Abstract
Anther and tapetum-specific genes are important for understanding male gametophyte development, as well as for their use in the development of barnase/barstar-gene based male sterility and restorer system for hybrid seed production. An essential component of the system is the availability of tapetum-specific promoters. In the present study, anther-specific genes were identified in cotton using microarray-based differential expression analysis, some of which show expression specific to the anthers at a stage where tapetum tissue was fully developed. Validation of the identified genes using RT-PCR and in situ hybridization identified one novel gene (AEG—Anther Expressing Gene) encoding a putative lipid binding protein as having a tapetum-specific expression. Further, three paralogs of the gene were identified in the cotton genome out of which the gene AEG1 (Anther Expressing Gene1) was found to express in the tapetum layer. Analysis of transgenic plants developed in cotton using 1.5 Kb promoter region of the of AEG1 gene fused upstream to the reporter gene β-glucuronidase revealed a broad window of expression of the AEG1 promoter in the tapetum tissue from the tetrad stage of anthers till the degeneration of the tapetum cells. Low levels of expression were also observed in the root tissues. Expression was not observed in the stem and leaves. The broad window of expression of AEG1 promoter in the tapetum tissue makes it a suitable candidate for the expression of the barstar gene for effective fertility restoration in the barnase/barstar system.
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Acknowledgements
Research work was supported by grants from the Department of Biotechnology (DBT) and NMITLI program of Council for Scientific and Industrial Research (CSIR), Government of India. KP acknowledges the fellowship from the Council for Scientific and Industrial Research, India. DP acknowledges an award of J. C. Bose fellowship (Department of Science and Technology, Government of India). The authors acknowledge the help of Dr. Anjana N Dev for editing the manuscript.
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Conceived and designed the experiments: DP KP PKB. Performed the experiments: KP (Microarray experiments and analysis, identification of genes, sequence analysis) AKS (development of transgenic cotton) AKM (development of promoter:reporter construct and in situ experiments). Analyzed the data: KP AKS DP PKB. Wrote the paper: KP AKS DP PKB.
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S1 Figure. Principal component analysis and correlation plot of the different samples used for the microarray experiments (PDF 1306 KB)
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Paritosh, K., Singh, A.K., Mehrotra, A.K. et al. Identification and characterization of the promoter of a gene expressing mainly in the tapetum tissue of cotton (Gossypium hirsutum L.). Plant Biotechnol Rep 12, 377–388 (2018). https://doi.org/10.1007/s11816-018-0501-z
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DOI: https://doi.org/10.1007/s11816-018-0501-z