Transgenic Soybean for Production of Thermostable α-Amylase.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'.

The Bethylidae are the most diverse of Hymenoptera chrysidoid families. As external parasitoids, the bethylids have been widely adopted as biocontrol agents to control insect pests worldwide. Thus far, the genomic information of the family Bethylidae has not been reported yet. In this study, we crystallized into a high-quality chromosome-level genome of ant-like bethylid wasps Sclerodermus sp. 'alternatusi' (Hymenoptera: Bethylidae) using PacBio sequencing as well as Hi-C technology. The assembled S. alternatusi genome was 162.30 Mb in size with a contig N50 size of 3.83 Mb and scaffold N50 size of 11.10 Mb. Totally, 92.85% assembled sequences anchored to 15 pseudo-chromosomes. A total of 10,204 protein-coding genes were annotated, and 23.01 Mb repetitive sequences occupying 14.17% of genome were pinpointed. The BUSCO results showed that 97.9% of the complete core Insecta genes were identified in the genome, while 97.1% in the gene sets. The high-quality genome of S. alternatusi will not only provide valuable genomic information, but also show insights into parasitoid wasp evolution and bio-control application in future studies.

Diurnal variation in the urban thermal environment and its relationship to human activities in China: a Tencent location-based service geographic big data perspective.

The main factor of the formation and deterioration in China's urban thermal environment is human activity, which is difficult to describe and measure. A new perspective on the effect of human activity on the urban thermal environment can be obtained by examining the interaction between location-based service (LBS) data and the urban thermal environment in China. However, relevant research is still limited. In this study, we used Tencent LBS data, Terra/Aqua MODIS land surface temperature (LST) data, and land use data to investigate the relationship between LBS and the urban thermal environment, specifically the LST and surface urban heat island intensity (SUHII) across China and its provinces. Our results showed that (1) in summer, the heat island effect was an issue in 94% of the urban areas in China, which was worse during the day. The high- and low-value periods of LBS data on a given day coincided with the acquisition times of MODIS LST products during the day and at night, respectively. (2) During both the day and at night, there was a significant connection between LBS data and the urban thermal environment in China. The highest correlation coefficient (r) between LBS data and the LST could reach 0.55 (p < 0.01) at the provincial level, and the highest correlation coefficient (r) between LBS data and the SUHII could reach 0.78 (p < 0.01) at the provincial level. (3) The urban thermal environment diurnal difference and LBS data exhibited a significant relationship. The ΔLBS diurnal differences were significantly positively related to the SUHII diurnal differences in China. The overall study findings revealed that LBS data constitute an important parameter to represent the human activity intensity when investigating the formation of the urban thermal environment in China.

Analysis of coastline changes under the impact of human activities during 1985-2020 in Tianjin, China.

The accurate evaluation of shoreline movement is a crucial aspect for managing highly dynamic coasts. This study employed Landsat TM and OLI data through the Digital Shoreline Analysis System model to quantify changes in the spatiotemporal distribution of Tianjin's coastline from 1985 to 2020. The results showed that the coastline length (CL) increased by 178 km and 151% over the past 36 years, with an average increase of 5.1 km/a. Accretion and erosion processes along the entire coast were observed at rates of 83.9% and 16.1%, respectively. Notably, the Tianjin Port Area and Nangang Industrial Zone showed remarkable changes in the shoreline in 2009. Night lights (NL) were used to display the intensity of human activity in this area, and the spatial heterogeneity of night light intensity was significant. Compared to the total night light (TNL) in 1985, it increased by 116% in 2020. The relationship between TNL and CL was then established and displayed a significant positive correlation (r = 0.91). With the increasing total night light, the growth of the CL presented changes with an initial slow increase, then rapid increase, and finally slow increase. In the second phase of TNL, the CL experienced a considerable increase due to anthropogenic activities such as land reclamation and port construction, fueled primarily by government policies during the period of 2005-2013. Subsequently, there was little change in the coastline. These findings provide valuable insights into spatiotemporal coastline monitoring programs and sustainable coastal management.

Chromosome-level Genome Assembly and Sex-specific Differential Transcriptome of the White-backed Planthopper, Sogatella furcifera.

