Isolation And Preliminary Characterization Of Proteins Interacting With CaWRKY40and CaWRKY3in Peper

Pepper(Capsicum annuum L.) is an important economic crop, the planting areasfor it is Top2in China,and is also a widely grown crop all of the world. However,various challenges by biotic or abiotic stresses have seriously negative effects on yieldand quality during pepper production. Cultivate and improve the stress tolerance ofnew varieties is an effective way to solve the problem of pepper diseases, whichdepends on the elucidation of he molecular mechanism. In recent years, studies haveshown that there are multiple ways to regulate the stress tolerance response in plant,which the salicylic acid-mediated systemic acquired resistance(SAR)is a hot topic inthe study,and is also a critical means of pepper stress tolerance response.Plant stress response requires resistance gene expression, transcription factorsthat regulate stress resistance gene expression play an important role in the regulation.In this study, the initial objects of study are CaWRKY40and CaWRKY3transcriptionfactor in salicylate resilience way, use the advanced yeast two-hybrid screening andidentification of its interacting proteins in order to establish a WRKY transcriptionfactor signaling pathways in disease resistance in pepper lay the foundation for theplant resistance breeding. The main results obtained in this experiment as follows:1. Use SMART and DSN homogenization technology to build a salicylic acid(SA) processing pepper yeast two-hybrid full-length homogenization cDNA library.The library has a titer of1.45×106, the average insert length is1.5-3kb, has goodeffect of homogenization, in line with the requirements of building a library of theyeast two-hybrid system.2. Use DUALhunter system to screen the library and achieve the CaWRKY40and CaWRKY3effectively interacting proteins,the number of the CaWRKY40interacting proteins is2, and the number of CaWRKY3interacting proteins is25. TheCaWRKY40s interacting proteins are vesicle-associated membrane Protein(VAMP),and thylakoid ADP/ATPcarrier protein, tentatively named them as CaVAMP andCaACP.Contrast to the VAMP protein research in Arabidopsis, to speculate CaVAMPmay be involved in disease resistance response. CaWRKY3′s interacting proteins are divided into five categories,chloroplast-related proteins, enzymes, specific regioncontaining protein, a special domain of the protein and the presumption unnamedprotein. The analysis show that most of the interacting proteins were associated withresistant and low phosphorus stress regulation, which are highly consistent with thefuction of CaWRKY3.3. In summary, a full-length normalized cDNA library of pepper under Salicylicacid treatment was constructed. In this library27positive clones were selected as baitwere CaWRKY40and CaWRKY3.The preliminary identification showed thesepositive clones may be involved in pepper response to different stress responses invariety ways. Isolation and identification of this positive clones has laid a solidfoundation for the construction and analysis of pepper signaling networks ofresistance.