Isolation and Characterization of New Members of class I Low-Molecular-Weight Heat Shock Proteins in Rice

• Main Authors: Chung-Yi Huang, 黃仲義
• Other Authors: Chu-Yung Lin
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/91056006694167721886

碩士 === 國立臺灣大學 === 植物學研究所 === 90 === There are 8 or 9 members of rice Class I low molecular weight heat shock protein (LMW HSP) genes according to 2D-PAGE and subsequent Western blotting analysis of the LMW HSP complex. Six members of Class I LMW HSP, Oshsp16.9A (pTS1), Oshsp16.9B, Oshsp16.9C, Oshsp17.3 (pTS3), Oshsp17.7 and Oshsp18.0 (pYL) have been isolated and characterized in previous study. This study was aimed to isolated and characterized the new members of rice Class I LMW HSP. Firstly, rice genomic DNA digested with EcoRI was ligated in λ ZAP vector (EcoRI/CIAP-treated) to construct the rice genomic library. Using the conserved DNA fragment encoding C-terminal 108 amino acids (C108) of Oshsp16.9A and gene specific fragment, 3’-untranslated regions (3’-UTR) of the 6 members as probes respectively, 18 positive clones were isolated. After restriction enzyme mapping and sequence analysis, only one genomic clone (designated as R2.6) was identified, which contained a putative new member of class I LMW HSP gene assigned as Oshsp17.9. Comparing the nucleotide sequence of Oshsp17.9 with Oshsp16.9A, Oshsp16.9B and Oshsp16.9C located on chromosome 1, the identities are 76.6%, 77.3% and 75.3%, respectively. On the other hand Oshsp17.9 shows higher identity of 91.8%, 90.8% and 93.2% with Oshsp17.3, Oshsp17.7 and Oshsp18.0, respectively. Several heat shock elements and one TATA box were deduced from 5’ upstream region of Oshsp17.9. The transcription initiation site of Oshsp17.9 was located on the 108th base upstream from the start codon and the 25th base downstream from the TATA box. Using 3’-UTR as probe, the transcription of Oshsp17.9 was induced after 41℃ heat shock treatment for 30 minutes, and reached to the highest level in one hour. Expression of Oshsp17.9 gene in E. coli was analyzed by western blot analysis after 2D-PAGE. We identified Oshsp17.9 as a new member of rice Class I LMW HSP with the molecular weight of 17.9 kD and pI value of 5.9.