Construction Of Normalized Hevea Latex CDNA Library And Screening Of Proteins Interacting With HbNIN2

In plants, invertases (Invs) are widely distributed and irreversibly catalyze the cleavage of sucrose into glucose and fructose. Invs play an important role in plant development, environmental stress response, and also other biological processes. Furthermore, plant Invs interact with different kinds of proteins, and form a complex network of proteins involved in multiple physiological processes. Previously, studies on rubber tree latex invertase have confirmed that this enzyme is an important enzyme playing a critical role in regulation of sucrose catabolism and rubber production, and have been identified to be a neutral or alkaline Invertase (N/A-Inv). Of the N/A-Inv isoforms identified in rubber tree latex, HbNIN2have been confirmed as the key member. However, no studies are available in that proteins interact with rubber tree N/C-Invs, and it is unclear whether some kind of proteins specificcally interact with HbNIN2and affect its functions.In this study, the normalized cDNA library of latex was constructed in yeast. The interaction proteins of HbNIN2were screened using the yeast two-hybrid system, and their characteristics were initially analyzed and validated. The full-length cDNA of the positive interactions were isolated by PCR technique, analyzed by bioinformatics, and preliminarily studied for their functions in the rubber tree. The main results of this study were as follows:1. The rubber tree latex normalized cDNA library has been constructed in yeast, with a library titer of1.8x108cfu/ml, and a recombination rate of about85%.2. For the first time,15candidate HbNIN2-interacting proteins were screened out by yeast two-hybrid technology, and experimentally verified.3. The full-length cDNAs of14candidate HbNIN2-interacting proteins were isolated, and bioinformatically classified as binding protein, enzyme, stress relative protein and other conserved hypothetical proteins.4. Successful expression of CBL-interacting serine/threonine-protein kinase (37)、 GTP binding protein (51)、 Clathrin coat assembly protein (72) and2conserved hypothetical protein (66,100), and several other interactions’protein were fulfilled in E. coli, and laid the foundation for further research on HbNIN2-interacting proteins.5. Analysis results of putative interacting proteins by functional prediction, indicated that they may participate in rubber laticifer biological processes, such as Ca2+signal pathway vesicle transport and stress response. This results indicated that the HbNIN2may be interacted with many proteins, which involved in biological processes such as latex regeneration regulation, environmental stress response, signaling pathway and regulation of growing development.