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Acta Biologica Hungarica
Authors: Xiaoyan Liu, Md. Asaduzzaman Khan, Jingliang Cheng, Chunli Wei, Lianmei Zhang, and Junjiang Fu

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

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Red coleoptile is an easily observed trait in Triticum aestivum and can provide some protection against stress. Here, TaMYB-A1 or TuMYB-A1, homologous to TaMYB-D1, which controls red coleoptile formation in the common wheat cultivar ‘Gy115’, was isolated from eight T. aestivum and 34 T. urartu cultivars. The genome sequence of TaMYB-A1 was 867 bp with an intron of 93 bp, which was similar to the MYBs regulating anthocyanin biosynthesis in T. aestivum but different from other MYB transcription factors regulating anthocyanin biosynthesis. TaMYB-A1 had an integrated DNA-binding domain of 102 amino acids and a transcriptional domain of 42 amino acids, which was responsible for regulating anthocyanin biosynthesis. TaMYB-A1 was assigned to the same branch as the MYBs regulating anthocyanin biosynthesis in a phylogenetic tree. A transient expression analysis showed that TaMYB-A1 induced ‘Opata’ coleoptile cells to synthesize anthocyanin with the help of ZmR. A non-functional allele of TaMYB-a1 existed in common wheat cultivars containing rc-a1. One single nucleotide was deleted 715 bp after the start codon in TaMYB-a1 compared with TaMYB-A1. The deletion caused a frame shift mutation, destroyed the DNA transcription activator domain, and resulted in TaMYB-a1 losing its ability to regulate anthocyanin biosynthesis in ‘Opata’ coleoptile cells. Those cultivars with functional TaMYB-A1 or TuMYB-A1 have red coleoptiles. The isolation of TaMYB-A1 should aid in understanding the molecular mechanisms of coleoptile traits in T. aestivum.

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Cereal Research Communications
Authors: L. Wei, S.G. Bai, X.J. Hou, J.M. Li, B. Zhang, W.J. Chen, D.C. Liu, B.L. Liu, and H.G. Zhang

Among 20 awnless Tibetan wheat cultivars analyzed by SDS-PAGE, the migration rate of an HMW-GS in XM001584 and XM001593, named 1BX23*. was shown to be slightly faster than 1Bx6. and slower than Bx7. Its nucleotide sequence was isolated based on homology clones. In a phylogenetic tree of 1Bx genes, 1Bx23* was apparently clustered with 1Bx23. Compared with 1Bx23. eight single nucleotide replacements caused four single amino acid replacements in 1Bx23*. The deletion of “G” at base pair 1463 and insertion of “A” at 1509 bps induced a 42-nucleotide frame shift. “GQRQQAGQWQRPGQ” was replaced by “DKGNRQDNGNDRDK”. The new segment cannot be found in other HMW-GSs, and it is very similar to a segment found in collagen. Moreover, an 18-nucleotide deletion made 1Bx23* six amino acids shorter than 1Bx23. The cultivar XM001593 had 28 chromosomes, which signifies that it was tetraploid wheat, and that the new HMW-GS 1Bx23* cannot be used directly for breeding in common wheat.

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Fusarium head blight (FHB), mainly caused by Fusarium graminearum , is a very serious disease in wheat and barley production area. FHB epidemics cause yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. Understanding the infection mechanism of F. graminearum plays an important role for the disease control. In present study, green fluorescence protein (GFP)-tagged were infected to wheat and barley varieties by single floret injection and screened via GFP signal. Results showed similar infection pattern of F. graminearum on both wheat and barley. Pathogen geminated in the inoculated spikelets, grew on the top of ovary or between lemma and palea, and extended towards and through rachis to the adjacent spikelets to infect the whole spike. When a spike of cultivar with FHB resistance was inoculated by F. graminearum , only the injected spikelet showed symptom at 6 days past inoculation (dpi). GFP signals indicated that F. graminearum colonized only in the inoculated spikelet and stop at the compact tissue of rachilla at 6 dpi. On the contrary, the diseased spikelets were up to 5 at 6 dpi in the spike of cultivars susceptible to FHB. F. graminearum extended through compact tissue to rachis and infected to the adjacent spikelets by spreading upward and downward to adjacent florets inter- and intra-cellularly in vascular bundles and cortical tissue of the rachis.

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In this study, we employed electron microscopy to investigate the cytogenetic and embryologic mechanisms of parthenogenesis induced in the 1BL/1RS male sterile lines of wheat. Analysis of the root tips and acid polyacrylamide gel electrophoresis indicated that all of the male sterile lines and their maintainer lines were 1BL/1RS translocation lines, whereas the restorer lines were non-1BL/1RS translocation lines. Furthermore, the chromosomes of 1BL/1RS wheat lines with T. aestivum cytoplasm and Aegilops cytoplasm (include Ae. kotschyi, Ae. ventricosa, Ae. variabilis) paired abnormally at different rates during meiotic metaphase I (MMI). The translocated segment size of the 1RS chromosome and the specific nuclear–alloplasm interaction impaired the pairing of homologous chromosome in the background of the specific Aegilops cytoplasm at MMI. In addition, the frequency of abnormal chromosomal pairing was directly affected by the frequency of haploid production induced by parthenogenesis. The results of this study provide significant insights into the mechanism of parthenogenesis, which is probably due to the abnormal fertilization of synergid cells in alloplasmic 1BL/1RS wheat.

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Cereal Research Communications
Authors: X. Zhang, Y. Chen, Y. Wei, W. Lu, H. Liao, Y. Liu, X. Yang, X. Li, L. Yang, L. Li, and R. Li

Partial abortion of gametes possessing S-5 j in S-5 i / S-5 j genotype at locus S-5 is responsible for hybrid sterility between indica and japonica subspecies in rice ( Oryza sativa L.), while a single wide compatibility (WC) allele S-5 n can restore normal hybrid fertility between the two groups. In this study, Pei’ai 64S, one of the most popular WC line widely used for subspecific hybrid rice breeding program in South China was studied for location of its S-5 locus. Twenty SSR (Simple Sequence Repeat) markers derived from Cornell SSR linkage map and 9 developed using sequences from GenBank database were employed to perform bulked segregant analysis of the mapping population derived from a three-way cross (Pei’ai 64S/T8//Akihikari) to tag fine location of the hybrid sterility locus, S-5 . This S-5 locus was mapped on chromosome 6 approximately 0.2 cM from GXR6 and RM276 SSR markers. This tight linkage of the markers and the S-5 locus would be very useful for efficient marker-assisted selection for WC varieties and for map-based cloning of the gene.

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