棉花抗黄萎病基因工程研究和防卫反应相关基因的克隆

系统获得性抗性(Systematic acquired resistance, SAR)是植物抵御病原菌侵染的最有效手段，利用基因工程技术导入SAR信号发生过程中的关键基因后，植物的SAR基因表达量提高并且对病原菌侵染的反应速度加快，因此植物的抗病性得以增强，与传统的抗病基因工程技术相比它对病原菌没有专一性，许多学者称之为广谱抗病基因工程，该领域已成为目前抗病基因工程研究的热点和前沿。 NDR1和NPR1基因在植物的SAR发生中起着重要的作用，前者功能定位在ROS(reactive oxygen species)的激活和随后的水杨酸(SA)诱导合成之间，突变株病原菌诱导后SA合成能力降低，SAR发生减弱，目前还没有对该基因进行过量表达分析的报道;后者功能定位在SAR信号转导级联反应之中的SA积累和随后的SAR基因表达之间。该突变株在病原菌侵染时不产生病程相关蛋白(PRs)，表现为感病，而对照抗病;过量表达该基因的转基因拟南芥对多种病原菌的侵染产生抗性，PR1等PRs蛋白的表达量也提高，异源表达该基因的水稻对白叶枯病的抗性也提高。本研究利用RT-PCR方法从拟南芥中克隆了这两种基因，序列分析表明拟南芥Wassilewskija生态型的NDR1基因与Columbia生态型相比，共有7处碱基不同，引起编码氨基酸变化4处，而NPR1基因与报道的Wassilewskija生态型来源的NPR1基因完全相同。 我们构建了35S启动子驱动的NDR1和NPR1基因的植物组成型高效表达载体，利用农杆菌介导法转化烟草，PCR和Southern鉴定外源基因已经整合到植物基因组中。抗病性分析显示过量表达NDR1和NPR1基因的烟草对晚疫病和赤星病的抗性都有明显提高，说明这两个基因的在其它植物中异源表达后，都能提高植物对多种病原菌的抗性。 本论文提出了利用这两个基因来培育抗黄萎病棉花的设想，一方面为解决这个“世纪性”难题积累新的资料，另一方面也为其它作物的抗病基因工程提供新的经验。利用35S启动子驱动的NDR1和NPR1基因的植物组成型表达载体分别对陆地棉品种石远321进行花粉管通道法转化。同时，还探讨了这两个基因在棉花中的共转化实验，希望它们的“协同增效”能进一步提高棉花的抗病性。对其中2001年夏天在南京注射所获得的5,000粒种子在三亚进行100 g/ml卡那霉素筛选，初步鉴定分别获得转NDR1和NPR1基因株系26和24棵，PCR进一步鉴定其中分别有12和7棵为转基因阳性，转基因频率分别为0.50％和0.27％，目前利用营养钵蘸根法对其二代进行抗枯、黄萎病鉴定，结果显示有转基因植株对枯、黄萎病的抗性都明显增强，进一步的鉴定正在进行中。2002年初海南注射分别获得转NDR1和NPR1基因以及共转化种子22,000、10,500和12,500粒种子，2002年夏在中国农科院植保所黄萎菌病圃筛选抗黄萎病单株，并利用100 g/ml卡那霉素初步筛选出了一批抗性植株，每种转基因株系随机挑选5株进行PCR鉴定，结果显示为阳性。进一步的抗黄萎病鉴定和筛选以及分子分析正在进行中。 同时，本文还探讨了病原菌诱导型启动子在广谱抗病基因工程应用的可能性。根据烟草的Pr1-a启动子已知序列设计引物，PCR扩增启动子序列后,构建病原菌诱导型NPR1基因植物表达载体，并对棉花进行转化，获得种子11,500粒，利用同上的筛选方法，获得了一致的结果，目前抗黄萎病鉴定、分子检测以及生物学分析正在进行中。 最后，鉴于抗生素标记在转基因植物的应用引起了许多“安全性”争论的事实，还构建了无筛选标记的表达载体对抗虫棉进行转化，这样在生产上可以直接获得抗虫棉抗黄萎病棉花新材料，也为其它作物抗病基因工程积累经验。 本研究还提出了一种较为有效的提取高质量棉花总RNA的方法，与原来一些棉花RNA纯化方法相比，该方法所用都为常规试剂，易于重复，质量高。并且利用获得的总RNA构建了黄萎菌激发子诱导的cDNA文库，滴度测定为1╳107pfμ/μg，插入片段大小在5 00~2 000 bp范围内。 鉴于NPR1基因研究的重要性，本研究还利用简并引物PCR技术从海岛棉和陆地棉的基因组中都分离到了NPR1基因的同源片段，大小都为208 bp，与拟南芥NPR1基因的相应部分的同源性分别为66％和65％，它们之间的同源性为87％，目前该基因的全长正在分离鉴定中。 多聚半乳糖醛酸酶抑制蛋白(PGIPs)在植物的防御反应中起着重要的作用，通过分析已知20余种pgip基因序列的保守区，设计简并引物，PCR扩增海岛棉（Gossypium barbadense）7124 cDNA文库，得到一条长561 bp的片段，序列测定后分析确认为pgip基因的一部分。根据此序列和棉花病原菌诱导的cDNA文库载体中已知部分设计RACE引物，扩增后，5’和3’RACE分别得到666bp和906 bp的片段。序列分析表明它具有完整的编码框，产物为330 aa的蛋白质。序列分析该蛋白具有10个串联的LRR(leucine-rich repeat)区，与柑桔(Citrus)和枳(Poncirus)的pgip基因的同源性分别为69.2%和68.7%。进一步PCR扩增得到该基因的全长阅读框，并且获得了相应的基因组片段，序列分析发现该基因没有内含子。这是从棉属植物中克隆的第一个pgip基因。

