Molecular Cloning And Expression Analysis Of Calnexin In Tomato

ER-resident proteins, especially chaperone proteins and folding enzymes are thekey to various roles of endoplamic reticulum in cell. Lectin-like chaperone proteins inER are mostly Calnexin and Calreticulin. Calnexin is a type I membrane protein,while Calreticulin is a soluble protein in ER. The amino acids sequence and secondarystructure of Calnexin in ER is similar to Calreticulin, and both have the similarmechanism to recognize substrate. Calnexin, Calreticulin and ERp57 constitute theCalnexin/Calreticulin cycle which can assist glycoproteins with N-linkedoligosaccharide to fold and assembly. Calnexin and Calreticulin are also the memberof ER quality control to make sure only native proteins to escape from ER. Moreover,Calnexin can deliver the misfolded proteins into cytoplasm to degrade by proteasome.Furthermore Calnexin is important Ca²⁺ storable proteins in ER. It probabalymodulate Ca²⁺ homeostasis of ER. Recent results indicated that lectin-like chaperoneplay role in apoptosis, for example the cleaved product of Calnexin which is cleavedby caspase family could lead to the attenuation of apoptosis. Calnexin paly importantroles in animal cell, but the expression and function of Calnexin in plant cell have lessresearch. In this experiment, a cDNA of 1964bp encoding a full-length calnexin was clonedfrom Lycopersicon esculentum, and designated as Lecnx61.0. From the analysis ofamino acids sequence, we found the N domain and P domain of calnexin in ER wassimilar in different species. The segment in cytoplasm of calnexin contained theretention signal of ER. A single copy of Lecnx61.0 was found in tomato genomicDNA by southern-blot analysis. Furthermore, the author researched the expression pattern of Lecnx61.0 in tomato.The results indicated that the expression of Lecnx61.0 in anther, ovary and seed wasdifferent. During the development of anther, the amount of LeCNX61.0 decreased. Inthe opening flower, no obvious signal was detected. Howerver, the amount ofLeCNX61.0 increased little by little during the development of ovary. During thegermination of seed the amount of LeCNX61.0 had no obvious change. Furthermore,the expression of Lecnx61.0 was induced by heat, cold and salty stress, but had noresponse to drought stress. And the protein of LeCNX61.0 only in the 2mm-long tipstissues made response to Ca2+ depletion.In order to find the role of Calnexin in the resistance to abiotic stress, Lecnx61.0was inserted into binary plant vector pROKII. The resulting plasmid, namedpROK-Lecnx61.0, was mobilized to Agrobacterium tumefaciens strain LBA4404 usedfor plant transformation. Lecnx61.0 gene was introduced into Lycopersiconesculentum by Agrobaterium tumefaciens-mediated transformation under the controlof 35S-CaMV promoter. The analysis of PCR and Western indicated that Lecnx61.0had been introduced into tomato and expressed correctly. But the effect ofoverexpressing Lecnx61.0 on the resistance to stress will be made further reseach.