Background: The white-backed planthopper (WBPH), Sogatella furcifera, causes great damage to many crops (mainly rice) by direct feeding or transmitting plant viruses. The previous genome assembly was generated by second-generation sequencing technologies, with a contig N50 of only 51.5 kb, and contained a lot of heterozygous sequences. Methods: We utilized third-generation sequencing technologies and Hi-C data to generate a high-quality chromosome-level assembly. We also provide a large amount of transcriptome data for full-length transcriptome analysis and gender differential expression analysis. Results: The final assembly comprised 56.38 Mb, with a contig N50 of 2.20 Mb and a scaffold N50 of 45.25 Mb. Fourteen autosomes and one X chromosome were identified. More than 99.5% of the assembled bases located on the 15 chromosomes. 95.9% of the complete BUSCO Hemiptera genes were detected in the final assembly and 16,880 genes were annotated. 722 genes were relatively highly expressed in males, while 60 in the females. Conclusion: The integrated genome, definite sex chromosomes, comprehensive transcriptome profiles, high efficiency of RNA interference and short life cycle substantially made WBPH an efficient research object for functional genomics.

Expression of a Cytochrome P450 Gene from Bermuda Grass Cynodon dactylon in Soybean Confers Tolerance to Multiple Herbicides.

Bermuda grass (Cynodon dactylon) is notoriously difficult to control with some commonly used herbicides. We cloned a cytochrome P450 gene from Bermuda grass, named P450-N-Z1, which was found to confer tolerance to multiple herbicides in transgenic Arabidopsis. These herbicides include: (1) acetolactate synthase (ALS) inhibitor herbicides nicosulfuron and penoxsulam; (2) p-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide mesotrione; (3) synthetic auxin herbicide dicamba; (4) photosynthesis inhibitor bentazon. We further generated transgenic soybean plants expressing P450-N-Z1, and found that these transgenic soybean plants gained robust tolerance to nicosulfuron, flazasulfuron, and 2,4-dichlorophenoxyacetic acid (2,4-D) in greenhouse assays. A field trial demonstrated that transgenic soybean is tolerant to flazasulfuron and 2,4-D at 4-fold and 2-fold the recommended rates, respectively. Furthermore, we also demonstrated that flazasulfuron and dicamba are much more rapidly degraded in vivo in the transgenic soybean than in non-transgenic soybean. Therefore, P450-N-Z1 may be utilized for engineering transgenic crops for herbicide tolerance.

A comparative study of land development patterns and regional thermal environments (RTEs) in typical urban agglomerations of China and America: A case study of Beijing-Tianjin-Hebei (BTH) and Boswash.

Currently, most regional thermal environment (RTE) studies in urban agglomerations focus on developing countries, especially China. However, there is still a lack of comparative studies on the RTEs of urban agglomerations between China and other developed countries, such as the United States. This paper used the Beijing-Tianjin-Hebei (BTH) agglomeration in China and Boswash (the highly urbanized area extending from Boston to Washington) in the United States as examples to investigate the differences in land development patterns, RTEs and their relationship between the agglomerations of China and America. The results showed that the land development patterns of BTH and Boswash were different, as evidenced by the spatial pattern of land development intensity (LDI) and impervious surface configuration. In terms of the RTE, the sub-high land surface temperature (LST) zones were aggregated in a large and compact patch in central and northern BTH. However, the sub-high zones of the cities in Boswash were relatively separate. Moreover, the land development pattern of Boswash showed a stronger relationship with the RTE than that of BTH did. Global Moran's I between the LDI and LST in Boswash was higher than that in BTH. In addition, the correlation between impervious surface configuration and LST in Boswash was stronger than that in BTH, and this correlation was more controlled by LDI in Boswash. This study also indicated that BTH should change the land development pattern to prevent the further expansion of aggregated sub-high LST zones and control the proximity of high LST zones in cities in central and southern BTH, however, Boswash should adopt some local heat management approaches (installing cool and green roofs and creating more green space) in the core areas to help reduce the very high temperatures in the already highly developed areas where the largest fraction of people live.

The Genetic Network of Forkhead Gene Family in Development of Brown Planthoppers.