河南省封邱县低产土壤上小麦黄叶病的发生及防治对策

河南省封邱县位于黄河下游的大冲积扇平原上。小麦是本县主要作物。本文主要根据野外调查结果讨论了小麦生理性黄叶病和土壤性质的关系。低产土壤包括盐土、碱土、风沙土及低肥力的两合土等。 主要论点及结论如下： l、调查了各种土壤上的小麦黄叶病。这种生理上的黄叶病导因于土壤的不良性质。次生盐渍化旱地上的小麦黄叶病是由于土壤中高量的可溶性盐分不但提高了土壤溶液的渗透势，而且损坏了根系的吸收功能，使得其得不到大量营养元素的补充而引起。次生盐渍化水浇地上的小麦黄叶病的原因是和土壤的水盐动态有关，田间高持水量和盐分使小麦根系黑，田间土体构型改变了盐分在土壤中的再分配。县西牛皮碱地区小麦黄叶病的原因是土壤结构、土壤高PH值。土壤结构呈层片状使小麦根系难以深扎、水稳性差，土壤高PH值损害作物的组织和吸收功能。 2、由于土壤的盐渍化，次生盐渍化的发生，由于土壤的水盐动态关系，土壤的微生物性质、土体构型及微地形差异都发生程度不同的黄叶病。本文还从大环境角度分析了次生盐渍化发生的原因是背河洼地边缘种稻引起地下水位抬高，从而导致大范围小麦生态环境条件恶化。 3、由于沙土、低肥力两合土的保水保肥能力较差，使小麦在主要发育期（拔节）产生生理性的黄叶病。 4、本文根据作者自己的调查结果认为，地下水在盐渍化作用中的影响是有条件的，土壤质地、微地形、土体构型、植被和降水蒸发比是重要因素;认为认为不同的土体构型中浅位粘层返盐效果最显著;认为盐渍土壤温度低是小麦黄叶病的重要原因。

Physiological and biochemical microcystin-RR toxicity to the Synechococcus elongatus

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 mug (.) L-1 significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F-v/F-m) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article. (C) 2004 Wiley Periodicals, Inc.

