Introduction
Wheat is one of the three most important crops in the world due to its value as a major food source and its unique suitability for bread production. Mutation breeding, in which ionizing radiations (IRs) are the most frequently used mutagens, has resulted in significant increases in the quality and yield of wheat during the past century (Ahloowalia and Maluszynski, Reference Ahloowalia and Maluszynski2001; Ahloowalia et al., Reference Ahloowalia, Maluszynski and Nichterlein2004; Liu et al., Reference Liu, Zanten, Shu and Maluszynski2004; http://mvgs.iaea.org/AboutMutantVarities.aspx). It is well known that IR cause clustered DNA damages, particularly double-strand breaks (DSBs) in the genome. Error-prone DSB repair processes have been inferred to play important roles in radiation-induced mutations in both mouse germ cells and mammalian somatic cells (Sankaranarayanan et al., Reference Sankaranarayanan, Taleei, Rahmanian and Nikjoo2013).
Both radiation mutagenesis studies and radiation cytogenetic studies indicate that non-homologous end joining (NHEJ) might be the principal DSB repair mechanism that underlies the origin of mutations (Kanaar et al., Reference Kanaar, Hoeijmakers and van Gent1998; West et al., Reference West, Waterworth, Sunderland and Bray2004; Weterings and Chen, Reference Weterings and Chen2008). Ku70/80 heterodimer plays an important role in this process, which functions both as a DNA-binding protein and as an allosteric activator (Lieber et al., Reference Lieber, Grawunder, Wu and Yaneva1997; Walker et al., Reference Walker, Corpina and Goldberg2001; Mari et al., Reference Mari, Florea, Persengiev, Verkaik, Bruggenwirth, Modesti, Giglia-Mari, Bezstarosti, Demmers, Luider, Houtsmuller and van Gent2006). The plant homologues of Ku70 and Ku80 were first cloned in Arabidopsis thaliana (Tamura et al., Reference Tamura, Adachi, Chiba, Oguchi and Takahashi2002). The atku80 mutant exhibited hypersensitivity to DNA-damaging agents (Friesner and Britt, Reference Friesner and Britt2003). We had cloned the complementary DNA sequences of TaKu70 and TaKu80 in wheat, the deduced amino-acid sequences of which shared high homology with Ku70 and Ku80 of other plants (Zhu et al., Reference Zhu, Gu, Guo, Zhao, Zhao, Shao and Liu2009). In the present study, we extended the genomic characterization of TaKu70 and TaKu80 genes and demonstrated the function of the TaKu70/TaKu80 heterodimer in vitro. We also analysed the response of TaKu70 and TaKu80 genes to IR in several wheat cultivars. The results indicated that TaKu70 and TaKu80 genes are required for DSB repair in wheat.
Materials and methods
Materials
The characterization of TaKu70 and TaKu80 genes of hexaploid Triticum aestivum L. cv. Chinese spring (AABBDD), tetraploid Triticum turgidum L. (AABB), and diploid Triticum monococcum L. (AA) and Aegilops Tauschii Coss. (DD) was carried out.
Amplification and cloning of sequences
Genomic DNA was extracted using the cetyltriethylammonium bromide (CTAB) method. Universal primers (Table S1, available online) were designed to amplify the genomic sequences using the FastStart High Fidelity PCR System (Roche, Mannheim, Germany). The amplified fragments were cloned into pGEM®-T (Promega, Madison, WI, USA) and sequenced at Sangon Biotech Company (Beijing, China). Specific primers (Table S1, available online) were used to determine the genome types.
Irradiation and growth conditions
Wheat seeds were subjected to γ-ray irradiation at doses of 100, 150 and 250 Gy (7.5 Gy/min) using a Co60 irradiator at Peking University (Beijing, China). The irradiated seeds were grown in a growth chamber at 21°C under a 16 h light–8 h dark cycle. The seedlings were harvested after 3 d for total RNA extraction.
Quantitative polymerase chain reaction (qPCR) analysis
Total RNA was extracted using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA). Real-time PCR and qPCR were carried out using the iScript™ cDNA Synthesis Kit and SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). As multi-internal controls, 18S rRNA (GenBank: JF489233) and actin (GenBank: AAW78915) were selected. The qPCR primers (Table S1, available online) were designed using Beacon Designer 7.9 (PREMIER, Palo Alto, CA, USA).
Yeast two-hybrid analysis
Two plasmids, pAD-TaKu80 (TaKu80-B) and pBD-TaKu70 (TaKu70-A), were constructed and transformed into Saccharomyces cerevisiae strain HF7c. Yeast strains transformed with the corresponding plasmid were examined on a histidine-deficient plate.
Electrophoretic mobility shift assays (EMSAs)
TaKu70 and TaKu80 glutatione S-transferase (GST) fusion proteins were prepared and analysed by EMSAs as described by Tamura et al. (Reference Tamura, Adachi, Chiba, Oguchi and Takahashi2002). IRDye800-labelled M13 oligonucleotide (LI-COR, Lincoln, NE, USA) was used as a single-stranded DNA (ssDNA) probe and that annealed with an antisense sequence was used as a double-stranded DNA (dsDNA) probe. EMSAs were carried out using the LI-COR 4300 system (LI-COR).
