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Song Y.,Northwest Agriculture and Forestry University | Song Y.,National Yangling Agricultural Biotechnology and Breeding Center | Song Y.,Yangling Branch of State Wheat Improvement Center | Song Y.,Wheat Breeding Engineering Research Center | And 31 more authors.
Euphytica | Year: 2015

We investigated the relationship between a pollen-specific protein homolog and pollen abortion in an SQ-1-induced male sterile line of wheat (Triticum aestivum L.). We previously determined that EST S414 (Ta20678) was obviously down-regulated in a line of Xinong 1376 wheat in which male sterility was induced by SQ-1 (a chemical hybridizing agent). In the present study, we obtained the full sequence of F8-1 from wheat. The open reading frame sequence of the F8-1 gene was 495 bp, encoding 165 amino acid residues with a predicted molecular weight of 18.2759 kDa and an isoelectric point of 4.86. This sequence was identified as a Secale cereale × Triticum durumF8-4 homolog with 100 % identity. The amino acids represent a conserved domain in pollen Ole e I that contains the consensus sequence Q-G-R-V-Y-C-D-T-C-R. The protein has a close evolutionary relationship with orthologs in S. cereale × T. durum and Triticum urartu, consisting of 9.09 % alpha helix, 24.85 % extended strand, and 66.06 % random coil. F8-1 was only expressed in anthers during the binucleate and trinucleate stages and expression gradually decreased from the binucleate stage to the trinucleate stage. The expression of F8-1 gene of physiological male sterility 1376 (PHYMS-1376) was lower than that of 1376 at both the binucleate and trinucleate stages. Subcellular localization showed that the F8-1 protein was expressed at the nucleus. RNAi tests showed that down-regulation of the F8-1 was associated with an increase in the percentage of pollen abortion. Therefore, F8-1 is a positive regulator of physiological male sterility. © 2015, Springer Science+Business Media Dordrecht.

PubMed | Yangling Branch of State Wheat Improvement Center
Type: Journal Article | Journal: Genetics and molecular research : GMR | Year: 2015

Although a number of studies have shown that chemical hybridizing agents (CHAs) affect anther growth and regulate cell-cycle progression, little is known about the molecular and cellular mechanisms involved. Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication, and in many other processes in eukaryotic cells. In this study, the open reading frame of TaPCNA, the PCNA in wheat (Triticum aestivum L.), was cloned by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was 792-bp long and encoded a protein with 234 amino acids. Alignment of the TaPCNA-predicted sequence revealed a high degree of identity with PCNAs from other plant species. A subcellular localization assay indicated that TaPCNA was localized in the nucleus. The TaPCNA was cloned into the prokaryotic expression plasmid pET32a, and the recombinant plasmid was transformed into BL21 (DE3). TaPCNA expression was induced by 0.5 mM isopropyl-beta-D-thiogalactopyranoside and verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot assays, which indicated that the fusion protein was successfully expressed. The gene involved in the G1-to-S transition, Histone H4, was downregulated by 1376- CIMS, which is a chemically induced male sterility line. However, a semi-quantitative RT-PCR revealed that TaPCNA expression was upregulated in 1376-CIMS. Our results suggest that CHAs (SQ-1) induce DNA damage in wheat anthers. DNA damage results in either the delay or arrest of cell-cycle progression, which affects anther development. This study will help to elucidate the mechanisms of SQ-1-induced male sterility.

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