Identification And Functional Analysis Of HbICE2 Interacting Protein In Hevea Brasiliensis

Hevea brasiliensis is not only the main commercial source of natural rubber,but also the important strategic resource of China.Rubber trees were successfully introduced to south China in the 1950s,however,as typical tropical tree species,they are often subjected to cold stress.Cold stress is one of the most devastating environmental factors that limit the geographic distribution and productivity of rubber trees in China.Understanding the mechanisms underlying rubber tree responses to low temperature stress is therefore an important aim.Hb ICE2（Inducer of CBF Expression 2）is a key transcription factor in the low temperature response signaling pathway of rubber trees.However,the proteins interacted with Hb ICE2 are still unclear,which seriously hampered the advance further research of molecular mechanism for cold resistance of Hevea brasiliensis.This study mainly clone and characterization of the candidate proteins that interacted with Hb ICE2 in Hevea brasiliensis.The main results are as follows:1.Hb NAE,Hbb HLH70,Hb1433,Hb FBA and Hb NTF4 genes were cloned from the c DNA of the leaves of Hevea brasiliensis,and the open reading frame lengths were1350bp,1074bp,783bp,1077bp,and 1221bp,respectively.2.Yeast two-hybrid and bimolecular fluorescence complementary experiments showed that both Hb NTF4 and Hbb HLH70 interact with Hb ICE2,and Hb NAE,Hb1433,Hb FBA and Hb ICE2 do not interact with Hb ICE2,suggesting that Hb NTF4 and Hbb HLH70 may interact with Hb ICE2,a key factor for low-temperature signaling,to regulate rubber trees low temperature response.3.Transcriptional activation assay showed that Hbb HLH70 had auto-activating activity in yeast cells,and its transcriptional activation activity was dependent on the N-terminus of Hbb HLH70.4.Amino acid sequence alignment of Hb NTF4 and Hbb HLH70 and other plant homologous genes show that Hb NTF4 and Hbb HLH70 have high homology with other plant homologous genes.5.We use the tobacco transient expression system and rubber tree protoplast transformation experiments to perform subcellular localization analysis,the results showed that Hb NTF4 was localized in the nucleus,cell membrane and cytoplasm;Hbb HLH70 was localized in the nucleus.6.The expression of Hb NTF4 and Hbb HLH70 in leaves,stems,latex,roots,and bark of rubber trees was analyzed by RT-PCR.The results showed that there was no tissue specificity for Hb NTF4 and tissue specificity for Hbb HLH70 expression in the leaves.The expression level was significantly higher than other tissues.7.The transgenic homozygous plants 35S::Hb NTF4 and 35S::Hbb HLH70 were treated with low temperature stress.It was found that the survival rate of transgenic plants was significantly higher than that of wild-type plants under low temperature stress.The results of cold stress-related physiological indicators showed that the transgenic plants35S::Hb NTF4 and 35S::Hbb HLH70 have lower levels of malondialdehyde,conductivity,and H₂O₂accumulation than wild type;while proline content is higher than wild type,these results indicate that 35S::Hb NTF4 and 35S::Hbb HLH70 Can improve the cold resistance of Arabidopsis.8.Prokaryotic expression analysis was performed on Hb NTF4 and Hbb HLH70.The results showed that Hb NTF4 was induced at 37°C,Hbb HLH70 at 28°C,and 0.2 mmol/L IPTG concentration for 8 hours.The expression was the highest.Hb ICE2 in turn regulates the protein activity of Hb ICE2;it provides an experimental basis for whether Hbb HLH70 protein and Hb ICE2 form a large protein complex in the nucleus to participate in the ICE-CBF cold signal pathway regulation network.The above research results show that under low temperature stress,Hb NTF4 and Hbb HLH70 of Hevea brasiliensis can interact with Hb ICE2,a key factor for low temperature signaling of Hevea brasiliensis,and regulate the expression of downstream stress-related genes to change the relevant indicators of the plant to improve the plant’s cold resistance.