Cloning of various promoters for foreign gene expression in Erwinia ananas

We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.     

Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors

ABSTRACT Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5' end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.     

The impact of global warming on plant diseases and insect vectors in Sweden

Cold winters and geographic isolation have hitherto protected the Nordic countries from many plant pathogens and insect pests, leading to a comparatively low input of pesticides. The changing climate is projected to lead to a greater rise in temperature in this region, compared to the global mean. In Scandinavia, a milder and more humid climate implies extended growing seasons and possibilities to introduce new crops, but also opportunities for crop pests and pathogens to thrive in the absence of long cold periods. Increased temperatures, changed precipitation patterns and new cultivation practices may lead to a dramatic change in crop health. Examples of diseases and insect pest problems predicted to increase in incidence and severity due to global warming are discussed.     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

2个适于水稻条斑病菌致病相关基因转录表达分析的启动子探针载体的构建

 水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)为稻黄单胞菌种下的致病变种,引起细菌性条斑病(bacterial leaf streak, BLS),对水稻安全生产构成严重威胁。为准确在水稻条斑病菌中进行致病相关基因的转录表达和调控分析,本研究构建了包含终止子、gusA报道基因和多克隆位点等启动子探针元件的载体pUTG01和pUTG14。选取Xoc的hrpF启动子,构建在pUTG01载体上,将其导入hrpX突变体RΔhrpX中,GUS活性测定结果显示,hrpF基因的表达显著减低,验证了hrpF受HrpX正调控,证实该载体可有效进行基因的转录表达分析;通过双质粒兼容共存策略,在hrpX突变体中同时实现了hrpX基因的功能互补和通过GUS活性定量测定hrpF基因的转录表达分析。该载体系统的建立,为后续分析稻黄单胞菌致病相关基因的表达调控提供了有效的工作系统。     

植物病毒与媒介昆虫互作促进其传播的研究进展

近年来植物病毒病频发,严重制约着农作物的产量与品质。绝大多数植物病毒依赖媒介昆虫进行传播,而传播的关键是病毒如何突破昆虫的肠屏障、唾液腺屏障和卵屏障等多个生物屏障。植物病毒一方面利用其外壳蛋白或非结构蛋白突破媒介昆虫的中肠屏障和唾液腺屏障;另一方面则与昆虫体内卵黄原蛋白、共生菌以及精子表面蛋白发生特异性互作,促进病毒跨越卵障碍,最终实现病毒在昆虫体内复制。此外,植物病毒还能通过侵染寄主植物影响其防御性状,间接改变媒介昆虫生理及其行为反应,促进病毒在植物间的传播。该研究对植物病毒突破昆虫生物屏障的分子机制,以及植物病毒-植物-媒介昆虫互作对于病毒传播的影响进行了综述,并对阻断病毒传播的方法进行展望。     

Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

Marker‐free,tissue‐specific expression of Cry1Ab as a safe transgenic strategy for insect resistance in rice plants

BACKGROUND: Rice is the major food resource for nearly half of the global population; however, insect infestation could severely affect the production of this staple food. To improve rice insect resistance and reduce the levels of Bt toxin released into the environment, the Cry1Ab gene was conjugated to the rice rbcS promoter to express Bt toxin in specific tissues of transgenic plants. RESULTS: Eight marker‐free, T₂ lines were separated from the T₀ cotransformants. Using RT‐PCR, high levels of Cry1Ab expression were detected in the leaf but not in the seed. The Cry1Ab protein level ranged from 1.66 to 3.31 µg g^?1 in the leaves of four transgenic lines, but was barely detectable in their seeds by ELISA. Bioassays showed that the mortality rate of silkworm larvae feeding on mulberry leaves dipped in transgenic rice flour and pollen was less than that of the positive control (KMD), and that their average weight was higher than that of KMD, suggesting that the Cry1Ab protein was not expressed in the seed and pollen. CONCLUSION: The transgene conferred a high level of resistance to insects and biosafety to the rice plants, which could be directly used in rice breeding. Copyright © 2012 Society of Chemical Industry     

Reference gene selection for qPCR gene expression analysis of rust-infected wheat

Real-time PCR (qPCR) is an effective method to quantify mRNA levels, but requires validated reference genes for data normalisation. The GeNorm-Plus algorithm was used to examine the expression stability of six candidate reference genes in resistant Avocet Yr1 wheat infected with Puccinia triticina, Puccinia striiformis and Puccinia graminis f.sp. tritici respectively. Results indicated that within the first 48 h after inoculation, the expression stability of the candidate reference genes differed between the three incompatible interactions. The geometric mean of ARF and RLI showed the best stability in P. triticina-infected wheat, CDC and RLI in P. striiformis-infected wheat and CDC, 18S and TUBB in P. graminis f.sp. tritici-infected wheat respectively. This clearly emphasised the need for reference gene validation for each different plant–pathogen interaction.     

