黏类小麦细胞质雄性不育相关基因cMDH的克隆与表达分析

为深入研究黏类小麦雄性不育的分子遗传机制,利用抑制差减杂交技术构建了黏类小麦育性相关基因的二核期SSH文库.经文库筛选后,得到一个在可育文库中表达的与胞质苹果酸脱氢酶基因同源的EST序列.以该EST序列为信息探针,经电子克隆、RT-PCR、PCR克隆与序列分析,获得了小麦胞质苹果酸脱氢酶(cytosolic malate dehydrogenases,cMDH)基因的cDNA与DNA序列,利用荧光定量PCR技术对该基因在不育株和可育株花药中的表达进行了分析,并比较了MDH在小麦不育株和可育株中的活性变化.结果表明,该基因的cDNA序列长1213 bp,编码333个氨基酸;DNA序列长2 908 bp,含有7个外显子和6个内含子;该基因在不育株和可育株花药发育3个时期(单核、二核和三核)的表达均表现为先升后降的模式,而且该基因在不育株花药发育的二核期和三核期的表达相对于可育株被明显抑制;MDH在小麦不育株和可育株中的活性变化趋势与定量结果一致.推测该基因在花粉发育早期表达,它的下调表达可能影响了小麦雄蕊发育过程中的能量供应而导致雄性不育.

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

Male sterility withAegilops kotschyi cytoplasmhas a great application potential in hybrid wheat (Triticum aestivum L.) breeding for its stable sterility and broad-spectrum of restoring gene resources. To furtherreveal 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. Furthermore, expression characteristics of the gene between male sterile and fertile anthers were analyzed via real-time PCR. In this study, the cDNA sequence was 1213 bp in length and the open reading frame encoded a peptide of 333 amino acids. The DNA sequence was 2908 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.