Molecular cloning and characterization of a rat sarcoma-like protein from brain (Rab) gene in sugar beet monosomic additional line M14

Suppression subtractive hybridization (SSH) and differential display reverse transcription PCR (DDRT-PCR) were performed in floral organs of Beta vulgaris monosomic addition line M14 (BvM14) (2n=18+1) and a control of Beta vulgaris L. (2n=18). One of the differential genes was designated as BvM14 rat sarcoma-like protein from brain, i.e., BvM14-Rab. The full length of BvM14-Rab was obtained by RACE. It had 1,051bp with an open reading frame of 609bp encoding 202 amino acids (GenBank ID DQ831861). Sequence analysis showed four conversed domains of small GTP-binding proteins and five conserved sequences of Rab protein in the deduced BvM14-Rab protein, which has an 82% homology with AtRabD2a. Real-time PCR demonstrated that BvM14-Rab was expressed ubiquitously in roots, stems, leaves, flowers, pistal and stamen tissues with six-fold high abundance in the stems at two days after flowering. The BvM14-Rab protein was found to be localized in plasma membrane, indicating BvM14-Rab may play an important role in protein constitution and substance trafficking. The pBI121-BvM14-Rab expression construct was transformed into tobacco (Nicotiana tabacum L.) SR-1 leaves. Positive transgenic plants were identified by western blotting with BvM14-Rab polyclonal antibody. Constitutive expression of the BvM14-Rab in tobacco led to a series of abnormal phenotypes, including easily exfoliated flower, reduced height, inflorescence and seed weight, implying potential involvement of BvM14-Rab in flower development and seed formation.