Molecular cloning and characterization of a proliferating cell nuclear antigen gene by chemically induced male sterility in wheat (Triticum aestivum L.).

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.

Relationship between male sterility and ß-1,3-glucanase activity and callose deposition-related gene expression in wheat (Triticum aestivum L.).

In previous studies, we first isolated one different protein ß-1,3-glucanase using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from normal wheat (Triticum aestivum L.) and chemical hybridization agent-induced male sterility (CIMS) wheat. In this experiment, ß-1,3-glucanase activity and the expression of a callose deposition-related gene, UDP-glucose phosphorylase (UGPase), were determinate in normal, CIMS, and genetic male sterility (GS) wheat. ß-1,3-glucanase activity was significantly different between the fertile and sterile lines during callose synthesis and degradation, but there was no difference between CIMS and GS wheat. The UGPase gene of callose deposition was highly expressed in the meiophase and sharply decreased in the tetrad stage. However, the expression of the UGPase gene was significantly different between the fertile and sterile lines. These data indicated that ß-1,3-glucanase activity and the expression of the UGPase gene play important roles in the male sterility of wheat. Consequently, pollen mother cells (PMCs) might degenerate at the early meiosis stage, and differences in UGPase gene expression and ß-1,3-glucanase activity might eventually result in complete pollen collapse. In addition, the critical period of anther abortion might be the meiosis stage to the tetrad stage rather than what we previously thought, the mononuclear period.

Homocysteine induces blood vessel global hypomethylation mediated by LOX-1.

Homocysteine (Hcy) is an independent risk factor of atherosclerosis through its involvement with the methionine cycle. In this study, we aimed to determine the blood vessel global methylation rate in Hcy-induced atherosclerosis in apolipoprotein-E-deficient (ApoE-/-) mice, and to explore the possible mechanism of this change in endothelial cells. ApoE-/- mice were divided into a hyperlipidemia (HLP) group, a hyperhomocysteinemia (HHcy) group, and an HHcy + folate + vitamin B12 (HHcy+FA+VB) group. Wild-type C57BL/6J mice were prepared as controls. Total Hcy, lipids, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) contents in serum were measured with an automatic biochemistry analyzer and high-performance liquid chromatography. Methylation of B1 repetitive elements in blood vessels was tested using nested methylation-specific-polymerase chain reaction (nMS-PCR). Endothelial cells (ECs) were pretreated with Hcy or by adding FA and VB. Lectin-like oxidized LDL receptor-1 (LOX-1) expressions were determined by quantitative PCR, Western blot, and nMS-PCR. The HHcy group displayed severe HLP and HHcy. SAM and SAH contents were also elevated in the HHcy group compared with other groups. Methylation of B1 repetitive elements was significantly increased in the HHcy group (0.5050 ± 0.0182) compared to the HLP (0.5158 ± 0.0163) and control (0.5589 ± 0.0236) groups. mRNA and protein expressions of LOX-1 increased (0.2877 ± 0.0341, 0.6090 ± 0.0547), whereas methylation expression decreased (0.5527 ± 0.0148) after 100 µM Hcy stimulation in ECs. In conclusion, Hcy-induced atherosclerosis was closely associated with induced hypomethylation status in the blood vessel, and this process was partially mediated by LOX-1 DNA methylation.