Wiley

Bacillus thuringiensis (Bt) là một loại vi khuẩn trong đất hình thành bào tử trong giai đoạn trì trệ của chu trình phát triển của nó. Các bào tử chứa các tinh thể, chủ yếu là một hoặc nhiều protein Cry và/hoặc Cyt (còn được gọi là δ-endotoxin) có hoạt tính diệt côn trùng mạnh và đặc hiệu. Các chủng Bt khác nhau tạo ra các loại độc tố khác nhau, mỗi loại ảnh hưởng đến một nhóm phân loại côn trùng hẹp. Do đó, độc tố Bt đã được sử dụng như thuốc trừ sâu dạng phun bề mặt để bảo vệ cây trồng, và gần đây hơn, các protein đã được biểu hiện trong cây trồng chuyển gen để tạo ra khả năng kháng sâu bệnh tự nhiên. Cây trồng chuyển gen Bt đã đạt được thành công áp đảo và mang lại lợi ích, dẫn đến sản lượng thu hoạch cao hơn và giảm việc sử dụng thuốc trừ sâu hóa học và nhiên liệu hóa thạch. Tuy nhiên, việc triển khai chúng đã thu hút một số chỉ trích, đặc biệt liên quan đến khả năng tiến hóa của các chủng côn trùng kháng thuốc. Ở đây, chúng tôi xem xét tiến bộ gần đây trong phát triển công nghệ Bt và các biện pháp đối phó đã được giới thiệu để ngăn ngừa sự phát triển của quần thể côn trùng kháng thuốc.

WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA‐inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA‐responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR‐protein phosphatase 2C‐ABA complex and the chloroplast envelope–located ABAR–ABA complex. In vivo and in vitro promoter‐binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well‐known ABA‐responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well‐characterized stress‐inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA‐mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA‐responsive signalling networks.

Transcription factors have been shown to control the activity of multiple stress response genes in a coordinated manner and therefore represent attractive targets for application in molecular plant breeding. We investigated the possibility of modulating the transcriptional regulation of drought and cold responses in the agriculturally important species, wheat and barley, with a view to increase drought and frost tolerance. Transgenic wheat and barley plants were generated showing constitutive (double 35S) and drought‐inducible (maizeRab17) expression of theTaDREB2andTaDREB3transcription factors isolated from wheat grain. Transgenic populations with constitutive over‐expression showed slower growth, delayed flowering and lower grain yields relative to the nontransgenic controls. However, both theTaDREB2andTaDREB3transgenic plants showed improved survival under severe drought conditions relative to nontransgenic controls. There were two components to the drought tolerance: real (activation of drought‐stress‐inducible genes) and ‘seeming’ (consumption of less water as a result of smaller size and/or slower growth of transgenics compared to controls). The undesired changes in plant development associated with the ‘seeming’ component of tolerance could be alleviated by using a drought‐inducible promoter. In addition to drought tolerance, bothTaDREB2andTaDREB3transgenic plants with constitutive over‐expression of the transgene showed a significant improvement in frost tolerance. The increased expression ofTaDREB2andTaDREB3lead to elevated expression in the transgenics of 10 otherCBF/DREBgenes and a large number of stress responsiveLEA/COR/DHNgenes known to be responsible for the protection of cell from damage and desiccation under stress.

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR‐associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide‐RNAs (gRNAs) as a DNA‐targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA‐targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9‐based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C‐terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C‐terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. Further, the dCas9:SRDX‐mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9 DNA‐targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.

