Analysis Of ECA1 And ACA2 Function By RNA Interference In Arabidopsis Thaliana

The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes. Until now, several Ca²⁺ pumps have been identified in plant, which fall into two major types: ER-type Calcium ATPase (ECAs) and Autoinhibted Calcium ATPase (ACAs).Here, we analysed the function of ECA1 and ACA2 by RNA interference in Arabidopsis thaliana. The results showed as followering:1. The RNAi vector containing ECA1 and ACA2 gene-specific sequences in the sense and antisense orientations was constructed: pGreen0029-ECA1-1-Sense-GFP-ECA1-1-Antisense(E1-G-E1); pGreen0029-ACA2-1-Sense-GFP-ACA2-1-Antisense(A2-G-A2).2. After confirmed by enzyme digestion, two constructs were transformed into Arabidopsis thaliana plant by Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic lines were obtained through kanamycin screening and PCR assays.3. The transcriptional product of ECA1 in transgenic lines was obviously reduced comparing with that of wild type (WT) by semi-quantity RT-PCR analysis. In addition, in the 11 E1-G-E1transgenic lines, there are some remarkable differences in ECA1 mRNA products of transgenic lines. Among the 12 A2-G-A2 transgenic lines, there are some remarkable differences in ACA2 mRNA products of transgenic lines besides that of A2-11.4. There was no significant difference between transgenic lines (E1-G-E1 and A2-G-A2) and wild type while growing on the 1/2MS medium. However, E1-G-E1 transgenic lines could not grow very well comparing with that of wild type on the stress mediums containing lower Ca²⁺ (0.2 mmol·L-1) or high Mn2+ (0.5 mmol·L-1). The tolerance to lower Ca²⁺ or high Mn2+ is consistent with the mRNA level of ECA1. On the other hand, A2-G-A2 transgenic lines have no significant difference with wild type on the stress mediums containing lower Ca²⁺ (0.2 mmol·L-1) or high Mn2+ (0.5 mmol·L-1).