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Ma X.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | Ma X.,South China Agricultural University | Zhang Q.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | Zhang Q.,Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions | And 49 more authors.
Molecular Plant | Year: 2015

Abstract CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. © 2015 The Author. Source


Chen L.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | Chen L.,Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms | Chen L.,South China Agricultural University | Liu Y.-G.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | And 2 more authors.
Annual Review of Plant Biology | Year: 2014

In plants, male sterility can be caused either by mitochondrial genes with coupled nuclear genes or by nuclear genes alone; the resulting conditions are known as cytoplasmic male sterility (CMS) and genic male sterility (GMS), respectively. CMS and GMS facilitate hybrid seed production for many crops and thus allow breeders to harness yield gains associated with hybrid vigor (heterosis). In CMS, layers of interaction between mitochondrial and nuclear genes control its male specificity, occurrence, and restoration of fertility. Environment-sensitive GMS (EGMS) mutants may involve epigenetic control by noncoding RNAs and can revert to fertility under different growth conditions, making them useful breeding materials in the hybrid seed industry. Here, we review recent research on CMS and EGMS systems in crops, summarize general models of male sterility and fertility restoration, and discuss the evolutionary significance of these reproductive systems. Copyright © 2014 by Annual Reviews. Source


Chen W.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | Chen W.,Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions | Chen W.,South China Agricultural University | Zeng D.,State Key Laboratory for Conservation and Utilization of Subtropical Agro Bioresources | And 19 more authors.
Biotechnology and Biotechnological Equipment | Year: 2016

Cloning of coding sequence (CDS) is an important step for gene function research. Here, we reported a simple and efficient strategy for assembling multiple-exon into an intron-free CDS from genomic DNA (gDNA) by an isothermal recombination reaction-based PCR (IRR-PCR) method. As an example, a 2067-bp full-length CDS of the anther-specific expression gene OsABCG15, which is composed of seven exons and six introns, was generated by IRR-PCR using genomic DNA of rice leaf as the template. Actually, this approach can be wildly applied to any DNA sequences assembly to achieve CDS cloning, gene fusion and multiple site-directed mutagenesis in functional genomics studies in vitro. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Source


Wang H.-Z.,South China Agricultural University | Wang H.-Z.,Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms | Chu Z.-Z.,South China Agricultural University | Chu Z.-Z.,Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms | And 18 more authors.
PLoS ONE | Year: 2015

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Nichelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Nichelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. © 2015 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

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