Study On Roles Of Diacylglycerol Kinase Gene Family In Rice Under Xylanase And NaCl Treatments By Transient RNA Interference System

Phospholipase C (PLC)/Diacylglycerol kinases (DGKs) signalling pathway is known as a major part of the phosphoinsiol signalling pathway. DGK is a pivotal enzyme that phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). Both DAG and PA are very important signaling molecules in plant cells. DGK is thought to regulate the balance between DAG and PA through catalyzing their interconversion. Therefore, DGK genes potentially affect a number of biological events including plant cell growth development, stress and hormone responses and disease resistance. Compared with the researches on the PLD pathway, the PLC-DGK pathway was reported in a less proportion, especially in rice. On the other hand, WRKY proteins are a large family of transcription factors that mainly participate in plant biotic and abiotic stress responses. OsWRKY71, a rice transcription factor, is demonstrated involved in rice defense response.In view of the above-mentioned facts, in this study, to analyze whether there is a difference expression characteristics of OsDGKs in biotic and abiotic stress conditions, we designed the primers for Real Time-PCR of cDNAs of OsDGKs gene family (OsDGK1～8), and their expression levels under treatments of Xylanase and salt were analyzed by semi-quantitative PCR and Real Time-PCR. Results showed that different individuals have different expression patterns in the above two stress situations, which illuminates their different functions. The expression of OsDGKl was rapidly activated within 6 h after Xylanase-treated rice protoplasts and maintained a high level of expression for a relatively long time period; the expression of OsDGK2 and OsDGK3 was also activated in Xylanase-treated rice protoplasts, but the expression mode and extent were distinct from OsDGK1; the expression abundance of OsDGK1 was consistently high. In addition, the expression patterns of OsDGKl,OsDGK2,OsDGK3,OsDGK7 in the NaCl-treated rice seedlings were also different from the expression patterns of them in the Xylanase-treated rice protoplast. These results suggest that OsDGKs play important roles in rice stress resistance responses.To investigate further, we also have established a rice seedling protoplasts system designed for the rapid characterization of large numbers of genes by transient double-stranded RNA interference (RNAi) which used for analysis of the relationship between diacylglycerol kinase gene family (OsDGKs) and transcription factor OsWRKY71 in rice stress resistance responses. We show that protoplasts transfection with an in vitro-synthesized dsRNA against OsWRKY71 within 6 h treatment with 150μg/ml Xylanase, resulted in a different degree of depletion of OsDGKs transcript; and dsRNA corresponding to the conserved sequence of OsDGKs-transfected protoplasts (RNAi) were collected after 6 h treatment with 150μg/ml Xylanase for RT-PCR analysis, the expression level of OsWRKY71 was dramaticly declined. These results suggest that there might be interaction between OsDGKs and OsWRKY71 in the Xylanase-related signalling pathway; but it seemed as if there was no interaction between them in the NaCl-related signalling pathway. And we also report that the protoplasts transfection with an in vitro-synthesized dsRNA against OsDGKs, resulted in a distinct depletion of the defense pathway marker gene OsNPRl and the salt-responsive gene OsCIPK15 transcript respectively in the different stress situation.In this study, we also have built an optimized system for isolation, transformation and gene silencing of rice leaf and stem-derived protoplasts, and improved the procedures for gene-specific and multiple gene interference of OsDGKs gene family in protoplasts by RNAi.Although the mechanism of interaction between OsDGKs, OsWRKY71 and the defence pathway marker gene is not fully explained clearly.But now at least we can confirm that there lie some correlations among OsDGKs and OsWRKY71 in rice defence response, and OsDGKs is demonstrated involved in rice biotic and abiotic responses.The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown. RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost-and space-efficient initial screening and selection of genes for subsequent in planta studies.