合上述结果,外源性羟基自由基不仅能损伤细胞壁、细胞膜等外部结构,也能损伤胞内结构和胞内生物大分子,从而导致G-细菌死亡
2、外源性羟基对G+细菌及芽孢的杀灭效应及作用机制。
致死效应研究表明,羟基也能有效杀灭枯草芽孢杆菌及芽孢,但完全杀灭它们所需的浓度分别为0.8mg/L和1.0mg/L,远高于杀灭大肠杆菌所需的浓度(0.4mg/L),这可能是由于枯草芽孢杆菌及芽孢的外壁比大肠杆菌厚造成的。电镜结果显示,不同浓度的羟基处理枯草芽孢杆菌时,菌体基本保持杆状,但其表面会出现褶皱,用0.2mg/L羟基溶液处理时,仅少数菌体表面出现褶皱,当羟基浓度提高至1.0mg/L时,所有菌体表面出现大量规则的竖条形纹路,超薄切片观察显示,胞内的拟核变模糊。对芽孢的扫描电镜观察表明,0.4mg/L羟基溶液不会引起芽孢表面结构的变化,而当羟基浓度提高到1.0mf/L时,芽孢体积大为缩小不足正常芽孢的1/9,并在芽孢全周布满凸起物。透射电镜结果显示,受损芽孢的外部结构呈解体状态,而芽孢内部呈深色,表明其致密度很高。由此推测,G+细菌的细胞壁和芽孢的芽孢壁等都是外源性羟基自由基的攻击靶标。
3、利用绿色荧光蛋白监测外源性羟基对大肠杆菌的杀灭效果。
研究采用基因工程技术制备了重组绿色荧光蛋白(EGFP),并构建了能组成型表达EGFP的工程菌E.coli-EGFP。离体研究表明,羟基溶液能有效淬灭重组EGFP的绿色荧光,当羟基浓度为0.2mg/L时,绿色荧光强度可降至48.25%,当羟基浓度增加到0.4mg/L以上时,绿色荧光基本消失。菌体研究表明,当羟基浓度为0.3mg/L时,工程菌的荧光强度可减至33.22%,当羟基浓度超过0.8mg/L时,荧光信号基本消失。结合工程菌的死亡率数据发现,用0.2-1.0mg/羟基溶液处理工程菌时,相对荧光强度与工程菌死亡率存在相关性,符合曲线方程：y=0.0009x2-0.0276x+0.0525(R2=0.9923)。该结果表明,借助工程菌胞内EGFP的荧光强度,可监测羟基溶液杀灭大肠杆菌是否彻底。
第二部分,农作物病原微生物的基因控制研究
培育和推广抗病品种是防治作物病害最经济、安全和有效的途径,其关键在于发掘和利用新的抗性资源。小麦白粉病是小麦三大病害之一,严重威胁着小麦生产。小麦野生近缘物种簇毛麦所携带的抗白粉病基因Pm21是目前最优秀的小麦白粉病抗源,但对其遗传基础尚缺乏足够的认识。论文借助比较基因组学高效开发携带Pm21基因的6VS缺失片段FL0.45-0.58及附近的CISP标记,构建高密度比较基因组图谱,进而采用RGA克隆法获得Pm21候选基因。研究的主要内容如下：
1、簇毛麦抗白粉病基因Pm21的精细定位
论文对已报道的4个6VS标记进行缺失定位,将携带Pm21基因的缺失片段FL0.45-0.58定位于标记Xcinau188和CINAU91之间。对现有的12个6VS标记在二穗短柄草基因组中进行电子定位,构建低密度比较基因组图谱。选择二穗短柄草第3号染色体短臂(3BdS)上连续的200个基因作为目标区,经共线性分析后,针对其中的69个基因设计了94对CISP引物,筛选到22个6VS特异性CISP标记。缺失定位分析表明,4个CISP标记定位于缺失片段FL0.58-1.0,10个定位于FL0.45-0.58,8个定位于FL0-0.45,从而将携带Pm21基因的FL0.45-0.58精细定位于CISP
标记6VS-CISP027与6VS-CISP145之间,并构建了高密度比较基因组图谱。2、采用RGA克隆法克隆Pm21候选基因
研究以植物R基因保守的NBS结构域作为种子序列,对二穗短柄草全基因组中的R基因进行鉴定,发现全基因组中存在299个R基因,其中3BdS染色体上有30个R基因,其结构以CC-NBS-LRR和NBS-LRR为主。根据高密度比较基因组图谱,将二穗短柄草的R5-R8基因视为簇毛麦Pm21基因的同源物,针对其保守序列设计简并引物,采用RGA克隆法从簇毛麦中获得3个R基因片段。缺失定位分析表明,仅Hv-R1基因定位于缺失片段FL0.45-0.58。利用RT-PCR技术进行转录分析,发现Hv-R1基因能组成型表达,并受白粉菌诱导增强表达。利用TAIL-PCR技术获得完整的Hv-R1基因,全长为5309bp,编码区为2568bp,其中内含子为250bp。Hv-R1基因编码的蛋白质为855个氨基酸残基,与二穗短柄草R5-R7基因的编码蛋白具有很高的同源性(72%-77%)。蛋白质结构域分析表明,簇毛麦Hv-R1基因为典型的CC-NBS-LRR类R基因。启动子模序分析发现,Hv-R1基因上游除了CAAT-box和(?)ATA-box,还有W-box、MYB和MYC转录因子识别位点等多种抗病相关的启动子模序,它们可能对Hv-R1基因起重要的调控作用。研究不仅为Pm21候选基因的功能鉴定奠定了良好的基础,也为克隆孤儿作物中的目的基因提供了新的思路和方法。
研究揭示了外源性羟基自由基杀灭大肠杆菌、枯草芽孢杆菌、芽孢的生物学效应及作用机理,并构建了能用于监测羟基杀菌效果的基因工程菌,为外源性羟基自由基控制农业有害微生物提供了重要依据。本研究还采用基于比较基因组学的RGA克隆法从簇毛麦中获得了Pm21候选基因,为其功能鉴定奠定了基础,也为利用该基因培育抗病品种、控制小麦抗白粉病提供了必要的依据。
China is a large agricultural country. Development of agriculture is the basis for economic growth. Agricultural harmful microorganisms can attack crops and contaminate agricultural products in various aspects of agricultural production. The harmful microorganisms will cause severe crop failures, quality decline of agricultural production and huge economic losses. They may also produce toxins and do harm to human and animals. Furthermore, Agricultural harmful microorganisms will contaminate the environment causing potential long-term risk to human health, even affecting sustained and stable development of economy.
