RNAi Vector Construction for Protein Disulfide Isomerase Gene and Seed Setting Characteristics in Offspring of Transgenic Rice under High Temperature Treatment

Abstract

To clarify the effect of PDI gene silence on rice yield traits and grain quality, we sub-cloned a 450 bp RNAi fragment from Nipponbare by RT-PCR, and successfully constructed a binary-vector pTCK303-RiOsPDI containing intron hpRNA (ihpRNA), then transformed it into the callus of wild type Nipponbare mediated by EHA105. The T-DNA region for PDI RNA interference in regenerating rice plants was integrated with rice genome via single copy in T0 generation transgenic plants, and showed a 3:1 genetic mode in T1 transgenic population, which could be conformed by PCR amplifying analysis and Southern blotting identification. The PCR and qRT-PCR for PDI gene expression in different organs showed that there were much lower level of PDI expression in grains, leaves, stem and sheath for transgenic plants compared with those for wild type Nipponbare, with almost 80% of the dropping in transgenic seeds. The influence of high temperature stress on seed setting traits, panicle agronomic traits and grain quality and also its relation to PDI gene expression in developing grains was further examined with T1 generation of transgenic plants imposed to two temperature treatments under the controlled temperature chambers, the results indicated that PDI silence transgenic plants had a remarkable lower seed setting rate, somewhat lower grain weight compared with its wild phenotype under the high temperature treatment in despite of no obviously varying with its wild phenotype under normal developmental condition, suggesting that PDI gene should be probably responsible for rice tolerance to high temperature stress. Moreover, there were lower translucence and higher chalky degree for transgenic plants than for Nipponbare, although no significant difference were observed in grain total protein and amylose content between PDI silence transgenic plant and its CK.