We identified 18 distinct Fox genes in the genome of the brown planthopper, Nilaparvata lugens, and further found a novel insect-specific subfamily that we temporarily named FoxT. A total of 16 genes were highly expressed in the eggs, while NlFoxL2 and NlFoxT are female- and male-specific genes, respectively. Large scale RNAi and RNA-seq analyses were used to reveal the functions and potential targets of NlFoxs. In the eggs, NlFoxA, NlFoxN1 and NlFoxN2 are indispensable to early embryogenesis by regulating different target genes; NlFoxG and NlFoxQ co-regulate NlSix3 for brain development; and NlFoxC, NlFoxJ1 and NlFoxP have complementary effects on late embryogenesis. Moreover, NlFoxA, NlFoxNl and NlFoxQ have pleiotropism. NlFoxA and NlFoxQ regulate the expression of NlCHS1 and cuticular proteins, respectively, thereby participating in the formation of cuticles. NlFoxN1, which regulates the expression of NlKrt9 is involved in the formation of intermediate filament frameworks. Our previous studies have revealed that NlFoxL2 and NlFoxO play important roles in chorion formation and wing polyphenism. Altogether, N. lugens Fox genes exhibit functional diversity in embryonic development and organogenesis. This comprehensive study combines genomics, transcriptomics and phenomics, thereby constructing a complex genetic network that spans the entire life cycle of the brown planthopper.

An analysis of the domestic resumption of social production and life under the COVID-19 epidemic.

Population migration and urban traffic are two important aspects of the socioeconomic system. We analyze the trends of social production and resumption of life after the coronavirus disease 2019 (COVID-19)-influenced Spring Festival in 2020 with statistics on reported cases of COVID-19 from China's National Health Commission and big data from Baidu Migration (a platform collecting population migration data). We find that (1) the distribution of COVID-19 cases throughout mainland China has a specific spatial pattern. Provinces in eastern China have more reported cases than those in western China, and provinces adjacent to Hubei have more confirmed COVID-19 cases than nonadjacent provinces. Densely populated regions with well-developed economies and transportation are more likely to have cluster infection incidents. (2) The COVID-19 epidemic severely impacts the return of the migrant population in the Spring Festival travel rush, as demonstrated by the significant reduction in the return scale, along with the extended timespan and uncertainty regarding the end of the travel rush. Among 33 provinces, special administrative regions, autonomous regions and municipalities, 23 of them (approximately 70%) have a return rate below 60%. Hubei, Hong Kong, Xinjiang, and Inner Mongolia have the lowest return rates (below 5%), whereas the return rates in Hainan and Shandong, 272.72% and 97.35%, respectively, indicate the best trend of resumption. Due to government regulations, the population return in densely populated and well-developed regions shows a positive trend. (3) The resumption of urban traffic is slow and varies greatly in different regions. The urban traffic conditions in 22 provinces and municipalities have a more than 60% level of resumption. Guizhou and Yunnan have the highest level of resumption of urban traffic, whereas Xinjiang, Hubei, and Heilongjiang have the lowest (29.37%, 35.76%, and 37.90%, respectively). However, provinces and municipalities with well-developed intercity traffic have a lower level of resumption, mainly because of regulatory methods such as lockdowns and traffic restrictions. The increased public awareness of epidemic prevention and the decreased frequency of outdoor activities are also two positive factors slowing the spread of the epidemic. (4) Time will be necessary to fully resume social production and life throughout China. Xining and Jinan have the highest levels of resumption, 82.14% and 71.51%, respectively. Urumqi and Wuhan are the cities with the lowest levels of resumption, only 0.11% and 0.61%, respectively. Currently, 12 of 33 provinces and municipalities have levels of resumption of more than 80%; among them, Guizhou, Yunnan, and Gansu have with the highest levels of resumption and have nearly resumed the 2019 levels of work and life, whereas Xinjiang and Hubei have the lowest resumption rates, only 0.09% and 7.57%, respectively. Thus, relevant government departments should focus more on densely populated and well-developed provinces and cities when applying epidemic prevention and work resumption methods. We reveal the general conditions of the epidemic and the population return scale across China, along with urban traffic conditions and the resumption of social production and life under COVID-19, providing a scientific basis for local governments to make further decisions on work resumption.

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity.

BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters. RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain. CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

Phytase is one of the most effective feed additives to increase the availability of phosphorus and minerals by catalyzing the hydrolysis of phytic acid. A modified appA gene (mappA) was transformed into soybean (Glycine max) under the control of a seed-specific promoter from common bean (Phaselous vulgaris). The soybean recombinant phytase showed optimal activity at pH 4.5 and 70 °C. A slight increase in enzyme activity occurred when the recombinant enzyme was pre-incubated with n-hexane. In addition, the phytase activity from our transgenic soybean does not reduce even after 2 hours of extraction with n-hexane at 55~65 °C. In conclusion, the oil extraction process using n-hexane does not inactivate the phytase expressed in the mAppA transgenic soybean, and the meal derived from the transgenic soybean processing can be used as feed supplement to livestock.

Characterization of transgenic rice expressing fusion protein Cry1Ab/Vip3A for insect resistance.

