Expression Profiling In Rice Panicle Under Heat Stress And Analysis Of Heat Inducible Promoter

With the increasing global warming, heat stress is becoming a major abiotic stress limiting plant growth and productivity around the world. Microarray has been widely used to systematically investigate the molecular reactions by which plants respond and adapt to complicated environment. Microarray was also applied to profile the tissue-specific and stress-specific gene expression. In present study, a time course gene expression profile of post-meiosis rice panicle under heat stress was performed by microarray to uncover the genome-wide gene response to heat stress. Stress-inducible promoters are considered to be ideal for driving specific gene expression for increased abiotic stress tolerance. Six highly heat responsive genes were identified from microarray data for their promoter analysis. The expression patterns of these six genes in different rice organs and under abiotic stresses were further confirmed by quantitative RT-PCR and three highly stress-inducible genes were selected for promoter-driven GUS expression analysis under heat and drought treatments in panicles and flag leaves. The major results are as followed:1. A time course whole genome rice gene expression profile in post-meiosis rice panicle under heat stress was obtained using Agilent44K rice microarray. A total of1815differentially expressed genes (DEGs) in rice panicle under heat treatment were identified.2. The results from GO retrieve and gene expression pattern analysis showed that the heat-responsive (HR) regulation-related genes in rice panicle are mainly classified as transcription factors, protein modification and degradation, phytohormones responsive factors, receptor kinases, and calcium-related proteins. The HR regulation-related transcription factors consist of bZIP, WRKY, NAC, C2H2, MYB, MADS, bHLH, and AP2/ERF families and more than half of these genes were up-regulated to a different extent under high temperature.3. The expression profile analysis showed that the complicated changes of ROS-related genes in rice panicle under heat stress are important in maintaining ROS balance. Ubiquitin-proteasome system (UPS) was enhanced under heat stress. HR UPS-related genes from E3families were up-regulated to different extent upon heat shock in rice panicle, suggesting that the UPS-related genes play important positive roles in panicle response to heat.4. Metabolism-related HR genes in rice panicle were mainly involved in cell wall formation, lipid metabolism and especially in secondary metabolisms. Most metabolism- related genes were slightly repressed under heat treatment, which suggested that the inhibition of metabolism in rice panicle under high temperature would be helpful for plant response to adverse environments.5. Six highly heat responsive genes were identified from microarray data for their promoter analysis. The expression pattern of these six genes in different rice organs and under abiotic stresses were analyzed by quantitative RT-PCR. Expression of these six genes were highly heat inducible, moderately responded to salt stress, polyethylene glycol (PEG) and abscisic acid (ABA), but little affected to cold treatment. Under normal conditions, expression of the six genes was detected in all the organs analyzed but at different levels.6. The promoter analysis of the three highly heat-inducible genes (OsHsfB2cp, PM19p and HSP90p) by PlantCARE and PLACE showed some stress response-related elements, such as G-box, GC-motif, HSE and ABRE in the three promoters. These three promoters were used to drive GUS gene expression in rice. GUS gene expression, histochemical staining and GUS activities in panicles and flag leaves of the transgenic rice plants was analyzed under heat and drought treatments. The three promoters exhibited similar high activity lever in rice leaf under heat, but OsHsfB2cp and PM19p showed much higher activities in panicles under heat stress. Our work confirmed that the OsHsfB2c and PM19promoters were highly heat inducible and further characterization and reconstruction of cis-elements in their promoters could lead to the development of highly effective heat-inducible promoters for plant genetic engineering.