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BackgroundAbiotic stresses due to climate change pose a great threat to crop production. Heat shock transcription factors (HSFs) are vital regulators that play key roles in protecting plants against various abiotic stresses. Therefore, the identification and characterization of HSFs is imperative to dissect the mechanism responsible for plant stress responses. Although the HSF gene family has been extensively studied in several plant species, its characterization, evolutionary history and expression patterns in the radish (Raphanus sativus L.) remain limited.ResultsIn this study, 33 RsHSF genes were obtained from the radish genome, which were classified into three main groups based on HSF protein domain structure. Chromosomal localization analysis revealed that 28 of 33 RsHSF genes were located on nine chromosomes, and 10 duplicated RsHSF genes were grouped into eight gene pairs by whole genome duplication (WGD). Moreover, there were 23 or 9 pairs of orthologous HSFs were identified between radish and Arabidopsis or rice, respectively. Comparative analysis revealed a close relationship among radish, Chinese cabbage and Arabidopsis. RNA-seq data showed that eight RsHSF genes including RsHSF-03, were highly expressed in the leaf, root, cortex, cambium and xylem, indicating that these genes might be involved in plant growth and development. Further, quantitative real-time polymerase chain reaction (RT-qPCR) indicated that the expression patterns of 12 RsHSF genes varied upon exposure to different abiotic stresses including heat, salt, and heavy metals. These results indicated that the RsHSFs may be involved in abiotic stress response.ConclusionsThese results could provide fundamental insights into the characteristics and evolution of the HSF family and facilitate further dissection of the molecular mechanism responsible for radish abiotic stress responses.

NAC (NAM, no apical meristem; ATAF, Arabidopsis transcription activation factor and CUC, cup-shaped cotyledon) proteins are among the largest transcription factor (TF) families playing fundamental biological processes, including cell expansion and differentiation, and hormone signaling in response to biotic and abiotic stresses. In this study, 172 RsNACs comprising 17 membrane-bound members were identified from the whole radish genome. In total, 98 RsNAC genes were non-uniformly distributed across the nine radish chromosomes. In silico analysis revealed that expression patterns of several NAC genes were tissue-specific such as a preferential expression in roots and leaves. In addition, 21 representative NAC genes were selected to investigate their responses to heavy metals (HMs), salt, heat, drought and abscisic acid (ABA) stresses using real-time polymerase chain reaction (RT-qPCR). As a result, differential expressions among these genes were identified where RsNAC023 and RsNAC080 genes responded positively to all stresses except ABA, while RsNAC145 responded more actively to salt, heat and drought stresses compared with other genes. The results provides more valuable information and robust candidate genes for future functional analysis for improving abiotic stress tolerances in radish.

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.

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