RESUMEN
The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.
Asunto(s)
Agrobacterium , Genética , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas , Genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Genética , Estrés Fisiológico , Nicotiana , Genética , Transformación GenéticaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.