There are many physical or chemical methods for controlling of agricultural harmful microorganisms; however, there are different defects of the methods in application. Sometimes, physical methods could not entirely kill microbes, especially bacteria producing spores. Chemical methods usually could cause environmental pollution.
In this study, we propose to apply environment-friendly technologies to control agricultural harmful microorganisms. We can utilize advanced oxidation technology (AOT) to sterilize the raw materials, wastes and excreta in agricultural production, or sterilize the progress rooms or storage room for agricultural products. We can also apply resistance gene for crop breeding, which contribute to controlling of plant pathogens and decreasing or avoiding environmental pollution and health risk causing by mass farm chemicals.
This paper includes the two following sections:control of harmful microorganisms using exogenous hydroxyl radicals and control of plant pathogens by disease resistance genes.
Section Ⅰ Control of Harmful Microorganisms Using Exogenous Hydroxyl Radicals
Advanced oxidation technology can produce hydroxyl radicals by reactions. Hydroxyl radical, a strong oxidant, can oxidate all kinds of organics and degraded them in to water, carbon dioxide and other harmless substances. In this paper, hydroxyl radicals were generated by generated by strong ionization discharge. As the models, Escherichia coli (Gram-negative bacteria), Bacillus subtilis (Gram-positive bacteria) and its spores were used to reveal the biological effects and mechanisms of exogenous hydroxyl radicals on agricultural harmful microorganisms. The major works are as the follows:
1. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-negative bacteria
E. coli were treated by exogenous hydroxyl radical solutions with different concentrantion. The results showed that0.4mg/L hydroxyl radical solution could entirely kill bacteria. It indicated that hydroxyl radical could efficiently kill Gram-negative bacteria. The assays on biomolecules (including DNA, RNA and protein) revealed that hydroxyl radical could enter the cell and destroy these biomolecules. The assays of cellular electrolytes leakage and MDA contents indicated that hydroxyl radical could lead to membranes damage. Cellular structures were also observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that cells were deformed and a great space appeared between the bacterial cell wall and membrane in the sample treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, bacteria kept rod-shaped; however, irregular wrinkles appeared on the surface of cells. Furthermore, the nucleoid changed vague, even disappeared in cells treated by hydroxyl radicals. The above results revealed that, exogenous hydroxyl radicals kill Gram-negative bacteria by damaging intracellular biomolecules and extracellular structures including membrane and cell wall.
