Screen The Proteins Interacting With HbJAZ Of Hevea Brasiliensis

Hevea brasiliensis is the main source of natural rubber. Laticifer is important organ of latex synthesis and storage in rubber trees.Mechanical damage or treatment of exogenous JA can induce differentiation and formation laticifer, suggesting that JA is the important signal molecules for regulation of differentiation of Hevea laticifer. JAZ proteins are important negative regulators in jasmonic acid related signalling pathways. JAZ can interact with JA-related transcription factors, thereby inhibit its transcriptional activity. However, the proteins interacted with JAZ proteins are still less known. In order to identify the Hevea proteins that interacted specifically with the JAZ protein, we used two strategies in this study:(1) constructing vector that expressed fusion protein of Hevea JAZ (HbJAZ1) and GST (Glutathione S-transferase) and prokaryotic expression of the fusion protein;(2) Constructing a bait vector HbJAZ1-pGBKT7and transformating into yeast strain Y2H gold. The following results were obtained:(1Successful amplification of HbJAZ1gene by RT-PCR. Sequencing results showed that PCR sequence was aligned with with HbJAZ1gene deposited in the Genbank (accession number GQ369508.1).(2) Construction of GST fusion expression vector of the HbJAZl gene by using pGEX-6p-1. The vector was transformed into E.coli stain BL21.(3) Screening the induced conditions of fusion protein expression.(4) Successful purification of HbJAZ1-GST fusion protein by using Glutathione-sepharose. Furhermore,HbJAZ1fusion protein was found to exist in pellet of lysised bacterial suspension and was possibly insoluble.(5) Successful constructing a bait vector pGBKT7-HbJAZ1,which was transformed into yeast strain Y2Hgold. Four interacted proteins were screened from the rubber latex cDNA library.This works would lay a foundation for isolation and further identification of the proteins interacted with HbJAZ1from the latex, which would help us to understand the regulation mechanisms of the JA signal pathway and further study of the mechanism of rubber biosynthesis.