Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

福建甘薯病毒病病原鉴定及主要病毒多样性

【背景】病毒病是甘薯的一种重要病害,给甘薯生产带来了严重的经济损失,而生产中甘薯病毒病病原种类复杂多样。【目的】明确福建甘薯病毒病种类、分布及流行,对主要病毒进行多样性分析。【方法】从福建主要甘薯种植区采集病毒病样品,利用PCR/RT-PCR的方法对采集的样品进行病原检测,获得病毒序列,利用MEGA 6.0构建系统进化树进行遗传分析。【结果】从福建7个甘薯产区鉴定12种甘薯病毒,包括9种RNA病毒:甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)、甘薯G病毒(Sweet potato virus G,SPVG)、甘薯C病毒(Sweet potato virus C,SPVC)、甘薯2号病毒(Sweet potato virus 2)、甘薯褪绿斑病毒(Sweet potato chlorotic fleck virus,SPCFV)、甘薯潜隐病毒(Sweet potato latent virus,SPLV)、甘薯轻型斑点病毒(Sweetpotatomildspeakingvirus,SPMSV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV),3种DNA病毒:甘薯卷叶病毒(Sweet potato leaf curl virus,SPLCV),甘薯无症状1号病毒(Sweet potato symptomless virus 1,SPSMV-1),甘薯杆状DNA病毒B (SPBV-B)。SPFMV、SPCSV、SPVG和SPLCV检出率最高,分别为50.28%、41.90%、35.75%和24.58%,CMV检出率最低,为2.79%,未检测到甘薯C-6病毒(SweetpotatoC-6)和甘薯轻型斑驳病毒(Sweetpotatomild mottle virus,SPMMV)。福建甘薯主要以2-6种病毒复合侵染为主,单一侵染率占14.39%,2种以上复合侵染占85.61%。福建SPFMV分离物存在EA、O和RC3种株系,SPCSV分离物存在WA1种株系,未发现EA株系,甘薯卷叶病毒分属于2个不同的株系群。【结论】福建甘薯病毒种类多样,以复合侵染为主,且存在多种株系,遗传结构复杂。     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

Phylogenetic diversity of nitrogen-fixing bacteria and the nifH gene from mangrove rhizosphere soil

Nine types of nitrogen-fixing bacterial strains were isolated from 3 rhizosphere soil samples taken from mangrove plants in the Dongzhaigang National Mangrove Nature Reserve of China. Most isolates belonged to Gammaproteobacteria Pseudomonas, showing that these environments constituted favorable niches for such abundant nitrogen-fixing bacteria. New members of the diazotrophs were also found. Using a soil DNA extraction and PCR-cloning-sequencing approach, 135 clones were analyzed by restriction fragment length polymorphism (RFLP) analysis, and 27 unique nifH sequence phylotypes were identified, most of which were closely related to sequences from uncultured bacteria. The diversity of nitrogen-fixing bacteria was assessed by constructing nifH phylogenetic trees from sequences of all isolates and clones in this work, together with related nifH sequences from other mangrove ecosystems in GenBank. The nifH diversity varied among soil samples, with distinct biogeochemical properties within a mangrove ecosystem. When comparing different mangrove ecosystems, the nifH gene sequences from a specific site tended to cluster as individual groups. The results provided interesting data and novel information on our understanding of diazotroph community diversity in the mangrove ecosystems.     

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft-inoculated with SPVD-affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme-linked immunosorbent assay (ELISA). Virus-free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

呼伦贝尔草原土壤固氮微生物nifH基因多样性与群落结构

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术及扩增产物序列分析方法比较研究了呼伦贝尔5种草地类型(线叶菊草原、贝加尔针茅草原、羊草草原、大针茅草原、克氏针茅草原)土壤固氮微生物nifH基因多样性及群落结构特征.结果表明,不同草地类型间土壤固氮微生物群落组成差异显著,羊草草原和大针茅草原相似性较高,相似度为67%,而其他3个草地类型间相似性较低,相似度均低于60%.羊草草原土壤nifH基因多样性最高,其次是克氏针茅草原、大针茅草原和贝加尔针茅草原,线叶菊草原最低.系统发育分析结果表明,呼伦贝尔草原土壤固氮微生物大部分属于变形菌门的α-变形菌纲,分别隶属于慢生根瘤菌属、中慢生根瘤菌属、红假单胞菌属、固氮螺菌属.5种草地类型中,克氏针茅草原土壤中联合固氮菌和自生固氮菌居多,而其他4个草地类型均以共生固氮菌为优势生态类型.     