2. Biological effects and mechanisms of exogenous hydroxyl radicals on Gram-positive bacteria
The study on lethal effects showed that exogenous hydroxyl radicals could also efficiently kill B. subtilis and its spores; however, the lethal concentrations were0.8mg/L and1.0mg/L, respectively, which were more higher than that (0.4mg/L) on E. coli. Under the SEM, treated B. subtilis cells were still rod-shaped. Like E. coli, wrinkles appeared on the surface of cells treated by0.2mg/L hydroxyl radical solution. After treated by1.0mg/L hydroxyl radical solution, wrinkles were more clear and regular. Under the TEM, the nucleoid become vague after treated by hydroxyl radicals. The SEM results showed that0.4mg/L hydroxyl radical solution could not lead to obvious change on the surface of spores. After treated by1.0mg/L hydroxyl radical solution, violent change was observed on the spores. Spores become small, about one ninth of the controls, and had many granules on the surface. The TEM results revealed that the surface structure of damaged spore were in disintegration and the inside of spore was showed in deep color, which indicated that the inside of spore with high-density. We proposed that the cell wall of Gram-positive bacteria and spore wall were also the major target attacked by hydroxyl radicals.
3. Tracing the lethal effect of hydroxyl radicals on E. coli by green fluorescence from EGFP.
Using genetic engineering method, recombinant enhanced green fluorescent protein (EGFP) was prepared and the engineered bacteria E. coli-EGFP strain was constructed in this study. In vitro, hydroxyl radical solution could efficiently decrease the green fluorescence intensity of purified recombinant EGFP. When treated by0.2mg/L hydroxyl radical solution, the fluorescence intensity was48.25%. When the concentration of hydroxyl radical reached to0.4mg/L, the fluorescence signal disappeared. In vivo, the fluorescence intensity deceased to33.22%after treated by0.3mg/L hydroxyl radical solution. When the concentration of hydroxyl radical reached to0.8mg/L, the fluorescence signal disappeared. Combined with the lethal effect on engineered bacteria, relative fluorescence intensity was related to mortality of engineered bacteria when the concentration range of hydroxyl radical reached was0.2to1.0mg/L. The data met the curve equation:y=0.0009x2-0.0276x+0.0525(R2=0.9923). This result indicated that the lethal effect of hydroxyl radical solution on engineered bacteria could be evaluated by fluorescence signal of EGFP.
Section Ⅱ Control of crop pathogens by disease resistance genes
Breeding and application of resistant cultivars is the most economical, safe and effective way to control crop disease. The key of this way is to discover and use new resistance resources. Wheat powdery mildew, one of the most severe diseases, is a great threat to wheat production. Haynaldia villosa, a wild relative of wheat, carres Pm21gene conferring the highest resistance to wheat powdery mildew. However, there is little known about Pm21gene. This paper attempts to develop CISP markers around6VS bin FL0.45-0.58carring Pm21gene based on comparative genomics and construct a high-density comparative genomic map. According to the map, we try to clone the Pm21candidate gene using the RGA method. The major works are as the follows:
1. Fine-localization of Pm21gene in H. villosa
In this paper, four reported6VS-specific markers were utilized to perform chromosome localization analysis by using the deletion materials. It indicated that chromosome bin FL0.45-0.58carring Pm21gene was between the marker Xcinau188 and CINAU91. The126VS markers reported could be localized to Brachypodium distachyon chromosomes. And a low-density comparative genomics map was constructed. In the target region of the short arm of B. distachyon chromosome3(3BdS), two hundred genes was selected to analyzed the colinearity between B. distachyon and rice. Among them,69genes with good colinearity were used to designed94CISP primers, and226VS-specific markers were founded. Four CISP markers were further localized on the6VS bin FL0.58-1.0, ten on FL0.45-0.58and8on FLO-0.45. So the bin FL0.45-0.58carring Pm21gene was between the CISP marker6VS-CISP027and6VS-CISP145. A high-density comaparative genomics map were further constructed.
