Screening For The Substrate Of E3 Ubiquitin-protein Ligase Related To Low Phosphorus Stress In Hevea Brasiliensis

Protein ubiquitination is a common posttranslational modification in eukaryotic cells,which play important roles in many physiological processes.The functional diversity and specificity of protein ubiquitination is achieved by the precise identification of key substrates by different E3 ubiquitin ligases.Studies shows that protein ubiquitination play important roles in the regulation of phosphorus uptake and transport,metabolism,hormone metabolism and root morphogenesis in response to low phosphorus stress in plants.In this study,the homogenized yeast two hybrid cDNA librarys of the aboveground part and underground part were constructed from tissue culture seedling of Reyan 7-33-97 rubber tree.The bait vectors of E3 ubiquitin ligases HbSINAT2 and HbTIR1 were constructed,which were obtained by SSH under low phosphorus stress.The proteins that interacted with HbSINAT2 or HbTIRl were identified using a yeast two-hybrid system search of Hevea cDNA librarys of the aboveground part and underground part,respectively.The full-length cDNA of the substrates were obtained by electronic cloning and analyzed by bioinformatics to explore the regulation function of the E3 ubiquitin ligases to phosphorus deficiency stress in rubber tree.The main research results were as follows:1.The homogenized Yeast Two-hybrid cDNA library of the aboveground part of rubber tree was established,had a library titer of 8×107cfu/mL,and the recombination rate of more than 0.5 KB inserts was 100%;2.The homogenized Yeast Two-hybrid cDNA library of the underground part of rubber tree was established,had a library titer of 7 × 107 cfu/mL,and the recombination rate of more than 0.5 KB inserts was 87%;3.Two bait vectors of E3 ubiquitin ligases HbSINAT2 and HbTIR1 were constructed,which were related to phosphorus-deficiency tolerance.4.Fifteen positive clones or ten positive clones were were preliminarily screened by yeast two-hybrid system,which may interact with HbSINAT2 or HbTIR1,respectively.5.The full-length cDNAs of 8 candidate HbSINAT2-interacting proteins were isolated,and bioinformatically classified as binding protein,stess relative protein and signal transduction protein.6.The full-length cDNAs of 5 candidate HbTIR1-interacting proteins were isolated,and bioinformatically classified as enzyme,regulatory protein and other conserved hypothetical protein.The results showed that HbSINAT2,HbTIR1 and their interacting proteins might be involved in the physiological processes of hormone metabolism,signal transduction and growing development under low phosphorus stress.