Dataset Information

A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice.

ABSTRACT: Inducible gene expression has emerged as a powerful tool for plant functional genomics. The estrogen receptor-based, chemical-inducible system XVE has been used in many plant species, but the limited systemic movement of inducer ?-estradiol in transgenic rice plants has prohibited a wide use of the XVE system in this important food crop. Here, we constructed an improved chemical-regulated, site-specific recombination system by employing the XVE transactivator in combination with a Cre/loxP-FRT system, and optimized a seed-soaking procedure for XVE induction in rice. By using a gus gene and an hpRNAi cassette targeted for OsPDS as reporters, we demonstrated that soaking transgenic seeds with estradiol solution could induce highly efficient site-specific recombination in germinating embryos, resulting in constitutive and high-level expression of target gene or RNAi cassette in intact rice plants from induced seeds. The strategy reported here thereby provides a useful gene activation approach for effectively regulating gene expression in rice.

SUBMITTER: Chen Z

PROVIDER: S-EPMC5566991 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice.

Chen Zaijie Z Cheng Qianqian Q Hu Chanquan C Guo Xinrui X Chen Ziqiang Z Lin Yan Y Hu Taijiao T Bellizzi Maria M Lu Guodong G Wang Guo-Liang GL Wang Zonghua Z Chen Songbiao S Wang Feng F

Frontiers in plant science 20170821

Inducible gene expression has emerged as a powerful tool for plant functional genomics. The estrogen receptor-based, chemical-inducible system XVE has been used in many plant species, but the limited systemic movement of inducer β-estradiol in transgenic rice plants has prohibited a wide use of the XVE system in this important food crop. Here, we constructed an improved chemical-regulated, site-specific recombination system by employing the XVE transactivator in combination with a Cre/<i>loxP</i ...[more]

PMID: 28871269

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Project description:BackgroundRice is not only an essential food but also a source of high quality protein. Polyploidy is an evolutionary trajectory in plants, and enhancing glutelin by polyploidization is an attractive strategy for improving the nutritional value of rice seeds and presents a great potential for enhancing the commercial value of rice. Elucidating the mechanisms underlying glutelin synthesis and accumulation in tetraploid rice is of great significance.ResultsTo enhance the nutritional value of rice, we developed tetraploid rice and evaluated the contents of various nutrient elements in mature seeds. The results revealed a significant increase in protein contents, including the total seed storage proteins, glutelins, and amino acids in tetraploid rice when compared with those in diploid rice. Tandem mass tag-based quantitative proteomic analyses of seeds revealed that glutelins regulated by several glutelin genes in 9311-4x were significantly up-regulated (≥1.5-fold), which was further verified by immunoblot analyses. In addition, temporal expression patterns of various glutelin subunits in different rice lines were investigated. The results revealed significant differences in the expression patterns between diploid and tetraploid rice seeds. Cytohistological analyses results revealed that the thickness of aleurone cell layers increased significantly by 32% in tetraploid rice, the structures of protein storage vacuoles (PSVs) in sub-aleurone cells were more diverse and abundant than those of diploid rice. Temporal expression and proteomic analyses results revealed that protein disulfide isomerase-like 1-1 expression levels were higher in tetraploid rice than in diploid rice, and that the gene responded to oxidative folding with increased levels of proglutelin and appropriate distribution of seed glutelins in tetraploid rice.ConclusionThe results of the present study revealed that polyploidization increased glutelin content by influencing glutelin biosynthesis, transport, and deposition, while variations in glutelin accumulation between tetraploid and diploid rice were largely manifested in the initial time, duration, and relative levels of various glutelin gene expressions during seed filling stages. These findings provide novel insights into improving the protein quality and nutritional value of rice seeds by polyploid breeding.

Project description:Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Dataset Information

A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice.

Publications

A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice.

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