2. Cloning of Pm21candidate gene using the RGA method.
As seed sequences, the conserved NBS domains of plant resistance (R) genes were used to perform BLAST analysis. In B. distachyon genome,299R genes were found. Among them,30R genes were on the3BdS chromosome. According to the high-density comaparative genomics map, only R5-R8gene locus corresponded to the region of6VS FLO.45-0.58in H. villosa. The conserved sequences of R5-R7were used to design degenerate primers, and three RGA fragments were PCR-amplified from H. villosa. Among them, only Hv-R1was localized on the bin FL0.45-0.58. Transcriptional analysis showed that Hv-R1constitutively expressed and the expression level was enhanced after inoculation by Blumeria graminis f. sp. tritici (Bgt). The full length sequence was obtained by TAIL-PCR. The result showed that Hv-R1gene was5309bp in length wih a250-bp intron and the coding region was2568bp in length. The predicted protein,855aa in length, had high homology (72%-77%) with R5-R7genes of B. distachyon. Protein domain analysis revealed that Hv-R1was with the classic CC-NBS-LRR domains. Promoter motif analysis indicated that CAAT-box, TATA-box, W-box, MYB and MYC transcription factor motif existed in the upstream of Hv-R1gene. These motifs might play critical roles in the expression regulation of Hv-R1gene. This study will contribute to revealing the function of Hv-R1gene and providing a new strategy for cloning of target gene in orphan crops.
These researches revealed the lethal effects and mechanisms of exogenous hydroxyl radicals on E. coli, B. subtilis and its spores, and constructed engineered bacteria with green fluorescence for fast tracing lethal effect of exogenous hydroxyl radicals. These results provided a basis for green controlling of agricultural harmful microorganism by using exogenous hydroxyl radicals. In this paper, a Pm21candidate gene, Hv-R1was obtained from H. villosa using a RGA cloning method combined with comparative genomics. This work contributed to discoving the function of the candidate R gene and made a basis for breeding applification of this gene in controlling wheat powdery mildew.
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68.柴丽娜,齐小明.冬小麦幼苗细胞膜透性的变化与品种抗旱性的关系及测定.北京农学院学报1999；14：60-63
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71.王颖臻,丁中涛,曹秋娥.偶氮胭脂红G共振光散射法测定羟自由基的研究及应用.分析试验室2007；26：68-71
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75. Skrlj N, Vidrih Z, Dolinar M. A universal approach for promoter strength evaluation supported by the web-based tool PromCal. Anal Biochem 2009;396:83-90
76. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 1992;111:229-233
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82.陈升位,陈佩度,王秀娥.利用电离辐射处理整臂易位系成熟雌配子诱导外源染色体小片段易位.中国科学C辑2008；38：215-220
83.庄丽芳,宋立晓,冯祎高.小麦EST-SSR标记的开发和染色体定位及其在追踪黑麦染色体中的应用.作物学报2008；34：926-933
84. Mullan DJ, Platteter A, Teakle NL, et al. EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci. Genome 2005;48:811-822
85. Song W, Xie C, Du J, et al. A "one-marker-for-two-genes" approach for efficient molecular discrimination of Pm12 and Pm21 conferring resistance to powdery mildew in wheat. Mol Breeding 2009;23:357-363
86.陈升位,陈佩度.簇毛麦6VS染色体短臂特异性EST标记的开发及缺失定位.麦类作物学报2010；30：789-795
87. Cao A, Wang X, Chen Y, Zou X, Chen P. A sequence-specific PCR marker linked with Pm21 distinguishes chromosomes 6AS,6BS,6DS of Triticum aestivum and 6VS of Haynaldia villosa. Plant Breeding 2006;125:201-205
88. Chen Y, Wang H, Cao A, Wang C, Chen P. Cloning of a resistance gene analog from wheat and development of a co-dominant PCR marker for Pm21. Plant Biol 2006;48:715-721
89.王春梅,别同德,陈全战等.簇毛麦6V染色体短臂特异分子标记的开发和应用.作 物学报2007;33：1595-1600
90.别同德.利用花粉辐射诱导小麦-亲缘物种属间染色体易位.In：南京农业大学;2007
91. Feltus FA, Singh HP, Lohithaswa HC, et al. A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops. Plant Physiol 2006;140:1183-1191
92. Zeid M, Yu JK, Goldowitz I, et al. Cross-amplification of EST-derived markers among 16 grass species. Field Crop Res 2010;118:28-35
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94. Liu YG, Mitsukawa N, Oosumi T, Whittier RF. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 1995;8:457-463
95. Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 2007;43:649-650,652,654 passim