Extreme Fuzzy Broad Learning System: Algorithm, Frequency Principle, and Applications in Classification and Regression.

As an effective alternative to deep neural networks, broad learning system (BLS) has attracted more attention due to its efficient and outstanding performance and shorter training process in classification and regression tasks. Nevertheless, the performance of BLS will not continue to increase, but even decrease, as the number of nodes reaches the saturation point and continues to increase. In addition, the previous research on neural networks usually ignored the reason for the good generalization of neural networks. To solve these problems, this article first proposes the Extreme Fuzzy BLS (E-FBLS), a novel cascaded fuzzy BLS, in which multiple fuzzy BLS blocks are grouped or cascaded together. Moreover, the original data is input to each FBLS block rather than the previous blocks. In addition, we use residual learning to illustrate the effectiveness of E-FBLS. From the frequency domain perspective, we also discover the existence of the frequency principle in E-FBLS, which can provide good interpretability for the generalization of the neural network. Experimental results on classical classification and regression datasets show that the accuracy of the proposed E-FBLS is superior to traditional BLS in handling classification and regression tasks. The accuracy improves when the number of blocks increases to some extent. Moreover, we verify the frequency principle of E-FBLS that E-FBLS can obtain the low-frequency components quickly, while the high-frequency components are gradually adjusted as the number of FBLS blocks increases.

PRMD: an integrated database for plant RNA modifications.

The scope and function of RNA modifications in model plant systems have been extensively studied, resulting in the identification of an increasing number of novel RNA modifications in recent years. Researchers have gradually revealed that RNA modifications, especially N6-methyladenosine (m6A), which is one of the most abundant and commonly studied RNA modifications in plants, have important roles in physiological and pathological processes. These modifications alter the structure of RNA, which affects its molecular complementarity and binding to specific proteins, thereby resulting in various of physiological effects. The increasing interest in plant RNA modifications has necessitated research into RNA modifications and associated datasets. However, there is a lack of a convenient and integrated database with comprehensive annotations and intuitive visualization of plant RNA modifications. Here, we developed the Plant RNA Modification Database (PRMD; http://bioinformatics.sc.cn/PRMD and http://rnainformatics.org.cn/PRMD) to facilitate RNA modification research. This database contains information regarding 20 plant species and provides an intuitive interface for displaying information. Moreover, PRMD offers multiple tools, including RMlevelDiff, RMplantVar, RNAmodNet and Blast (for functional analyses), and mRNAbrowse, RNAlollipop, JBrowse and Integrative Genomics Viewer (for displaying data). Furthermore, PRMD is freely available, making it useful for the rapid development and promotion of research on plant RNA modifications.

Genome editing for plant synthetic metabolic engineering and developmental regulation.

Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.

Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation.

Plant height is an important agronomic trait for lodging resistance and yield. Here, we report a new plant-height-related gene, OsUBR7 in rice (Oryza sativa L.); knockout of OsUBR7 caused fewer cells in internodes, resulting in a semi-dwarf phenotype. OsUBR7 encodes a putative E3 ligase containing a plant homeodomain finger and a ubiquitin protein ligase E3 component N-recognin 7 (UBR7) domain. OsUBR7 interacts with histones and monoubiquitinates H2B (H2Bub1) at lysine148 in coordination with the E2 conjugase OsUBC18. OsUBR7 mediates H2Bub1 at a number of chromatin loci for the normal expression of target genes, including cell-cycle-related and pleiotropic genes, consistent with the observation that cell-cycle progression was suppressed in the osubr7 mutant owing to reductions in H2Bub1 and expression levels at these loci. The genetic divergence of OsUBR7 alleles among japonica and indica cultivars affects their transcriptional activity, and these alleles may have undergone selection during rice domestication. Overall, our results reveal a novel mechanism that mediates H2Bub1 in plants, and UBR7 orthologs could be utilized as an untapped epigenetic resource for crop improvement.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Molecular farming using transgenic rice endosperm.

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences.

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5' tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Oxidatively Induced Selective Carbon-Carbon Bond Formation From Isolated Rhodium(III) Complexes.

This work focuses on oxidatively induced regioselective intramolecular C-C bond formations based on the RhIII complexes synthesized from dirhodium(II) trifluoroacetate with 2-arylpyridines. With the selection of electron-donating groups on the arene rings of 2-arylpyridines, the unusual meta-ortho C-C bond-forming was favored, which led to the formation of meta-substituted 2-arylpyridine homocoupling dimers. On the contrary, the electron-withdrawing groups have tendency to occur conventional ortho-ortho bond-forming, resulting in the formation of new RhIII complexes possessing the intriguing RhIII (TFA)3 fragment. Preliminary mechanistic experiments suggest that the sequential oxidation of RhIII occurred in the reaction.

Late-Stage Macrocyclization of Bioactive Peptides with Internal Oxazole Motifs via Palladium-Catalyzed C-H Olefination.

Oxazole is an important pharmacophore and exists in the backbone of many bioactive peptide natural products and peptidomimetics. Efficient methods for the synthesis and direct functionalization of complex oxazole-containing peptides are in high demand. Herein, we report the late-stage site-selective functionalization of oxazole-containing peptides via palladium-catalyzed δ-C(sp2)-H olefination of phenylalanine, tryptophan, and tyrosine residues. This strategy utilizes oxazole motifs as internal directing groups and provides access to oxazole-containing peptide macrocycles with bioactivities.

Prospects and progress on crocin biosynthetic pathway and metabolic engineering.

Crocins are a group of highly valuable apocarotenoid-derived pigments mainly produced in Crocus sativus stigmas and Gardenia jasminoides fruits, which display great pharmacological activities for human health, such as anticancer, reducing the risk of atherosclerosis, and preventing Alzheimer's disease. However, traditional sources of crocins are no longer sufficient to meet current demands. The recent clarification of the crocin biosynthetic pathway opens up the possibility of large-scale production of crocins by synthetic metabolic engineering methods. In this review, we mainly introduce the crocin biosynthetic pathway, subcellular route, related key enzymes, and its synthetic metabolic engineering, as well as its challenges and prospects, with a view to providing useful references for further studies on the synthetic metabolic engineering of crocins.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality.

Nutrient deficiencies in crops are a serious threat to human health, especially for populations in poor areas. To overcome this problem, the development of crops with nutrient-enhanced traits is imperative. Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components. Compared with agronomic practices and conventional plant breeding, plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients, phytonutrients, and/or bioactive components in crops. In this review, we discuss recent progress in the field of plant synthetic metabolic engineering, specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways, with a focus on improving traits related to micronutrients, phytonutrients, and bioactive components. Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.