Oilseed rape (Brassica napus) was selected as an example of a polyploid crop, and the Solexa sequencing system was used to generate approximately 20 million expressed sequence tags (ESTs) from each of two cultivars: Tapidor and Ningyou 7. A methodology and computational tools were developed to exploit, as a reference sequence, a publicly available set of approximately 94 000 Brassica species unigenes. Sequences transcribed in the leaves of juvenile plants were aligned to approximately 26 Mb of the reference sequences. The aligned sequences enabled the detection of 23 330–41 593 putative single nucleotide polymorphisms (SNPs) between the cultivars, depending on the read depth stringency applied. The majority of the detected polymorphisms (87.5–91.2%) were of a type indicative of transcription from homoeologous genes from the two parental genomes within oilseed rape, and are termed here ‘hemi‐SNPs’. The overall estimated polymorphism rate (~0.047%–0.084%) is consistent with that previously observed between the cultivars analysed. To demonstrate the heritability of SNPs and to assess their suitability for applications such as linkage map construction and association genetics, approximately nine million ESTs were generated, using the Solexa system, from each of four lines of a doubled haploid mapping population derived from a cross between Tapidor and Ningyou 7. Computational tools were developed to score the alleles present in these lines for each of the potential SNPs identified between their parents. For a specimen region of the genome analysed in detail, segregation of alleles largely, although not entirely, followed the pattern expected for genomic markers.

The growth and yield of many crops, including cotton, are affected by water deficit. Cotton has evolved drought specific as well as general morpho‐physiological, biochemical and molecular responses to drought stress, which are discussed in this review. The key physiological responses against drought stress in cotton, including stomata closing, root development, cellular adaptations, photosynthesis, abscisic acid (ABA) and jasmonic acid (JA) production and reactive oxygen species (ROS) scavenging, have been identified by researchers. Drought stress induces the expression of stress‐related transcription factors and genes, such as ROS scavenging, ABA or mitogen‐activated protein kinases (MAPK) signalling genes, which activate various drought‐related pathways to induce tolerance in the plant. It is crucial to elucidate and induce drought‐tolerant traits via quantitative trait loci (QTL) analysis, transgenic approaches and exogenous application of substances. The current review article highlights the natural as well as engineered drought tolerance strategies in cotton.

The past couple of decades have witnessed global resurgence of herbal‐based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time‐consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH‐psbA, ITS, trnL‐F, 5S‐rRNA and 18S‐rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next‐generation sequencing and high‐resolution melting curve analysis have paved the way for successful species‐level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode‐based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need‐based transcriptomics and proteomics.

One of the major concerns of the general public about transgenic crops relates to the mixing of genetic materials between species that cannot hybridize by natural means. To meet this concern, the two transformation concepts cisgenesis and intragenesis were developed as alternatives to transgenesis. Both concepts imply that plants must only be transformed with genetic material derived from the species itself or from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector‐backbone sequences should be absent. Intragenesis differs from cisgenesis by allowing use of new gene combinations created by in vitro rearrangements of functional genetic elements. Several surveys show higher public acceptance of intragenic/cisgenic crops compared to transgenic crops. Thus, although the intragenic and cisgenic concepts were introduced internationally only 9 and 7 years ago, several different traits in a variety of crops have currently been modified according to these concepts. Five of these crops are now in field trials and two have pending applications for deregulation. Currently, intragenic/cisgenic plants are regulated as transgenic plants worldwide. However, as the gene pool exploited by intragenesis and cisgenesis are identical to the gene pool available for conventional breeding, less comprehensive regulatory measures are expected. The regulation of intragenic/cisgenic crops is presently under evaluation in the EU and in the US regulators are considering if a subgroup of these crops should be exempted from regulation. It is accordingly possible that the intragenic/cisgenic route will be of major significance for future plant breeding.

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p‐coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross‐linking these components. A controlled reduction of FA level or of FA cross‐linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.

Using plants as production factories for therapeutic proteins requires modification of their N‐glycosylation pattern because of the immunogenicity of plant‐specific sugar residues. In an attempt towards such humanization, we disrupted the genes for α1,3‐fucosyltransferase and β1,2‐xylosyltransferase in Physcomitrella patens by homologous recombination. The single Δfuc‐t and Δxyl‐t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild‐type moss in morphology, growth, development, and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF₁₂₁, into the culture medium. N‐Glycan analysis, however, revealed the absence of 1,3‐fucosyl and/or 1,2‐xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant‐made glycosylated biopharmaceuticals with nonallergenic N‐glycans.