Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.

Phenotypic and genetic dissection of the contents of important metallic elements in hybrid rice grown in cadmium-contaminated paddy fields.

Rice (Oryza sativa L.) is a staple food that feeds over half of the world's population, and the contents of metallic elements in rice grain play important roles in human nutrition. In this study, the contents of important metallic elements were determined by ICP-OES, and included cadmium (Cd), zinc (Zn), manganese (Mn), copper (Cu), iron (Fe), nickel (Ni), calcium (Ca), and magnesium (Mg) in brown rice, in the first node from the top (Node 1), in the second node from the top (Node 2), and in roots of 55 hybrids and their parental lines. The heritability of metallic element contents (MECs), the general combining ability (GCA) for MEC, and the correlation between MECs in different organs/tissues of hybrids were also analyzed. The results indicated that: (1) there was a positive correlation between the contents of Cd and Zn in nodes and roots, but a negative correlation between the contents of Cd and Zn in brown rice of the hybrids(2) the GCA for MECs can be used to evaluate the ability of the parental lines to improve the metal contents in brown rice of the hybrids(3) the contents of Cd, Zn, Ca, and Mg in brown rice were mainly affected by additive genetic effects(4) the restorer lines R2292 and R2265 can be used to cultivate hybrids with high Zn and low Cd contents in the brown rice.

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

Bis(vinylsulfonyl)piperazines as efficient linkers for highly homogeneous antibody-drug conjugates.

Disulfide re-bridging strategy has demonstrated significant advantages in the construction of homogeneous antibody drug conjugates (ADCs). However, a major issue that disulfide scrambling at the hinge region of antibody leads to the formation of "half-antibody" has appeared for many re-bridging linkers. We present bis(vinylsulfonyl)piperazines (BVP) as efficient linkers to selectively re-bridge disulfides at the antigen-binding fragment (Fab) regions and produce highly homogeneous conjugates with a loading of two drugs without disulfide scrambling. We also found that optically active (S)-configuration linkers led to more sufficient conjugation compared with (R)-configuration. The BVP-linked ADCs demonstrated superior efficacy and antigen-selectivity in vitro cytotoxicity.

A novel mass spectrometry-cleavable, phosphate-based enrichable and multi-targeting protein cross-linker.

Chemical cross-linking mass spectrometry (XL-MS) is a powerful technology for obtaining protein structural information and studying protein-protein interactions. We report phospho-bisvinylsulfone (pBVS) as a novel water-soluble, MS-cleavable, phosphate-based enrichable and multi-targeting cross-linker. In this approach, the fragmentation of pBVS cross-linked peptides occurs in situ through retro-Michael addition. The phosphate group is successfully used as a small affinity tag to isolate cross-linked peptides from the highly abundant non-cross-linked peptides. In addition, the linker targets multiple types of amino acid residues, including cysteine, lysine and histidine. This method was applied to cross-link bovine serum albumin (BSA), myoglobin and Lbcpf1 demonstrating the ability to yield accurate and abundant information to facilitate protein structure elucidation.

H2O-Regulated Chemoselectivity in Oxa- Versus Aza-Michael Reactions.

A H2O-regulated chemoselective addition in oxa- and aza-Michael reactions for aminoalcohols and mixtures of structurally similar alcohols and amines was reported. The oxa-Michael reactions might be kinetically controlled, and the reactions to produce O-selective products were slowed by the addition of water. The electrophilicity of Michael acceptors and the steric hindrance of Michael donors also affect the ratios of O/N products. This method offers novel ideas over conventional metal-catalyzed or ligand-induced strategies.

N-Methyl- N-phenylvinylsulfonamides for Cysteine-Selective Conjugation.

Use of N-methyl- N-phenylvinylsulfonamides to perform chemoselective modification of cysteine-containing peptides and proteins is reported. Probes linked to the drug were applicable to prepare antibody-drug conjugates (ADCs). The drug-antibody ratio for ADCs was controlled by rationally tuning the electron deficiency and linker hydrophilicity of the probes.