Abstract
When facing upon various kinds of stresses such as heat, cold, wounding, heavy metals, and water deficit, organisms usually synthesize molecular chaperons to cope with stress. The seventy kilodalton heat shock protein (HSP70) and the constitutively expressing heat shock cognate (HSC70) are conserved among all organisms. In addition to stress-induction, HSP/HSC70 have been found to be up-regulated at specific development stages in the absence of stress. Previous studies in our laboratory showed that a HSP/HSC of about 71 kDa is expressed in germinating mung bean (Vigna radiata) seedlings. In order to investigate the expression profile of Hsc70 in mung bean, a cDNA library was constructed from mRNAs isolated from mung bean seedlings at 3 days after germination (dag). Three heat shock protein genes were isolated from the library and designated as VrHsc70-1, VrHsc70-2, and VrHsc70-3. The expression profiles of these genes were analyzed with Northern blots analyses and RNase protection assays using the [□-32P] UTP l led antisense RNA as gene-specific probes. The levels of VrHsc70-1, VrHsc70-2 and VrHsc70-3 mRNAs increased from 0 dag to 3 dag and maintained until 7 dag. During embryogenesis, the mRNA levels of these three genes was substantial from 0 to 15 days after anthesis (daa) and declined remarkably at 25 daa. The expression of VrHsc70-1 mRNA was significantly induced by cold (4oC) and heat (37oC) treatments, while VrHsc70-2 and VrHsc70-3 mRNAs were only slightly induced by heat. Our results indicate that the expression of these three genes might not be related to seed maturation and desiccation during late embryogenesis. Although these three genes are constitutively expressed, the expression of VrHsc70-1 is apparently regulated by temperature stress.Abstract ••••••••••••••••••••••••••••••ii謝誌•••••••••••••••••••••••••••ivList of Figures •••••••••••••••••••••••••••vAbbreviations •••••••••••••••••••••••••••viTable of Contents••••••••••••••••••••••••••viiIntroduction ••••••••••••••••••••••••••••1Materials and Methods •••••••••••••••••••••••81. Plant growth •••••••••••••••••••••••••••82. Stress treatments •••••••••••••••••••••••••83. Total cellular RNAextraction ••••••••••••••••••••94. cDNA library construction •••••••••••••••••••••115. cDNA library screening •••••••••••••••••••••••206. Mini-scale plasmid DNA preparation •••••••••••••••••237. DNA sequencing••••••••••••••••••••••••••248. Large-scale plasmid DNA preparation•••••••••••••••••259. Amplifying the 5’ end of cDNA by reverse transcription and polymerase chain reaction (PCR) ••••••••••••••••••••••••••2710.Amplifying the 3’ untranslated sequences by polymerase chain reaction••••2811. Synthesis of single stranded RNA probes by in vitro transcription••••••2912. Northern blot analysis•••••••••••••••••••••••• 3013. RNase protection assay•••••••••••••••••••••••31Results ••••••••••••••••••••••••••••••33Discussion •••••••••••••••••••••••••••••39Figures ••••••••••••••••••••••••••••••42References ••••••••••••••••••••••••••••53