Three-Dimensional-Percolated Ceramic Nanoparticles along Natural-Cellulose-Derived Hierarchical Networks for High Li+ Conductivity and Mechanical Strength.

Solid polymer electrolytes for safe lithium batteries are in general flexible and easy to process, yet they have limited ionic conductivity and low mechanical strength. Introducing nano/microsized fillers into polymer electrolytes has been proven effective to address these issues, while formation of a percolated network of fillers for efficient Li+ conduction remains challenging. In this work, composite polymer electrolyte with 3D cellulose/ceramic networks is successfully developed using natural cellulose fibers and Li+-conducting ceramic nanoparticles. Monodisperse ceramic nanofillers first form interconnected networks driven by the self-assembly of hybrid cellulose fibers. The hierarchical cellulose skeleton provides spatial guidance for ceramic fillers and firmly supports the whole structure. After polymer electrolyte infusion, the resultant hybrid electrolyte affords both 3D continuous Li+ pathways for high Li+ conductivity and sufficient mechanical strength for dendrite suppression. This cellulose-confined particle percolation approach enables efficient and strong solid electrolytes for lithium batteries.

Precision Imprinted Nanostructural Wood.

Wood is a ubiquitous material, widely used in human society, that features naturally abundant, aligned longitudinal cells (e.g., tracheids in softwood and fibers/vessels in hardwood) with diameters of ≈50-1000 µm. Here, the realization of, fine patterns on a wood surface ranging in size from 40 nm to 50 µm by precision imprinting is described. The precision imprinting is enabled by releasing cellulose fibril aggregates from the bondage of lignin through the delignification process, then imprinting in wet condition and fixing the designed configuration in the dry state. Various precision structures on a wood surface using imprinting technology, including dot arrays, lines, triangular features, and other complex patterns, are successfully demonstrated. Even multiscale structures with nanosized lines on the surface of micrometer hemiballs can be acquired. As a proof of concept, the use of surface-imprinted wood as a microlens array (MLA), which exhibits superior imaging ability and thermal stability even at a high temperature up to 150 °C compared with traditional polystyrene MLA, is demonstrated. This precision imprinted wood may open new possibilities toward environmentally friendly devices and applications in optics, biology, electronics, etc.

Isotropic Paper Directly from Anisotropic Wood: Top-Down Green Transparent Substrate Toward Biodegradable Electronics.

Flexible electronics have found useful applications in both the scientific and industrial communities. However, substrates traditionally used for flexible electronics, such as plastic, cause many environmental issues. Therefore, a transparent substrate made from natural materials provides a promising alternative because it can be degraded in nature. The traditional bottom-up fabrication method for transparent paper is expensive, environmentally unfriendly, and time-consuming. In this work, for the first time, we developed a top-down method to fabricate isotropic, transparent paper directly from anisotropic wood. The top-down method includes two steps: a delignification process to bleach the wood by lignin removal and a pressing process for removing light-reflecting and -scattering sources. The resulting isotropic, transparent paper has high transmittance of about 90% and high haze over 80% and is demonstrated as a nature-disposable substrate for electronic/optical devices. Adjusting the pressing ratio used changes the density of the resulting paper, which tunes the microstructure-related properties of the isotropic, transparent paper. This top-down method is simple, fast, environmentally friendly, and cost-effective, which can greatly promote the development of paper-based green optical and electronic devices.

Transcriptome profiling revealed novel transcriptional regulators in maize responses to Ostrinia furnacalis and jasmonic acid.

Chewing insects cause severe yield losses in crop production worldwide. Crop plants counteract chewing insects by transcriptionally promoting a repertoire of defense gene products that are either toxic to, or attractive to the natural enemies of, pest insects. However, the complexity of the transcriptional reprogramming in plant defense response against chewing insects is still not well understood. In this study, the genome-wide early responses in maize seedlings to Asian corn borer (ACB, Ostrinia furnacalis) and also to jasmonic acid(JA), the pivotal phytohormone controlling plant defense response against herbivory, were transcriptionally profiled by RNA-Seq. Clustering of differentially expressed genes (DEGs) along with functional enrichment analysis revealed important biological processes regulated in response to ACB infestation and/or jasmonic acid. Moreover, DEGs with distinct expression patterns were differentially enriched with diverse families of cis-elements on their promoters. Multiple inventories of differentially expressed transcription factors (DETFs) in each DEG group were also analyzed. A transient expression assay using transfected maize protoplastswas established to examine the potential roles of DETFs in maize defense response and JA signaling, and this was used to show that ZmNAC60, an ACB- and JA-inducible DETF, represented a novel positive regulator of JA and defense pathway genes. This study provided a comprehensive transcriptional picture for the early dynamics of maize defense responses and JA signaling, and the identification of DETFs offered potential targets for further functional genomics investigation of master regulators in maize defense responses against herbivory.

Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system.

