Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus

Rice ragged stunt disease, caused by rice ragged stuntoryzavirus (RRSV), was first discovered in 1976–1977 inIndonesia and Philippines [1]. Subsequently the diseasewas found in most rice-growing countries in south-easternand far-eastern Asia [2] and may inflict heavy loss on thecrop. RRSV is the type species of the genus Oryzavirus in thefamily Reoviridae. The virus particle is icosahedral witha diameter of about 65–70 nm and the genome consistsof 10 double stranded RNA (dsRNA) segm…     

Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

Cryopreservation of monogonont rotifers

Development of techniques to maintain viable rotifer clones in a frozen state would preserve the genotype and reduce routine maintenance for those clones not being actively studied. To this end we have frozen Brachionus plicatilis in dimethyl sulfoxide at concentrations ranging from 6% to 18%. Survival rates decreased as the endpoint temperature was reduced from ?20 °C to ?45 °C, but did not decrease when the temperature was further reduced to ?196 °C (liquid nitrogen). Only 2% of the individuals survived freezing in liquid nitrogen.     

来源于葡萄球菌的N-乙酰神经氨酸裂合酶的基因克隆及性质

葡萄球菌Staphylococcus hominis来源的N-乙酰神经氨酸裂合酶基因shnal(GenBank Accession No.EFS20452.1)构建至pET-28a质粒并在大肠杆菌中得到表达.通过目的蛋白的纯化和酶学性质研究发现,ShNAL是一个四聚体,裂解方向的最适反应pH为8.0;合成方向的最适反应pH为7.5,最适反应温度为45℃.在45℃下孵育2h对ShNAL的活力基本无影响,高于45℃时,活力迅速下降.该酶在pH 5.0～10.0的环境中比较稳定,4℃下放置24 h酶的残余活力在70％以上.ShNAL对N-乙酰神经氨酸(Neu5Ac)、N-乙酰甘露糖胺(Man)和丙酮酸(Pyr)的Km值分别是(4.0±0.2) mmol/L、(131.7±12.1)mmol/L和(35.14±3.2) mmol/L,kcat/Km值分别为1.9 L/(mmol·s)、0.08 L/(mmol·s)和0.08 L/(mmol·s).     

Low temperature acclimation to chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling temperature tolerance of rice roots was improved with increasing duration of LT-PT, but LT-PT longer than 12 h gave no additional improvement. Alcohol dehydrogenase activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h. Chilling tolerance was also improved by exogenously applied ethanol. These results suggest that LT-PT may increase chilling tolerance in rice roots owing to ethanol accumulation in the roots and LT-PT acclimation to chilling temperature may occur within 12 h.     

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and ?20?°C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18?kDa, as observed on SDS?CPAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at ?20?°C.     

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20?^°C, ～85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15?mg which was ～3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37?^°C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.     

Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni²⁺-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.     

The effect of temperature on the growth,development and survival of Macropetasma africanus (Balss) (Penaeoidea:Penaeidae) larvae reared in the laboratory

Macropetasma africanus (Balss) has been successfully spawned and its larvae reared under controlled laboratory conditions. The relationship between egg number (E) and female total length (L) was E = 18.59 L^2.11. An experiment was designed to test the effect of temperature on larval development, survival and growth. Temperature effected larval development time, from 13–15 days at 25°C, to 25 days at 15°C (nauplius 1 to post-larva). Mortality was low for the naupliar stages at 25, 22 and 18°C, while at 15°C only 52% of the larvae reached nauplius 6. Mortality was highest from nauplius 6 to protozoea 1 (17, 21, and 18% at 25, 22, and 18°C, respectively), but decreased considerably for all temperatures once the mysis stage was reached. Overall survival rates from nauplius 1 to post-larva decreased with decreasing temperature (65, 54, 48, and 39% at 25, 22, 18, and 15°C respectively). Temperature also significantly affected larval growth. At 25°C mean total length was significantly (P < 0.05) larger than at 15°C (protozoea 2 to post-larva), while from protozoea 3 to post-larva total length differences were significantly different (P < 0.05) between 18 and 25°C. M. africanus has a major spawning peak in summer, suggesting that there may be a selective advantage to reproducing during the warmer months.     

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between ?12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (?12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (?12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C–40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.     