云南甘薯病毒的检测及主要病毒的多样性分析

[目的]明确云南甘薯病毒的种类,并对主要病毒进行遗传多样性分析.[方法]利用PCR/RT-PCR技术,对采自云南16个县、市的279个甘薯样品进行扩增、测序,对所得序列应用分子生物学软件MEGA 5进行系统发育分析.[结果]除普洱和祥云的样品中未检测到任何病毒外,其余14个县、市的123个甘薯样品中共检测到甘薯褪绿斑病毒(SPCFV)、甘薯羽状斑驳病毒(SPFMV)、甘薯卷叶病毒(SPLCV)、甘薯C病毒(SPVC)、甘薯G病毒(SPVG)和甘薯病毒2号(SPV2)等6种病毒.其中SPVG的检出率最高,达39.1％,为云南甘薯病毒的优势种,SPFMV和SPVC的检出率分别为26.9％和24.7％,而SPLCV检出率最低,仅为0.4％.在所检测的样品中未发现甘薯褪绿矮化病毒(SPCSV)和甘薯轻斑驳病毒(SPMMV).云南甘薯病毒多数为2-5种病毒复合侵染,占总样品数的31.9％,其中2-3种病毒复合侵染现象最为常见,单一病毒侵染占总样品数的12.2％.检出率比较低的SPCFV、SPLCV和SPV2未发现单独侵染现象.[结论]云南甘薯上发生的SPFMV分离物存在EA株系和O株系,未发现RC株系,另有两个分离物同EA、O、RC之间的亲缘关系均较远,有可能是一个新的株系;SPVC和SPVG分离物均可分为3个不同的组,大部分SPVG云南分离物属于Ⅰ组.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Cloning a peanut resveratrol synthase gene and its expression in purple sweet potato

A resveratrol synthase gene was cloned from the peanut plant (Arachis hypogaea) by RT-PCR and was transformed into purple sweet potato (Ipomoea batatas) by Agrobacterium-mediated transformation. Stem sections were infected with bacterial solution of OD₆₀₀ = 0.4 for 20 min and then cocultured for 2 days. Infected explants were cultured on MS media containing 50 mg/l kanamycin, 0.02 mg/l NAA and 1 mg/l 6-BA for bud induction or containing 75 mg/l kanamycin, 1.0 mg/l NAA and 0.1 mg/l 6-BA for root formation. The bud and root induction rates were 37.5 and 25.0%, respectively. 105 regenerated plants were obtained, with 11 positive plants by PCR and Southern blotting analyses. A high level of resveratrol glucoside (340 μg/g dry weight), but no resveratrol, was detected in the transformed plants by HPLC. This study also provides a stable genetic transformation and plant regeneration method for metabolic modification of purple sweet potato.     

The incidence of sweet potato virus disease and virus resistance of sweet potato grown in Uganda

Sweet potato virus disease (SPVD) was common (25–30% average incidences), and farmers recognised it as an important disease, in sweet potato crops in southern Mpigi, Masaka and Rakai Districts in Uganda, but SPVD was rare in Soroti and Tororo Districts. Whiteflies, which are the vector of sweet potato chlorotic stunt crinivirus (SPCSV) a component cause of SPVD, were correspondingly common on sweet potato crops in Mpigi and rare on crops in Tororo. However, aphids, which are the vectors of sweet potato feathery mottle potyvirus (SPFMV), the other component cause of SPVD, were not found colonising sweet potato crops, and itinerant alate aphids may be the means of transmission. Different sweet potato cultivars were predominant in the different districts surveyed and four local cultivars obtained from Kanoni in S. Mpigi, where whiteflies and SPVD were common, were more resistant to SPVD than four cultivars from Busia in Tororo District, where whiteflies and SPVD were rare. However, nationally released cultivars were even more resistant than the local cultivars from Kanoni. Yield results and interviews with farmers indicated that farmers in S. Mpigi were making compromises in their choice of cultivars to grow, some key factors being SPVD susceptibility, and the yield, taste, and marketability, duration of harvest and in-ground storability of the storage roots. These compromises need to be included in an assessment of yield losses attributable to SPVD.     

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.     

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×10⁶ were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation. Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998