Cloning and Expression Analysis of cMDH Gene Related to Cytoplasmic Male Sterile Wheat with Aegilops kotschyi Cytoplasm

Male sterility with Aegilops kotschyi cytoplasm has a great application potential in hybrid wheat(Triticum aestivum L.) breeding for its stable sterility and broad-spectrum of restoring gene resources.To further reveal the genetic mechanism of male sterile with Ae.kotschyi cytoplasm,we employed a male sterile line ms(Kots)-90-110(A) and its near isogenic line BC4F1(fertility restored by rk5451) to construct sterile and fertile cDNA libraries by suppression subtractive hybridization(SSH) in binucleate stage of anther development.Comparative analysis of differentially expressed EST sequences revealed that one EST highly similar to cytosolic malate dehydrogenases gene was identified from the fertile SSH-cDNA library.Then,the EST sequence was used as a querying probe to blast the Genbank databases.Based on the assembled homologous cDNA sequence,both cDNA and DNA sequences encoding a cytosolic malate dehydrogenases were isolated and characterized by PCR and sequence analysis.Fur-thermore,expression characteristics of the gene between male sterile and fertile anthers were analyzed via real-time PCR.In this study,the cDNA sequence was 1 213 bp in length and the open reading frame encoded a peptide of 333 amino acids.The DNA sequence was 2 908 bp in length,which contained seven extrons and six introns.According to expression analysis,the expression of this gene in fertile anthers was much higher than that in sterile anthers at binucleate and trinucleate stage during anther development.The trend of MDH activity was consistent with the quantitative results between fertility and sterility.Therefore,the gene is conjectured to be an early expression gene and its down-regulated expression may affect energy supply during stamen growth in sterile line anthers resulting in male sterility in wheat.