植物病毒经介体昆虫唾液腺水平传播至寄主韧皮部机制的研究进展及展望

很多植物病毒经介体昆虫以持久循回型的方式水平传播至寄主韧皮部致病，而唾液腺是介体昆虫持久传毒的重要器官，也是植物病毒在介体昆虫内循回需要克服的最后一道防线。持久性植物病毒要完成水平传播，必须突破昆虫唾液腺屏障的阻碍，因此病毒和介体昆虫间形成了“攻”与“守”的较量与对决。揭示持久性植物病毒克服昆虫唾液腺屏障，实现水平传播的机制，对病害控制具有重要意义。该文着眼于介体昆虫唾液腺在持久传毒过程中的重要功能，回顾了虫传植物病毒突破介体昆虫唾液腺侵入屏障和释放屏障的分子机制，探讨了昆虫唾液蛋白通过调节植物或昆虫的适应性和行为促进或抑制病毒水平传播的功能，为制定阻断介体昆虫传播植物病毒途径的防控策略提供理论依据。     

Assessing wind and mammals as seed dispersal vectors in an invasive legume

While some plants have modified seed structures to facilitate dispersal, many lack such specialised adaptations, making their mode of dispersal unclear. This can be particularly problematic for predicting shifts in species ranges or tracking the spread of invasive plants. As an example, the seed size and shape of the invasive legume, Lespedeza cuneata, suggest that wind and attachment to animals are not important for dispersal, yet populations can spread surprising distances within a few years. Using a series of experiments conducted in the laboratory and field, we tested wind and mammal fur as mediators of seed dispersal. To test wind dispersal, seed traps were arranged radially around a patch of L. cuneata and seeds were collected following dispersal. Attachment to mammal fur was tested by fitting pelts of a deer, coyote and raccoon to artificial torsos and determining seed retention in both the field and the laboratory. Laboratory trials also examined the influence of wet versus dry conditions. Our results showed that wind direction strongly influenced dispersal distance and seeds were readily dispersed by mammal fur. The number of seeds retained was species specific, depending on fur depth and mammal size, with seed retention increasing under wet conditions. Together, these results suggest that both wind and mammal fur contribute to the movement of L. cuneata across grasslands. Consequently, both dispersal vectors should be considered when designing and implementing control strategies.     

Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal O-dealkylases

Methoxy-, ethoxy-, propoxy-, and butoxyresorufin were prepared and tested as substrates for the fluorometric assay of O-dealkylation by the mixed-function oxidase system in house flies, Musca domestica L. Methoxyresorufin proved to be the most suitable substrate because of the favorable reaction rates. This sensitive assay can be performed with a minimum of microsomal protein (<0.3 mg/ml) in 5 min or less. The apparent K_m and maximum velocity were calculated as 2.88 μM and 0.27 nmol of resorufin produced/min/mg of microsomal protein, respectively. The O-dealkylation reaction required O₂ and NADPH and was inhibited by CO.     

Bioresmethrin-induced alterations in the ultrastructure of neurosecretory cells of insect corpora cardiaca

The ultrastructure of the neurosecretory cells of the corpora cardiaca (CC) of Locusta migratoria and Rhodnius prolixus was altered following incubation of the isolated glands in 1 μM bioresmethrin. The insecticide induced formation of large vacuoles in the soma and extensive exocytosis in the axon terminals of treated cells. Pretreatment of the CC with 1 μM tetrodotoxin blocked the action of bioresmethrin. Bioresmethrin affected the distribution and shape of mitochondria in neurosecretory cells. In Rhodnius, numerous mitochondria were found clustered in vacuoles in neurosecretory cells of the CC treated with bioresmethrin. Some of these mitochondria were abnormally elongated while others showed signs of fission. The results are discussed in relation to the action of bioresmethrin on neurosecretory cells.     

转cry2Ah-vp基因玉米的抗虫性鉴定

为获得新型抗虫转基因玉米,将通过群体筛选获得的具有抗虫性的转cry2Ah-vp基因玉米VP1-5采用PCR、Southern blot、实时荧光定量PCR(qPCR)、酶联免疫吸附测定(ELISA)等方法进行阳性植株鉴定、拷贝数分析、转录水平和翻译水平分析,同时通过室内和田间生物活性测定鉴定转基因玉米VP1-5对东方黏虫Mythimna separata和亚洲玉米螟Ostrinia furnacalis的抗性。结果表明,在转基因玉米VP1-5中cry2Ah-vp基因已整合到玉米基因组,以单拷贝的形式插入;cry2Ah-vp基因在转基因玉米VP1-5不同部位组织中均可以正常转录,在灌浆期叶片中的mRNA表达量最高,相对表达量为32.67,在灌浆期穗轴中的mRNA表达量最低,相对表达量为3.74;Cry2Ah-vp蛋白在转基因玉米VP1-5的6叶期各组织中表达量均较高,其中在叶片中的表达量达到2 155.18 ng/g FW,在抽雄期花丝中的表达量最高,达到2 165.86 ng/g FW;且转基因玉米VP1-5对东方黏虫有很高的杀虫活性,接虫3 d后幼虫死亡率达到100.00%;对亚洲玉米螟幼虫也有明显的生长抑制作用。表明转基因玉米VP1-5可作为玉米抗虫育种和害虫防治的种质资源。     

Potential for exploiting plant genes to genetically engineer insect resistance,exemplified by the cowpea trypsin inhibitor gene

Over the 400 million or so years of their co-evolution, plants have evolved some highly effective strategies to combat herbivorous insects. Amongst these natural defence mechanisms are some which depend on a primary gene product for effect. These are currently suitable for producing insect-resistant, transgenic crop plants by genetic engineering. One such mechanism involves the protease inhibitors from cowpea (Vigna unguiculata), which are anti-metabolic to a wide range of insects. Cowpea trypsin inhibitor (CpTI) genes have been cloned and transferred to tobacco plants. Those transgenic tobacco plants which express physiological levels of CpTI have enhanced resistance to a wide range of insect pests. The insecticidal characteristics of these plants compare favourably with those of transgenic plants expressing the B. thuringiensis endotoxin gene. Some possible advantages of, and prospects for, using plant-derived ‘insect-resistance’ genes in this way are discussed.     

Transient expression of pepper Bs2 gene in Citrus limon as an approach to evaluate its utility for management of citrus canker disease

The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.