In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF) incorporating two report genes, Green Fluorescent Protein (GFP) and ß-glucuronidase (GUS), separated by 2A (or modified 2A), LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP) with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target localization in maize protoplast transformed by LP4/2A polyprotein construct and it implied the LP4/2A linker peptide could alleviate the inhibition of 2A processing by the carboxy-terminal region of upstream protein of 2A when translocated into the ER.

In vitro template-change PCR to create single crossover libraries: a case study with B. thuringiensis Cry2A toxins.

During evolution the creation of single crossover chimeras between duplicated paralogous genes is a known process for increasing diversity. Comparing the properties of homologously recombined chimeras with one or two crossovers is also an efficient strategy for analyzing relationships between sequence variation and function. However, no well-developed in vitro method has been established to create single-crossover libraries. Here we present an in vitro template-change polymerase change reaction that has been developed to enable the production of such libraries. We applied the method to two closely related toxin genes from B. thuringiensis and created chimeras with differing properties that can help us understand how these toxins are able to differentiate between insect species.

[Review of transgenic crop breeding in China].

The development history and fundamental experience of transgenic crops (Genetically modified crops) breeding in China for near 30 years were reviewed. It was illustrated that a scientific research, development and industrialization system of transgenic crops including gene discovery, transformation, variety breeding, commercialization, application and biosafety assessment has been initially established which was few in number in the world. The research innovative capacity of transgenic cotton, rice and corn has been lifted. The research features as well as relative advantages have been initially formed. The problems and challenges of transgenic crop development were discussed. In addition, three suggestions of promoting commercialization, speeding up implementation of the Major National Project of GM Crops, and enhancing science communication were made.

The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles.

Over the past two decades, Zea mays (maize) has been established as a model system for the study of indirect plant defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles, mainly composed of sesquiterpenes, to attract the natural enemies of the herbivores. This is associated with a swift transcriptional induction of terpene synthases such as TPS10; however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure. Here, by exploiting the finding that the maize TPS10 promoter retained its full responsiveness to herbivory in Arabidopsis, we identified the region from -300 to -200 of the TPS10 promoter as both necessary and sufficient for its herbivore inducibility through 5' deletion mapping. A high-throughput screening of an Arabidopsis transcription factor library using this promoter region as the bait identified seven AP2/ERF family transcription factors. Among their close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Meanwhile, EREB58 was also responsive to Jasmonate. In vivo and in vitro assays indicated that EREB58 promotes TPS10 expression by directly binding to the GCC-box within the region from -300 to -200 of the TPS10 promoter. Transgenic maize plants overexpressing EREB58 constitutively over-accumulate TPS10 transcript, and also (E)-ß-farnesene and (E)-α-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles was abolished in EREB58-RNAi transgenic lines. In sum, these results demonstrate that EREB58 is a positive regulator of sesquiterpene production by directly promoting TPS10 expression.

[Optimized condition for protoplast isolation from maize, wheat and rice leaves].

Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100 x g 2 min and the protoplasts yield was 7x106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100 x g 2 min and the protoplasts yield was 6 x 10(6) cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000 x g 2 min and the protoplasts yield was 6x10(6) cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations.

Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis.

In Bacillus thuringiensis, a novel N-acetylmuramoyl-L-alanine amidase gene (named cwlB) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA) formed one transcriptional unit. 5' rapid amplification of cDNA ends (5'-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, P(cwlA), which is located upstream from the cwlA gene and that the transcription start site is a single 5'-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of P(cwlA) was controlled by σ(K). Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis.

Chloroplast-targeted expression of the codon-optimized truncated cry1Ah gene in transgenic tobacco confers a high level of protection against insects.

KEY MESSAGE: The study of insect-resistant transgenic tobacco provides a good foundation for the further application of the cry1Ah gene in other important crops. To improve transgene expression levels and insect resistance, the coding sequence of the novel Bacillus thuringiensis insecticidal gene cry1Ah (truncated cry1Ah) was modified according to the codon bias of the plant by increasing its GC content from the original 37 % to 48, 55, and 63 % (designated m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah, respectively). In addition, the m3-cry1Ah gene was linked with a transit peptide sequence for chloroplast-targeted expression (designated ctp-m3-cry1Ah). Four plant expression vectors were constructed harboring m1-cry1Ah, m2-cry1Ah, m3-cry1Ah, or ctp-m3-cry1Ah. A total of 23 transgenic tobacco lines were produced with the four constructs by Agrobacterium tumefaciens-mediated transformation. PCR, Southern hybridization, quantitative RT-PCR and ELISA indicated that the cry1Ah gene was not only integrated into the tobacco genome, but was also successfully expressed at the mRNA and protein levels. The Cry1Ah protein level in ctp-m3-cry1Ah plants reached 4.42 µg/g fresh weight, which was a 2- to 10-fold increase over the levels observed in m1-cry1Ah, m2-cry1Ah, and m3-cry1Ah plants and resulted in the highest resistance to Helicoverpa armigera based on bioassays. Our results demonstrated that combining the codon optimization of cry1Ah gene with the targeting of Cry1Ah protein to the chloroplasts conferred a high level of protection against insects. The results of our experiments in tobacco, an important model system, provide a good foundation for enhancing the insecticidal efficacy of staple crops.

Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001.

Three cry9 genes, cry9Da4, cry9Eb2, and cry9Ee1, were cloned from Bacillus thuringiensis strain T03B001 using a high-resolution melting analysis method. All three cry9 genes were overexpressed in Escherichia coli Rosetta (DE3), and the expressed products Cry9Eb2 and Cry9Ee1 were shown to be toxic to Plutella xylostella and Ostrinia furnacalis, but not to Helicoverpa armigera or Colaphellus bowringi. The bioassay of Cry9Eb2 and Cry9Ee1 against Cry1Ac-resistant P. xylostella strains indicated that both novel Cry9 toxins exhibited no cross-resistance with Cry1Ac. Cry9Eb2 and Cry9Ee1 can be applied not only for P. xylostella and O. furnacalis control, but also for the Cry1Ac-resistance management of pests.

Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73.

Bacillus thuringiensis is a Gram-positive bacterium that produces intracellular protein crystals toxic to a wide variety of insect larvae. We report the complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 from the Centre OILB (Institut Pasteur, France), which belongs to serotype 3ab and is toxic to lepidopteran larvae.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela.

KEY MESSAGE: Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.

Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation.

The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.

Weak transcription of the cry1Ac gene in nonsporulating Bacillus thuringiensis cells.

The cry1Ac gene of Bacillus thuringiensis subsp. kurstaki HD-73 (B. thuringiensis HD-73) is a typical example of a sporulation-dependent crystal gene and is controlled by sigma E and sigma K during sporulation. To monitor the production and accumulation of Cry1Ac at the cellular level, we developed a green fluorescent protein-based reporter system. The production of Cry1Ac was monitored in spo0A, sigE, and sigK mutants, and these mutants were able to express the Cry1Ac-green fluorescent protein fusion protein. In nonsporulating B. thuringiensis HD-73 cells, low-level expression of cry1Ac was also observed. Reverse transcription-PCR and Western blotting results confirmed that the cry1Ac promoter has low activity in nonsporulating B. thuringiensis cells. A beta-galactosidase assay demonstrated that the transcription of the cry1Ac gene during exponential and transition phases is positively regulated by Spo0A. Additional bioassay results indicated that spo0A and sigE mutants containing the cry1Ac-gfp fusion exhibited insecticidal activity against Plutella xylostella larvae.

A multicomponent sugar phosphate sensor system specifically induced in Bacillus cereus during infection of the insect gut.

Using a previously developed Bacillus cereus in vivo expression technology (IVET) promoter trap system, we showed that spsA, a gene of unknown function, was specifically expressed in the larval gut during infection. Search for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) as an activation signal. Analysis of the spsA-related 5-gene cluster indicated that SpsA is part of a new sugar phosphate sensor system composed of a 2-component system (TCS) encoded by spsR and spsK, and 2 additional downstream genes, spsB and spsC. In B. cereus, American Type Culture Collection (ATCC) 14579, spsRK, and spsABC are separate transcriptional units, of which only spsABC was activated by extracellular G6P. lacZ transcriptional fusions tested in mutant and complemented strains showed that SpsRK, SpsA, and SpsB are essential for the transcription of spsABC. Deletion mutant analysis showed that SpsC is essential for the G6P uptake. gfp-transcriptional fusions showed that these genes are required for host-activated expression, as well. This sugar phosphate sensor and transport system is found in pathogenic Bacillus group and Clostridia bacteria and may be important for host adaptation. Our findings provide new insights into the function of 2-component sensor systems in host-pathogen interactions, specifically in the gut.

Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor.

Bacillus thuringiensis Cry8Ea toxin is specifically toxic to larvae of the Asian cockchafer, Holotrichia parallela. Here we investigated the mechanism of transcriptional regulation of the cry8Ea1 gene. Reverse transcription-PCR (RT-PCR) results indicated that cry8Ea1 and an upstream gene (orf1) were cotranscribed. Transcriptional fusions with the lacZ gene demonstrated that transcription of the cry8Ea1 gene started from two promoters: P(orf1), which is located upstream of the orf1 gene, and P(cry8E), located in the intergenic region mapping between orf1 and cry8Ea1. Of the known, similar orf1-cry operons, this is the first report of the existence of a promoter in the intergenic region between the orf1 and cry genes. The transcriptional activity of P(orf1) was found during sporulation in B. thuringiensis subsp. kurstaki HD-73 and was almost abolished in the sigE mutant, while the transcriptional activity of P(cry8E) was detected after the end of the exponential phase in HD-73 and was considerably lower in the sigH mutant. The transcription start sites generated by the two cry8Ea1 promoters were determined by the 5' -SMARTer rapid amplification of cDNA ends (RACE) method. The -35 and -10 regions of P(orf1) and P(cry8E) showed high sequence similarity with the σ(E) and σ(H) promoters, respectively. These results indicated that P(orf1) is controlled by the σ(E) factor and P(cry8E) by the σ(H) factor.

Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis.

In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction-high resolution melt (PCR-HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR-HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.