Temperature-dependent development and oviposition models of Leptocorisa chinensis Dallas (Hemiptera: Alydidae)

Leptocorisa chinensis Dallas (Hemiptera: Alydidae) is known to cause pecky rice by sucking panicles of milk stage of rice. Based on its continuous spread and expanded damage area in Asian countries such as Japan with high reproduction potential, it is highly likely that L. chinensis will become an important rice pest in the near future. However, limited information is available to predict its distribution and occurrence. Thus, the objective of this study was to develop models for their development and oviposition. We investigated the development of L. chinensis immatures (from egg to adult) at 11 constant temperatures ranging from 16.2 to 35.3 °C and the oviposition of female adults at five constant temperatures ranging from 22.3 to 35.3 °C in this study. For L. chinensis immatures, the lower developmental threshold temperature, optimal developmental temperature, upper developmental threshold temperature were 12.7, 32.3, and 37.6 °C, respectively. The highest survival rate of immatures was observed at temperature of 25.2 °C and the highest mean total fecundity was 585.8 at 28.0 °C. This study provides basic information for the ecology of L. chinensis. It is applicable to forecast the phenology of its populations in the fields and to predict its future distribution under global warming.     

Stage-specific effects of temperature and dietary protein on growth and survival of Manduca sexta caterpillars

The effects of temperature and dietary protein concentration on growth and survival of Manduca sexta L. (Lepidoptera: Sphingidae) caterpillars during different larval stages were examined. Sets of caterpillars were raised from hatching at one of five constant temperatures (18, 22, 26, 30 or 34°C) and on one of two artificial diets (low or high protein concentration). Mass gain, duration (development time) and mean growth rate were measured for each caterpillar for the 1st to 3rd stadia, the 4th stadium, and the 5th stadium. Temperature significantly affected mass gain during each larval stage, resulting in smaller mass gains at higher temperatures at each stage. This effect was strongest at high temperatures during the 5th stadium. Temperature significantly affected durations of each larval stage, but the effect varied among stages: for example, the duration of stadia 1–3 decreased continuously with increasing temperature, whereas the duration of the 5th stadium was shortest at 26–30°C and increased at lower and higher temperatures. The effect of temperature on mean growth rate changed dramatically across larval stages: maximal growth rate occurred at 34°C during the 1st to 3rd stadia, at 30°C during the 4th stadium and at 26°C during the 5th stadium. Higher dietary protein concentration significantly decreased the duration of stadia 1–3 and of the 4th stadium, but had no significant effect on the duration of the 5th stadium. Temperature and dietary protein had little effect on mortality rates during any larval stadium, with one exception: mortality during the 5th stadium increased dramatically at temperatures of 30 and 34°C. These results demonstrate that the effects of temperature and dietary protein concentration on growth, development and survival in M. sexta vary markedly in different larval stadia during development; 5th instar caterpillars are particularly sensitive to higher temperatures.     

Environmental modification of nest-building in the white-footed mouse, Peromyscus leucopus

Exposure of Peromyscus leucopus to low ambient temperature (5°C versus 26°C) during a 5-day test resulted in the building of larger nests. The weight of cotton used by the animal was employed as an index of nest size. Animals which had been acclimated to 5°C for 6 weeks prior to testing built larger nests at 5°C and smaller nests at 26°C than did warm-acclimated mice. In addition, warmacclimated P. leucopus maintained for 6 weeks under short photoperiod (LD9:15; L=light, D=dark) built larger nests at both 5°C and 26°C than did animals maintained under long photoperiod (LD 16:8). This pattern of response to environmental conditions approximating winter (low ambient temperature, short photoperiod) indicates that nesting is a component of the physiological-behavioural complex of cold adaptation.     

Involvement of nitric oxide in acquired thermotolerance of rice seedlings

The role of nitric oxide (NO) in thermotolerance acquired by heat acclimation (38°C) was investigated. Results showed that 38°C acclimation, on the one hand, obviously reduced hydrogen peroxide (H₂O₂) and MDA contents and ion leakage degree in rice leaves; however, on the other hand, it increased the survival of rice (Oryza sativa L.) seedlings under 50°C heat stress. Application of nitric oxide donor, sodium nitroprusside (SNP), prior to 38°C acclimation dramatically increased the acquired thermotolerance. To elucidate the role of endogenous NO in acquired thermotolerance, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a specific NO scavenger) was used (scavengers are used to control the level of both exogenous and endogenous NO). Results showed that PTIO pretreatment resulted in the elimination of acquired thermotolerance induced by 38°C acclimation in rice seedlings. Nitric oxide (NO) release measurement indicated that there was indeed an abrupt elevation in the NO content in 40 min after 38°C acclimation, proving the involvement of NO in acquired thermotolerance inducement in rice seedling.