Fingerprinting methods to approach multitrophic interactions among microflora and microfauna communities in soil

The soil functioning and the response of the biota to external perturbations such as organic input are based on multitrophic interactions among a wide range of organisms. However, the various components of the soil microflora and microfauna are rarely addressed in ecological studies. We have developed a molecular approach based on terminal restriction fragment length polymorphism (T-RFLP) analysis to assess the community structures of protozoa and nematodes, together with bacteria and fungi that share the same soil environment. Two soils were characterized by a specific fingerprint for each of the four groups of organisms, showing the potential of all T-RFLP procedures to differentiate the community structures. The response of the soil biota to organic inputs was addressed using T-RFLP fingerprints together with physiological profiles of bacteria communities and global microbial activities and densities. Although the impact of compost or manure on the soil biota was only slightly noticeable from the global parameters measured, it was obvious from the community level analyses. However, the different components of the soil biota were altered to various extents, depending on the group of organisms and the soil–organic matter combination. The potential of the T-RFLP strategy to analyze simultaneously different biotic groups from the same soil DNA extract will facilitate the more systematic integration of eukaryotic organisms in ecological studies to investigate multitrophic interactions among the microflora and microfauna in relation with soil processes.     

胡杨基因组片段转化拟南芥表型研究

[目的]本研究旨在探索与挖掘胡杨基因组大片段的潜在功能,发掘具有潜在育种价值的胡杨基因簇。[方法]利用已构建的胡杨基因组BIBAC文库,采用花序浸染法,将胡杨基因组大片段78A2D10导入模式植物拟南芥基因组中。采用抗性筛选、分子检测及表型观察等方法鉴定、分析转化型植株。[结果]共获得15株特异表型的转化植株。与野生型相比,转化型植株主侧茎生长受到抑制,莲座叶面积增大近3倍,叶片数量增多,叶边缘皱缩,抽薹推迟约13周,株高增加近32.0 cm,侧茎发育成次生莲座,植株寿命延长约7周。[结论]胡杨基因组片段78A2D10可延长植株营养生长期及植株寿命,据此推测该基因片段可能与营养生长有关。     

Characterization of tuber blight‐suppressive soils from four provinces of the Ecuadorean Andes

Late blight, caused by the oomycete Phytophthora infestans, is a threat to potato‐cropping systems worldwide. In the Ecuadorian Andes, despite a high late blight incidence in foliage, tuber blight is rare. In this work, the hypothesis that Ecuadorian Andean soils are naturally suppressive to P. infestans tuber infection was evaluated. Soils from four potato‐growing regions were assessed for disease suppressiveness by determining the effects of soil heat treatment on P. infestans sporangia and their ability to infect potato slices after 1, 8, 15 and 30 days of exposure to soils. Tuber infection after inoculation with P. infestans‐infested soils was consistently lower during the evaluation period compared with heat‐treated soils. Fresh, untreated soils affected germination and viability of P. infestans sporangia in a site‐dependent manner. In addition, the effect of heat treatment on soil bacterial communities was assessed through terminal restriction fragment length polymorphism analysis of the 16S rDNA gene region. Heat treatment disrupted bacterial community composition, and a subset of terminal restriction fragments (TRF) was either positively or negatively correlated with tuber infection. Bacterial TRF negatively correlated with tuber infection corresponded in fragment size to taxa with known ability to inhibit pathogens and promote plant growth. Finally, bacterial isolates obtained from untreated soils, which inhibited P. infestans growth in vitro, represented 22–47% of isolates recovered, and matched classes predicted by the TRFs. This work represents a first step in understanding the mechanisms behind the low incidence of tuber blight in Andean potato‐cropping systems.     

两种柑橘线粒体DNA的获得与AFLP分析

为了研究柑橘种属间线粒体基因组的遗传多样性以及温州蜜柑胞质雄性不育的分子基础，采用了两个柑橘品种‘国庆1号’温州蜜柑和无核‘冰糖橙’的胚性愈伤组织作为提取线粒体的材料。不连续蔗糖梯度超速离心法纯化线粒体。提取的线粒体完整，具很强的荧光活性。mtDNA片段在20kb左右，且无降解。叶绿体探针rubisco L、核探针45 s rRNA Southem blotting没有检测到叶绿体DNA与核DNA，而线粒体探针apt6杂交获得一条5kb大小的带，说明用此提取方法可获得无污染的mtDNA。4对AFLP引物共得到98条带，其中14条带有多态性。     

Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的最小活性区研究

为了确定Cry9Aa3蛋白对亚洲玉米螟(Ostrinia furnacalis)和小菜蛾(Plutella xylostella)的最小活性区,根据NCBI BLAST比对结果与SWISS-MODEL同源建模结果,设计了12对截短引物,以cry9Aa3全长序列为模板,扩增出了12种不同截短片段,与pEB载体重组后的阳性克隆,转入大肠杆菌(Escherichia coli)菌株Rosetta(DE3)诱导表达,提取蛋白。对小菜蛾和亚洲玉米螟的生物学活性测定,发现截短蛋白Cry9Aa3-45~700、Cry9Aa3-1~654,对小菜蛾和亚洲玉米螟有较高的活性,其中截短蛋白Cry9Aa3-45~700对小菜蛾和亚洲玉米螟的LC50分别为5.27、7.56μg/g,截短蛋白Cry9Aa3-1~654对小菜蛾和亚洲玉米螟的LC50分别为7.76、7.79μg/g,与阳性对照无显著差异,而截短蛋白Cry9Aa3-1~653和Cry9Aa3-46~700(LC50200μg/g)对幼虫几乎丧失生物学活性。这说明Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的杀虫活性区相同,最小活性区位于45I和654P之间。     

人类活动对村级景观的影响

村落具有人类活动所造成的许多景观特征 ,是研究人为干扰对景观影响的良好场所。本文以研究点与村落中心间的距离作为人为干扰强度的划分指标 ,把村落按重度人为干扰、中度人为干扰和轻度人为干扰三个等级分为中心区、过渡区和基质区 ,分析人类活动与村级景观特征的相互关系。结果表明 :随着人为干扰强度的增加村级景观的斑块密度指数、破碎化指数、多样性指数、相对丰富度指数和均匀度指数均相应增大 ;中度人为干扰使得上述五种指数值最大 ;但中度干扰时景观优势度指数最小 ;随着干扰强度的增强 ,与人们生活密切相关的景观类型的分形维数减小 ,斑块的相似性增强     

FMDV免疫活性串联片段FB与T4噬菌体SOC基因的融合表达

将FMDV免疫活性串联片段FB的cDNA片段插入表达质粒pSOC的EcoR Ⅰ位点,获得重组质粒pSOC-FB.用pSOC-FB转化E.coli BL21（DE3）,获得表达FB蛋白的工程菌BL-SOC-FB.在1 μg/ml IPTG诱导之下,BL-SOC-FB表达了融合蛋白SOC-FB,其分子量为19 ku.SDS-PAGE检测表明,融合蛋白SOC-FB的最大表达量为细菌总量的24.5%.Western blot分析表明,大肠杆菌表达的SOC-FB能与FMDV阳性血清发生特异性反应.     

Actions of cathepsins on troponin T during postmortem aging of porcine muscle

In this study, we examined the contribution of the cathepsins (cathepsin D and crude cathepsins containing cathepsins B and L) to troponin T degradation during postmortem aging. The action of cathepsin D on troponin T was examined at various pHs (pH 4.0–6.5). The degradation of intact troponin T was observed at pH 4.0, but not observed at pH 5.5 and 6.5. As a result of the degradation of troponin T, the 30 kDa fragment was not generated in any pH condition. The action of the crude cathepsins on troponin T was also examined at various pHs (pH 4.0–6.5). The intact troponin T was degraded at pH 4.0 and the 30 kDa fragments were observed. These 30 kDa fragments disappeared during further incubation. On the other hand, at pH 5.5 and 6.5, the intact troponin T was degraded and the 30 kDa fragment was accumulated. These results suggested that the cathepsin D scarcely contributed to the degradation of troponin T during postmortem aging, but crude cathepsins containing cathepsins B and L were partially involved in the degradation of troponin T and the generation of 30 kDa fragments.     

利用家蚕细胞表达重组蝎毒素蛋白及其杀虫效果分析

用天然毒素蛋白杀灭害虫是农林病虫害防治的一个重要发展方向。利用家蚕核型多角体病毒(BmNPV)多角体蛋白基因的部分序列与蝎毒素蛋白基因(BmK ITa1)融合表达,SDS-PAGE分析通过融合表达策略构建的含有蝎毒素蛋白基因的重组病毒具有较高的蝎毒素蛋白表达水平,重组病毒vBmBac(polh-)PH5B/35B-BmK ITa1.6His,在家蚕BmN细胞内的BmKITa1蛋白表达水平分别为0.4、0.2 mg/mL。以家蚕和棉铃虫为供试昆虫,探讨融合蝎毒素蛋白的杀虫效果,结果表明:经口添食含融合蝎毒素蛋白的BmN细胞液对不同龄期家蚕幼虫均具有毒性,尤其对低龄幼虫的毒性较强,蚁蚕添食后的死亡率达80%;给1龄期的家蚕和棉铃虫幼虫经口添食含有不同剂量融合蝎毒素蛋白的BmN细胞破碎液,当添食剂量在2.8μg/头时,家蚕的死亡率即达100%,棉铃虫的死亡率达74%。研究结果提示:BmNPV多角体蛋白编码基因的部分序列与蝎毒素蛋白基因融合表达,可提高蝎毒素蛋白的表达水平,且重组病毒表达的蝎毒素蛋白具有较好的杀虫活性,在较低剂量下即可对低龄昆虫产生良好的杀灭效果。