Management of resistance development of insect pests is of great importance for continued utilization of Bt crop. The high-dose/refuge and pyramid (gene stacking) strategy are commonly employed to delay the evolution of insect resistance. Due to the anticipated difficulty for deployment of mandatory refuge for transgenic crops in China, where the size of farmer is quite small, stacking of genes with different modes of action is a more feasible strategy. Here we report the development of transgenic rice expressing a fusion protein of Cry1Ab and Vip3A toxin. Analysis of trypsin proteolysis suggested that the fusion protein is equivalent to the combination of Cry1Ab and Vip3A protein. The transgenic plants expressing the fusion protein were found to be highly resistant to two major rice pests, Asiatic rice borer Chilo suppressalis (Lepidoptera: Crambidae) and rice leaf folder Cnaphalocrocis medinalis (Lepidoptera: Crambidae), while their agronomic performances showed no significant difference compared to the non-transgenic recipient rice. Therefore, the transgenic rice may be utilized for rice pest control in China.

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance.

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.

Effects of Vip3AcAa+Cry1Ac Cotton on Midgut Tissue in Helicoverpa armigera (Lepidoptera: Noctuidae).

To determine cellular changes caused by the chimeric protein Vip3AcAa against Helicoverpa armigera, we used transmission electron microscopy to examine ultrastructural changes in midgut cells of third-instar larvae of Cry1Ac-susceptible H. armigera after feeding on an artificial diet containing the Vip3AcAa toxin. Midgut epithelial cells of Cry1Ac-resistant H. armigera larvae that had fed on an artificial diet containing Vip3AcAa or on Bt cotton expressing Vip3AcAa+Cry1Ac were also examined using optical microscopy and hematoxylin-eosin staining. In the midgut cells of H. armigera larvae fed with Vip3AcAa, microvilli were swollen and broken; inner cristae of the mitochondria were indistinct and vacuolated; endoplasmic reticulum was swollen, fractured, and disordered; boundaries of karyotheca in the nucleus were indistinct and chromatin underwent pyknosis and was pressed close to the karyotheca. Histopathological changes and the time of onset in midgut tissues of H. armigera larvae fed on Vip3AcAa or Cry1Ac were similar. Vip3AcAa and transgenic cotton expressing Vip3AcAa+Cry1Ac caused the goblet cell cavity and microvilli pathological changes in the midgut epithelial cells of the Cry1Ac-susceptible and Cry1Ac-resistant H. armigera larvae that eventually killed the larvae.

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

Bitrophic and Tritrophic Effects of Transgenic cry1Ab/cry2Aj Maize on the Beneficial, Nontarget Harmonia axyridis (Coleoptera: Coccinellidae).

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China.

Characterization of Xylanase and Cellulase Produced by a Newly Isolated Aspergillus fumigatus N2 and Its Efficient Saccharification of Barley Straw.

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9 U/mL) and CMCase (5.61 U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65 °C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65 °C. The FPase was optimally active at pH 5.0 and 60 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.

Effects of Transgenic Bt Rice on Nontarget Rhopalosiphum maidis (Homoptera: Aphididae).

The effects of three transgenic Bacillus thuringiensis (Bt) rice lines, KMD1, KMD2, and G8-7, on biological parameters and population dynamics of nontarget insect, Rhopalosiphum maidis (Fitch) (Homoptera: Aphididae), were investigated in the laboratory and field. No significant differences were found between Bt and non-Bt rice lines for aphid survival. The developmental time of R. maidis that fed on KMD1 and KMD2 did not differ significantly from those of the individuals feeding on the parental variety Xiushui11, but significantly prolonged developmental time was observed on G8-7 as compared with its parental variety Xiushui110. Aphid fecundity was significantly higher on Bt than on parental rice. A 2-yr field survey indicated that Bt rice did not significantly affect the population dynamics of R. maidis in comparison with non-Bt rice. Additionally, guttation droplets of Bt rice and aphids feeding on Bt rice were analyzed for presence of Cry1Ab using ELISA. No Cry1Ab protein was found in aphid adults feeding on Bt rice lines both in the laboratory and field. By using the guttation droplets from the top of rice seedlings, we designed a novel method to collect phloem sap, and found that relatively low concentrations were detected in the guttation droplets from Bt rice lines. In conclusion, although the Bt rice lines tested in this study stimulate the fecundity of R. maidis, the aphid population density did not increase in Bt rice fields.

Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment.

Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co(2+), increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase.