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Development of an mPing-based Activation Tag for Crop Insertional Mutagenesis

ABSTRACT: Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize novel genes associated with crop traits. To meet this need, the mPing element from rice was developed as an activation tag designed to induce overexpression of nearby genes. Addition of promoter sequences to mPing resulted in a decrease in overall transposition rate, however, this effect was negated by using a hyperactive version of mPing called mmPing20. These mPing-based activation tags were found to induce expression of nearby reporter genes in transient assays. Transgenic soybean plants carrying mPing-based activation tags and the appropriate transposase protein expression constructs showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag, indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the system.

ORGANISM(S): Glycine max

PROVIDER: GSE145631 | GEO | 2020/05/31

REPOSITORIES: GEO

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Project description:Purpose: Tag-based approach for global gene expression analysis has been revolutionised with the advent of next generation sequencing (NGS) technology. The aim of the present study is to present comprehensive view of differentially expressed genes under cold and freeze stress in seabuckthorn (Hippophae rhamnoides L.) Methods: DeepSAGE, a tag based approach, was used to identify differentially expressed genes under cold and freeze treatments in seabuckthorn (Hippophae rhamnoides L.). The 30 days old plantlets, at six leaves stage, were subjected to cold stress (CS) at 4°C and freeze stress (FS) at -10°C treatment for 6 hr. The seedlings grown at 28°C were taken as control (CON). Total RNA from all the three samples was isolated. Illumina Gene Expression Sample Prep Kit and Solexa Sequencing Chip (Flowcell) were used for tag preparation and the main instruments used for sequencing included Illumina Cluster Station and Illumina HiSeqTM 2000 System. Bioinformatics analysis resulted in to high number of differentially expressed genes under cold and freeze stress. Results: 36.2 million raw tags including 13.9 million distinct tags were generated from three leaf tissue libraries (control, cold stress and freeze stress). After removing low quality tags, 35.5 million clean tags including 7 million distinct clean tags were obtained. In total, 11922 differentially expressed genes (DEGs) were identified including 6539 up regulated and 5383 down regulated genes. Conclusions: DeepSAGE data of seabuckthorn provided useful resource and reference dataset for further functional genomics analysis in seabuckthorn and other important crops. The present study implicated a large number of genes with different biological functions expressing differentially in response to cold and freeze stress treatment. Isolation and further characterization of these genes will help researchers in understanding their role in cold and freeze tolerance in seabuckthorn and may provide important gene resources to be exploited for the development of stress tolerant crop plants in future.

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Development of an mPing-based Activation Tag for Crop Insertional Mutagenesis

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