捷安肽素对采后柑桔青绿霉菌抑制效果及作用机理研究

捷安肽素是一种由枯草芽孢杆菌（Bacillus subtilis）ZK 产生的抗真菌多肽。本文以柑桔青霉菌（Penicillium italicum）和绿霉菌（Penicillium digitaum）为供试真菌，研究了捷安肽素的抑菌性能及作用机理，为捷安肽素开发为有效的生物杀菌剂提供理论依据。全文共分两部分：第一部分：捷安肽素对柑桔青霉菌和绿霉菌抑制效果研究。采用琼脂扩散法测定捷安肽素对柑桔青霉菌和绿霉菌的抑菌活性。53.9 µg/mL 捷安肽素对绿霉菌和青霉菌的抑菌圈直径分别为26.7mm 和24.1mm。结果表明捷安肽素能够抑制柑桔青绿霉菌的生长，柑桔绿霉菌比青霉菌对捷安肽素敏感。在柑桔果实上，研究了不同浓度、不同接入时间的捷安肽素对柑桔青霉病和绿霉病的防治效果，并与常用化学杀菌剂抑霉唑、咪鲜胺、甲基硫菌灵和多菌灵作比较。53.9 µg/mL捷安肽素处理柑桔果实，柑桔青霉病和绿霉病发病率分别为5.0 %和5.3 %,比对照低95.0 %和94.7 %；柑桔青霉病和绿霉病的病情指数分别为1.87 和2.18，比对照低73.73 和97.82。结果表明，捷安肽素能够有效地防治柑桔青绿霉病。与对照相比，捷安肽素先于或后于柑桔青绿霉菌接入时，对柑桔青绿霉菌均有抑制作用，但抑制效果随接入间隔时间的增长而降低。第二部分：捷安肽素对绿霉菌作用机理研究。首先在光学显微镜和透射电镜下观察捷安肽素处理后绿霉菌菌丝表面形态结构与菌丝体内超微结构的变化。形态观察发现，捷安肽素处理24h以内，绿霉菌菌丝结构无变化。捷安肽素作用36h后，绿霉菌菌丝不规则缢缩和膨大。48h后，在绿霉菌菌丝顶端、中部、末端的多处细胞均可发生畸形的球状结构，这种畸变结构随处理的延长而增加，致使细胞成为捻珠状。处理72 h后，畸变球形细胞开始断裂离解。处理96h后，镜下几乎无完整菌丝，成单个的球状细胞，部分细胞出现破裂。而对照菌丝表面光滑，结构完整。通过透射电镜观察发现，与对照相比，捷安肽素处理后，绿霉菌细胞壁、细胞膜轮廓模糊不清，细胞质外泄。推测捷安肽素能够使绿霉菌细胞膜通透性发生改变。进一步实验利用紫外-可见分光光度计检测捷安肽素作用后绿霉菌胞外液紫外吸光度的变化，表明捷安肽素作用于绿霉菌菌丝后，细胞内蛋白质、核酸缓慢泄漏。通过Atomscan Advantage单道扫描等离子体发射光谱仪(ICP)测定捷安肽素作用后菌丝体内K+浓度的改变，结果表明捷安肽素作用于柑桔绿霉菌1h内，菌丝体内K+含量迅速下降，为对照绿霉菌K+含量的37.53 %，1 h后菌丝体内K+含量变化趋于平缓。K+的迅速泄漏，以及蛋白质、核酸的泄漏表明捷安肽素通过迅速改变绿霉菌细胞膜通透性，使绿霉菌菌丝生长受到抑制。Jiean-peptide produced by Bacillus subtilis ZK has broad-spectrumresistance to plant pathogens. In this study, we investigated the antifungal propertyand the possible antifungal mechanism of jiean-peptide against two commonphytopathogenic fungi of citrus fruits: blue molds (P. italicum) and green molds (P.digitatum).The paper involved two parts:Part 1 is the study of the antifungal property of jiean-peptide against blue moldsand green molds of citrus fruits. The in vitro inhibition effect of jiean-peptide againstblue molds and green molds was detected by agar diffusion method. The diameters ofinhibition zones of green molds and blue molds are 26.7mm and 24.1mm respectivelyby treating with 53.9 µg/mL jiean-peptide. It shows that jiean-peptide effectivelyinhibits the both phytopathogenic fungi, and it is more effective for inhibiting greenmolds than blue molds. The effectiveness of jiean-peptde to inhibit green molds andblue molds in vivo was investigated compared with four conventional fungicides thatare imazalil, prochloraz, carbendazin and methylthiophanate. The result is that the incidences of the blue mold disease and green mold disease are 5.0 % and 5.3 %, thedisease severities are 1.87 and 2.18 respectively when citrus are inoculated with 53.9µg/ml jiean-peptide. The decay incidences and disease severities were significantlyreduced by treating with jiean-peptide compared with the control. The results indicateJiean-peptide is effective for controlling blue molds and green molds on citrus. Theoptimized inoculation time was also investigated. When inoculated with jiean-peptideat 0 h, 6 h, 12 h, 24 h and 48 h before or after pathogens’ inoculation, Jiean-peptidecan suppress the occurrence of blue molds and green molds compared with the control, but the effect of later inoculation decreases compared with the inoculation at the sametime.In Part 2, we investigated the possible antifungal mechanism against greenmolds of citrus. At first, we observed the exterior morphological changes andultrastructural changes of blue molds under light microscopy (LM) and transmissionelectron microscopy (TEM). Compared with untreated control cells which aregenerally uniform in shape, the appearances of treated hyphae change obviously. Itshows that some cells of hyphae irregularly shrink or enlarge when cultured for 36h.When the treating time of jiean-peptide increases, the aberrance of the hyphaebecomes more obvious, and hyphae exhibit the moniliform appearances. Finally, thereis no intact hypha leaved except only single cells, and some of which appear fractured.By transmission electron microscopy (TEM) observation, we find that the outline ofthe cell wall and the cell membrane of hyphae are blurry, and the cytoplasma oozesout. The observation result under LM and TEM suggests that jiean-peptide mightchange the permeability of the cell membrane. So we conducted further experiment todetect the change of permeability when the cells of blue molds were treated withjiean-peptide. And the effect of jiean-peptide on non-growing cells of blue molds wastested. By the spectrophotometer measurement, we found that compounds with lightabsorption at 260 nm and 280 nm were released and amounts increased within 12 hcompared with the control. Moreover, by the ICP measurement, the leakage of K+occurred immediately in the presence of jiean-peptide within 1 h, but with nearly nofurther change after 1 h. All these results indicate that jiean-peptide could change themembrane permeability of blue molds immediately and result in leaking nucleotides,proteins and K+ from cells.