Cloning Of Heat Shock Protein90 Gene From Rice And Screening For Interacted Proteins

A series of physiological and metabolic responses caused by environmental stresses such as low temperature,high temperature,drought,and high salt,etc,were represented by reversible inhibition on the metabolism and growth,and heavily irreversible damage leading to death.Plants can make use of the already existing proteins or some new synthesis proteins to maintain the metabolism which carry through only under normal condition.These proteins,called plants adversity proteins,are very important for plants to tolerate adversity stress.Researching on these proteins is an important research content of molecular biology,proteomics and physiological responses of environmental stresses,and the central problem is the biological function of these proteins.With permeating of molecular biology theory and technique recently,research on plants adversity proteins has a great progress and some genes encoding plants adversity proteins have been isolated. Understanding physiological mechanism of plants adversity proteins comprehensively and deeply,and cognizing the physicochemical properties and biological function of plants adversity proteins inside plant bodies are very important.It is helpful to cognize the adaptive mechanism of tolerating adversity stress,and screen and cultivate new adverse-resistant varieties.Heat shock proteins,a class of conserved adversity protein,are induced by adversity stress,especially by high temperature.Hsps are one of major molecular chaperones discovered to date,and participate in and promote directly synthesis of primary peptide chains and fold and assembly of multiple subunit complexes of some proteins.At present, Hsps have been studied more in animals,but less in plants.In this study,we take Hsp90 from rice as the study object.We clone OsHsp90 gene and by methods of Yeast two-hybrid system,screen interacted proteins.It is helpful for further research on function,especially action mechanism of OsHsp90.The results are showed as below.1.Using Fluorescent Differential Display(FDD) method,the mRNA expression in rice roots stems and leaves was compared under low temperature and drought stress and normal condition.One interest fragment from OsHsp90 was isolated by combining the H. A.Yellow-PAGE(contained 0.1%H.A.Yellow) separation and Large Matrix Screening methods.The full-length cDNA,obtained by RACE-PCR,contained 2408 bp bases which encode a protein of 699 amino acid residues.The residues from 26 AA to 35 AA is Hsp90 family signature,and the residues from 28 AA to 182 AA is ATPase domain,the residues from 185 AA to 699 AA is Hsp90 domain(GeneBank access number is AB246888).2.Using Yeast two-hybrid system method,we screened OsHsp90 interacted proteins, especially interacted proteins which can combine with the region without ATPase domain. 13 positive colonies were screened on a strict criteria.Among them there were 3 colonies which can combine with the region without ATPase domain.After sequencing,amino acid sequence alignment showed that there were 4 interacted proteins discovered:(1) rice proline rich protein,containing 416 AA residues.The residues from 54 AA to 412 AA is rich in proline region;(2) rice putative carbamoyl phosphate synthetase small subunit, containing 462 AA residues.The residues from 75 AA to 222 AA is carbamoyl phosphate synthetase domain,and the residues from 261 AA to 462 AA is transglutaminaseâ… domain. (3) rice aminotransferaseâ…£,containing 361 AA residues.The residues from 244 AA to 273 AA is aminotransferaseâ…£signature,and the residues from 68 AA to 343 AA is aminotransferaseâ…£domain.(4) a rice putative protein,there isn't any functional domain existed and its function is unknown.3.According to the characteristics of interacted proteins,we discuss the roles of OsHsp90 in the process that interacted proteins display their main physiological function.