Results and discussion
Three types of TaKu70 and TaKu80 genes are present in hexaploid wheat (T. aestivum L.)
Based on previously published cDNA sequences of TaKu70 and TaKu80 genes (Zhu et al., Reference Zhu, Gu, Guo, Zhao, Zhao, Shao and Liu2009), four and three sets of universal primers were designed to amplify genomic sequences in hexaploid T. aestivum L. cv. Chinese spring (genomes AABBDD). Three types of sequences were found for TaKu70 and TaKu80 genes, and it was assumed that each belonged to a different genome. Using specific primers designed based on the differences to amplify target fragments in the genomic DNA of T. monococcum, T. turgidum, Ae. tauschii and T. aestivum, the genome types of different sequences were determined. The sizes of TaKu70 genes ranged from 10,089 bp for genome A (TaKu70A) and 10,712 bp for genome D (TaKu70D) to 10,899 bp for genome B (TaKu70B). Comparison between genomic and cDNA sequences confirmed the presence of 19 exons and 18 introns in all the genomes (Fig. 1(a)). The sizes of TaKu80 genes ranged from 5337 bp for genome A (TaKu80A) and 5856 bp for genome D (TaKu80D) to 5936 bp for genome B (TaKu80B). Comparison between genomic and cDNA sequences confirmed the presence of 12 exons and 11 introns in all the genomes (Fig. 1(b)). Differences found between the exons of TaKu70 and TaKu80 genes were due to single-nucleotide substitutions, and no insertions or deletions were detected. The size of the introns exhibited a slight variation between the sequences due to single-nucleotide substitutions and insertions/deletions.
Fig. 1 Molecular characteristics of the Ku70 and Ku80 homologues of hexaploid wheat (Triticum aestivum L.). (a) and (b) Genomic organization of TaKu70 and TaKu80 genes. A total of 19 and 12 exons (black boxes) and 18 and 11 introns (lines) were identified for TaKu70 and TaKu80, respectively. (c) Yeast two-hybrid assay. The growth of yeast cells harbouring the recombinant plasmids for both AD-TaKu80 and BD-TaKu70 revealed the protein–protein interaction between TaKu70 and TaKu80 proteins (AD-Ku80+BD-Ku70). Neither negative control (T+Lam) nor BD-Ku70 (AD+BD-Ku70) could activate the reporter gene. (d) Electrophoretic mobility shift assay. Neither TaKu70 (lane 1) nor TaKu80 (lane 2) alone could affect the mobility of probes, suggesting that neither protein alone bound to DNA. The combination of TaKu70 and TaKu80 induced a substantial decrease in the mobility of the double-stranded DNA (dsDNA) probe (lanes 5 and 6), without affecting that of the single-stranded DNA (ssDNA) probe (lanes 3 and 4).
TaKu70 and TaKu80 form a functional heterodimer in vitro
Ku protein has been thought to always exist and function as a heterodimer, which is essential for DSB repair process (Liang et al., Reference Liang, Romanienko, Weaver, Jeggo and Jasin1996; Jin and Weaver, Reference Jin and Weaver1997; Gell and Jackson, Reference Gell and Jackson1999). In the yeast two-hybrid system, only the yeast strain transformed with both pAD-TaKu80 and pBD-TaKu70 grew on the histidine-deficient medium (Fig. 1(c)). In EMSAs, neither TaKu70 nor TaKu80 alone could visibly affect the mobility of the ssDNA and dsDNA probes. By contrast, the combination of TaKu70 and TaKu80 led to a significant decrease in the mobility of the dsDNA probe, but not in that of the ssDNA probe (Fig. 1(d)). These results indicate that TaKu70 and TaKu80 form a functional heterodimer that exhibits the ability to bind to dsDNA in vitro.
Expression of TaKu70 and TaKu80 genes is induced by IR
To determine whether the expression of TaKu70 and TaKu80 genes is regulated by induced DSBs, seeds of several wheat cultivars were treated with γ-ray irradiation. Different doses of IR (100–250 Gy) caused a marked arrest in seedling growth in a dose-dependent manner. The expression of TaKu70 and TaKu80 genes was detected in the seedlings of the IR-irradiated seeds and their controls by qPCR. Tanscriptional up-regulation of both TaKu70 and TaKu80 genes occurred under IR treatment in a dose-dependent manner in all the examined cultivars (Fig. 2(a) and (b)). Consistent with the role of the Ku70/80 heterodimer in NHEJ (Walker et al., Reference Walker, Corpina and Goldberg2001), TaKu70 and TaKu80 genes appeared to be associated with the repair of the IR-induced DSBs during wheat seed germination.
Fig. 2 Transcriptional activation of TaKu70 and TaKu80 genes in response to ionizing radiation (IR). (a) and (b) Transcriptional levels of TaKu70 and TaKu80 genes were up-regulated by IR in a dose-dependent manner in different wheat cultivars.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S1479262114000367
Acknowledgements
This study was supported by the National Natural Science Foundation Research Program (grant no. 11305261), the National 973 Program (grant no. 2014CB138101), and the National 863 Program (grant no. 2012AA101202) and the International Atomic Energy Agency project (CRP15651 and RAS5056).
