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Multigene engineering of medium-chain fatty acid biosynthesis in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system.

ABSTRACT: Medium-chain fatty acids (MCFAs) are an ideal feedstock for biodiesel and a range of oleochemical products. In this study, different combinations of CnFATB3, CnLPAAT-B and CnKASI from coconut (Cocos nucifera L.) were coexpressed in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system. Transgenic lines expressing different combinations of these genes were designated FL (FatB3 + LPAAT-B), FK (FatB3 + KASI) and FLK (FatB3 + LPAAT-B + KASI). The homozygous seeds of transgenic Arabidopsis thaliana expressing high levels of these genes were screened, and their fatty acid composition and lipid contents were determined. Compared with its content in wild-type A. thaliana, the lauric acid (C12:0) content was significantly increased by at least 395%, 134% and 124% in FLK, FL and FK seeds, respectively. Meanwhile, the myristic acid (C14:0) content was significantly increased by at least 383%, 106% and 102% in FL, FLK and FK seeds, respectively, compared to its level in wild-type seeds. Therefore, the FLK plants exhibited the best effects to increase the level of C12:0, and FL expressed the optimal combination of genes to increase the level of 14:0 MCFA.

SUBMITTER: Zheng Y

PROVIDER: S-EPMC7367956 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Multigene engineering of medium-chain fatty acid biosynthesis in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system.

Zheng Yusheng Y Chen Lizhi L Zhu Zhiyong Z Li Dongdong D Zhou Peng P

3 Biotech 20200717 8

Medium-chain fatty acids (MCFAs) are an ideal feedstock for biodiesel and a range of oleochemical products. In this study, different combinations of CnFATB3, CnLPAAT-B and CnKASI from coconut (Cocos nucifera L.) were coexpressed in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system. Transgenic lines expressing different combinations of these genes were designated FL (FatB3 + LPAAT-B), FK (FatB3 + KASI) and FLK ...[more]

PMID: 32714735

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Project description:Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

Project description:Cre/loxP recombination is a well-established technique increasingly used for modifying DNA both in vitro and in vivo. Nucleotide alterations can be edited in the genomes of mammalian cells, and genetic switches can be designed to target the expression or excision of a gene in any tissue at any time in animal models. In this study, we propose a system which worked via the Cre/loxP switch gene and DsRed/emGFP dual-color fluorescence imaging. Mesenchymal stem cells (MSCs) can be used to regenerate damaged tissue because of their differentiation capacity. Although previous studies have presented evidence of fusion of transplanted MSCs with recipient cells, the possibility of fusion in such cases remains debated. Moreover, the effects and biological implications of the fusion of MSCs at the tissue and organ level have not yet been elucidated. Thus, the method for determining this issue is significant and the models we proposed can illustrate the question. However, the transgenic rats exhibited growth slower than that of wild-type rats over several weeks. The effects on the stemness, proliferation, cell cycle, and differentiation ability of bone marrow-derived rat MSCs (BM-rMSCs) from the models were examined to ensure our design was appropriate for the in vivo application. We demonstrated that MSC surface markers were maintained in DsRed and Cre transgenic rMSCs (DsRed-rMSCs and Cre-rMSCs, respectively). A WST-8 assay revealed decreased proliferative activity in these DsRed-rMSCs and Cre-rMSCs; this result was validated through cell counting. Furthermore, cell cycle analysis indicated a decrease in the proportion of G1-phase cells and a concomitant increase in the proportion of S-phase cells. The levels of cell cycle-related proteins also decreased in the DsRed-rMSCs and Cre-rMSCs, implying decelerated phase transition. However, the BM-rMSCs collected from the transgenic rats did not exhibit altered adipogenesis, osteogenesis, or chondrogenesis. The specific markers of these types of differentiation were upregulated after induction. Therefore, BM-rMSCs from DsRed and Cre transgenic models can be used to investigate the behavior of MSCs and related mechanisms. Such application may further the development of stem cell therapy for tissue damage and other diseases.

Project description:The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

Dataset Information

Multigene engineering of medium-chain fatty acid biosynthesis in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system.

Publications

Multigene engineering of medium-chain fatty acid biosynthesis in transgenic <i>Arabidopsis thaliana</i> by a <i>Cre/LoxP</i> multigene expression system.

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