The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis.
The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis.
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DOI：
10.1186/1756-0500-3-245
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发表时间：
2010-09-29
影响因子：
1.8
通讯作者：
Mayes S
中科院分区：
文献类型：
--
作者：
Devisetty UK;Mayes K;Mayes S
真核生物的减数分裂重组需要大肠杆菌RecA蛋白的两个同源物：Rad51和Dmc1。这两种蛋白在单链DNA的结合、同源性搜索、链入侵和链交换中发挥重要作用。拟南芥、水稻、玉米等植物的减数分裂重组已经得到了很好的研究，RAD51和DMC1的同源基因已经被鉴定。然而，由于缺乏完整的序列信息，以及在六倍体小麦基因组中存在多个拷贝，对面包小麦RAD51和DMC1基因的遗传分析一直受到阻碍。在本研究中，我们确定了TaRAD51和TaDMC1同源物分别位于六倍体小麦的第7组和第5组染色体上。TaRAD51和TaDMC1同源基因的序列比较分析显示，两者在核苷酸和氨基酸水平上存在有限的序列差异。事实上，将预测的TaRAD51和TaDMC1的氨基酸序列与其他真核生物的氨基酸序列进行比较，揭示了高度的进化保守性。尽管TaRAD51和TaDMC1在核苷酸水平上具有高度的序列保守性，但研究人员开发了针对TaRAD51和TaDMC1 cdna的基因组特异性引物，以评估减数分裂过程中单个同源物的表达模式。QRT-PCR分析显示，TaRAD51和TaDMC1 cDNA同源物的表达主要局限于减数分裂组织，在减数分裂重组发生的前期I阶段表达水平升高。这两种链交换蛋白的三个同源物（TaRAD51和TaDMC1）都在小麦中表达。面包小麦含有TaRAD51和TaDMC1同源物各3个表达拷贝。虽然在TaRAD51的三个cDNA同源物和TaDMC1的三个同源物之间检测到差异，但根据我们的三维结构预测分析，预测的氨基酸取代不太可能对蛋白质结构产生影响。通过QRT-PCR确定，TaRAD51和TaDMC1的三个同源物的表达水平存在差异，如果这些差异反映在蛋白质水平上，那么面包小麦可能更依赖于一个特定的同源物来实现完全育性，而不是所有三个同源物。
Meiotic recombination in eukaryotes requires two homologues of the E. coli RecA proteins: Rad51 and Dmc1. Both proteins play important roles in the binding of single stranded DNA, homology search, strand invasion and strand exchange. Meiotic recombination has been well studied in Arabidopsis, rice, maize and the orthologues of RAD51 and DMC1 have been characterized. However genetic analysis of the RAD51 and DMC1 genes in bread wheat has been hampered due to the absence of complete sequence information and because of the existence of multiple copies of each gene in the hexaploid wheat genome. In this study we have identified that TaRAD51 and TaDMC1 homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the TaRAD51 and TaDMC1 homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of TaRAD51 and TaDMC1 and those of other eukaryotes reveal a high degree of evolutionary conservation. Despite the high degree of sequence conservation at the nucleotide level, genome-specific primers for cDNAs of TaRAD51 and TaDMC1 were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the TaRAD51 and TaDMC1 cDNA homoeologues was largely restricted to meiotic tissue, with elevated levels observed during the stages of prophase I when meiotic recombination occurs. All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat. Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues. While differences were detected between the three cDNA homoeologues of TaRAD51 as well as the three homoeologues of TaDMC1, it is unlikely that the predicted amino acid substitutions would have an effect on the protein structure, based on our three-dimensional structure prediction analyses. There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.