Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

摘要

Interspecific hybridization in the genus Phaseolus, conducted to introgress desired traits into common bean (Phaseolus vulgaris L.), leads to the abortion of immature embryos, usually at early developmental stages. Little is known about the physiological responses of embryo dysfunction in P. vulgaris during the early stages of embryogenesis and the genes that are involved in these responses. Identification of the genes involved in Phaseolus embryogenesis may provide information that will help to understand the molecular basis of Phaseolus embryo dysfunction. To investigate the genes expressed during Phaseolus seed development, we constructed a suppression subtractive hybridization (SSH) library using cDNA from abortive seed development as the driver and those from normal seed development as tester. The differentially expressed cDNA fragments were identified by differential screening. We identified 72 unique ESTs of which we selected 12 candidates on the basis of their redundancy. These candidates were subjected to a validation procedure based on the study of their expression level by real-time PCR. Sequence analysis revealed that most of the differentially expressed genes are related to metabolism and regulation such as protein synthesis. Some genes also encoded transcription factors. These genes showed high mRNA transcript levels in seed tissues and little or no expression in other tissues (root, stem, flower, leaf, and cotyledon). Seven genes were chosen and their expression profile during seed development in P. vulgaris was analyzed by real-time PCR using RNA preparations originating from different seed development stages. This study revealed hitherto unknown genes putatively involved in dicotyledonous embryogenesis, which serve as a starting point for understanding Phaseolus embryogenesis.