甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达  

cDNA Cloning and Expression of a Cytosolic Small Heat Shock Protein Gene (CaHSP18) from Capsicum annuum

作　　者：郭尚敬[1] 陈娜[1] 郭鹏[1] 孟庆伟[1] 

机构地区：[1]山东农业大学生命科学学院

出　　处：《植物生理与分子生物学学报》2005年第4期409-416,共8页Journal Of Plant Physiology and Molecular Biology

基　　金：国家重点基础研究发展规划项目(No.G1998010100);国家自然科学基金项目(No.30370854)资助。~~

摘　　要：用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779bp的cDNA基因序列,包含一个480bp开放阅读框,编码159个氨基酸。Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族。Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导。原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力。Full length 779 bp cytosolic clsss Ⅰ sHSP cDNA was cloned from heat-shocked sweet pepper leaves using a PCR approach with degenerate primers designed from conserved motifs found in a number of plant cytosolic clsss Ⅰ sHSP genes, named CaHSP18. Its accession number is AY284925. CaHSP18 was high identified with Nicotiana tabacurn （Fig. 1）. A multigene family was found in sweet pepper genomic DNA by Southern-blot analysis （Fig.3）. Northern blot revealed that the expression of CaHSP18 in sweet pepper was induced by heat treatment and was significantly different between different tissues （Fig.4）. The transcription of CaHSP18 in leaves and stems were higher than those in roots. The expression of CaHSP18 could be detected after chilling treatment at 4℃ for 2 d. Recombinant CaHSP18 was overexpressed in Escherichia coli to study its possible function under heat and chilling stress. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that CaHSP18 was expressed in Escherichia coli （Fig.6）. The growth of wild type and transformed cells was similar at 37℃ （Fig.5）.Upon transfer from 37℃ to 50℃, a temperature known to cause cell autolysis, those cells that accumulated CaHSP18 showed improved viability compared with the control lines （Fig.7）. To test the hypothesis that sHSPs may be involved in protection against chilling stress, the viability of recombinant cells at 4℃ was studied. The sweet pepper CaHSP18 significantly enhanced cell survivability （Fig.8）. These results indicate that plant cytosolic clsss I sHSP have protective functions not only against heat stress but also against chilling stress.

关 键 词：甜椒 小分子量热激蛋白 基因克隆 基因表达 

分 类 号：S641.3[农业科学—蔬菜学]