甘蔗与蔗茅杂交亲本及后代材料的抗旱性鉴定

研究旨在筛选出优良抗旱育种材料,为今后甘蔗抗旱育种和抗旱栽培技术提供重要的理论依据。采用盆栽干旱处理,测定蔗茅野生种无性系、甘蔗栽培品种（系）及其杂交后代共12份材料苗期与抗旱相关的8项生理生化指标,利用极点排序法对供试材料的抗旱性进行了综合评价。结果表明：干旱胁迫下,12份供试材料的Pro、MDA、SS及PMP均呈上升趋势,SP、Chl和SOD活性呈下降趋势,CAT活性有升有降。极点排序法分析表明,12个供试材料的抗旱强弱为：‘蔗茅99-2’>‘蔗茅99-1’>‘蔗茅99-4’>‘蔗茅I91-8’>‘蔗茅99-3’>‘蔗茅90-29’>‘滇蔗01-58’>‘滇蔗04-14’>‘滇蔗02-39’>‘蔗茅II91-2’>‘新台糖10’>‘崖城89-9’,说明蔗茅优质抗旱性状可以通过基因渗透方式传递给后代。在苗期干旱胁迫下,‘蔗茅99-2’在渗透调节等方面表现出积极的生理响应。     

Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.     

草鱼三元病原菌融合子的构建及生物学特性

采用细胞融合技术首次将肠型点状产气单胞菌菌株和鱼害粘球菌菌株融合，构建二元融合菌株AM-1，再将AM-1与荧光假单胞菌菌株融合，构建三元融合菌株AM-2。对AM-1和AM-2菌株的形态结构、生理生化特性、致病性、抗原性、抗体效价进行分析。结果表明，AM-1活菌椭圆形，无鞭毛，死菌短杆状，AM-2菌株短杆状，有鞭毛，它们的生理生化性状介于3原始亲本菌株之间；用AM-2菌株对当年健康草鱼种进行人工感染攻毒，被攻毒草鱼种具有烂鳃病、赤皮病及肠炎病的部分症状；AM-2菌株抗原的受免草鱼种对荧光假单胞菌、鱼害粘球菌和肠型点状产气单胞菌菌株的免疫保护率分别达64.90％、68.00％、67.00%，抗体效价最高达1∶256、1∶256、1∶512。测定了AM-1和AM-2菌株及亲本DNA含量并进行了琼脂糖凝胶电泳分析，AM-1和AM-2菌株DNA含量明显高于任一原始亲本。图4表6参23     

5种常规中草药联用对鱼害粘球菌的药效研究

根据K－B法、横盘法测定大黄、黄芩、黄柏、金银花、穿心莲独用和以大黄为基础两两联用时抑制鱼害粘球菌的效果。结果表明：单独作用时,穿心莲无抑菌效果,大黄、黄芩、黄柏、金银花的MIC分别为：0．098mg/l,1.56mg/l,6.25mg/l,0.196mg/l。两两联用时,大黄与黄芩、大黄与金银花有协同作用,而大黄与黄柏则出现拮抗作用。     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

黏细菌降解黄曲霉毒素B_1的产酶条件优化

本研究对一株具有降解黄曲霉毒素B1活性的黏细菌的产酶条件进行了优化。通过单因素实验和正交实验得到最优化的培养基以及培养条件。优化后的培养基为:酵母粉7 g/L、CaCl2.2H2O为1 g/L、MgSO4.7H2O为0.5 g/L、VB12为750μg/L;并确定其最佳培养条件为:初始pH 7.5、接种量20%、发酵时间50 h、发酵温度30℃。在上述最佳培养基和最佳发酵条件下,该菌株降解黄曲霉毒素B1的能力为78.2%。     

不同信息化学物质对4种松树小蠹虫的野外诱集效果研究

为了对小蠹虫进行野外监测和防治,采用人工合成的缓释性信息化合物诱芯和国产漏斗式诱捕器,研究了α-蒎烯(Alphapinene,AP)、壬醛(nonanal,NL)、反式马鞭草烯醇(Trans-verbemol,,TV)和桃金娘烯醇(myrtenol,MT)信息化学物质对黄色梢小蠹、纵坑切梢小蠹、横坑切梢小蠹和红松根小蠹4种松树小蠹虫的林问诱集效果.结果表明:信息化学物质处理对4种小蠹虫的诱集效果显著,2AP诱芯对黄色梢小蠹的诱集量(17.5头)显著高于CK和其他处理,诱集效果最好,是CK的25倍;信息化学物质处珲对纵坑切梢小蠹、横坑切梢小蠹和红松根小蠹的诱集量显著高于CK.不同信息化学物质处理间诱集量差异不显著;2AP+NL+TV诱芯对纵坑切梢小蠹的诱集量最高,诱集效果是CK的612倍:2AP+NL+TV诱芯对横坑切梢小蠹的诱集量最高,诱集效果是CK的1085倍:2AP+NL+MT+TV诱芯对红松根小蠹引诱效果最好.是对照的136倍.2005-2006年,千山风景区黄色梢小蠹和横坑切梢小蠹的虫口密度较大.     

甘蔗与蔗茅属间杂交F1代真实性的鉴定

采用体细胞染色体计数和RAPD分子标记的方法鉴定“甘蔗×蔗茅”属间杂交种F1代的真实性。结果表明:杂种F1代材料“01/47,01/85,01/120”的2n=80～82,染色体遗传方式为“n 2n”;用110个随机引物进行RAPD扩增,63个引物可获得双亲的RAPD多态性,其中3个引物(OPC-19,OPE-2,OPF-4)可在杂种F1代材料01/64和01/120的RAPD扩增标记中显示出双亲的特征谱带,分别为父本3500bp和母本1300bp、父本1350bp和母本900bp、父本550bp和母本900bp。     

5种笛鲷属鱼类的遗传多样性及分子标记

运用RAPD及SSR技术对笛鲷属的画眉笛鲷(Lutjanus vitta)、金带笛鲷 (L. fulvus)、金焰笛鲷 (L. fulviflamma)、千年笛鲷(L. sebae)和星点笛鲷(L. stellatus)等5种鱼的遗传多样性及其分子标记进行了研究。RAPD研究结果表明, 5种鱼的平均多态性位点比率(P)依次为89.30%、86.70%、92.11%、86.47%和86.00%; 种内两个体间平均遗传距离(D)分别为0.3431，0.2130，0.3121, 0.1825和0.1775；平均遗传多样性指数(Hi)分别为0.1634、0.1095、0.1353、 0.1022和0.1024。引物OPA8和OPP10的扩增产物中得到9个RAPD标记，分别为OPA8-413bp、OPA8-140bp、OPP10-418bp、OPA8-697bp、OPP10-526bp、OPA8-361bp、OPP10-449bp、OPA8-311bp和OPP10-599b，可鉴别5种鱼。SSR研究结果显示, 5种鱼的有效等位基因数依次为1.9610、3.3793、3.2957、1.7893和3.6591；群体的杂合度(H)分别为0.332、0.462、0.593、0.367和0.676；多态信息含量(PIC)分别为0.302、0.438、0.554、0.332和0.641。 在11个微卫星位点上得到6个微卫星标记，分别为13 Prs229-115 bp、4 Lca43-212 bp、4 Lca43-240 bp、13 Prs229-288 bp、19 Prs275-156bp和7Lca91-118 bp可用于鉴别5种鱼。