Studies On The Interaction Between The Latex Invertase And 14-3-3 Proteins In Hevea Brasiliensis

The latex invertase in Hevea brasiliensis (para rubber tree) belongs to a kind of neutral/alkaline invertase (N/A-Inv), using sucrose as the only substrate, and plays a key role in the regulation of sucrose metabolism and latex regeneration. Our earlier results reveal that a member of the neutral/alkaline invertase gene family,i.e.HbNIN2, encodes the major latex invertase, and the regulation of its gene expression and enzymatic activity are closely related to tapping- and ethylene-stimulated latex production.To further characterize the molecular regulation of HbNIN2’s enzymatic activity,HbNIN2 was used as a bait to screen the HbNIN2-interacting factors by yeast two-hybrid,and investigate their regulatory modes on HbNIN2. The main findings are as follows:1. A normalized cDNA library of latex was constructed, and screened for the HbNIN2-interacting proteins by yeast two-hybrid. Among the finally affirmed interacting proteins, 43.8% turned out to be 14-3-3 proteins.2. A total of fourteen 14-3-3 protein (HbGF14) genes were isolated in H. brasiliensis by homology cloning methods, which are divided into two subtypes: epslion and non-epslion.3. Of the five N/A-Invs expressed in the latex, only HbNIN2, the predominant isoform, interacted positively with eleven of the fourteen HbGF14s as examined by the yeast two-hybrid technique.4. Using the Escherichia coli expressional system, large amounts of soluble His-tagged HbGF14 proteins and active HbNIN2 enzymes were obtained. Of the 11 HbNIN2-interacting HbGF14 proteins, only two (HbGF14-1 and HbGF14-12) revealed to markedly enhance the enzymatic activity of E. coli expressed HbNIN2 protein.5. Using the Pichia pastoris expressional system, His-tagged active HbNIN2 protein was produced, and large amounts of active HbNIN2 protein was obtained by the method of ammonium sulfate precipitaion. Of the 11 HbNIN2-interacting HbGF14 proteins, only HbGF14-1 could enhance the enzymatic activity of yeast-expressed HbNIN2 protein,whereas six, viz. HbGF14-2, HbGF14-4, HbGF14-9, HbGF14-10, HbGF14-13 and HbGF 14-14, significantly repressed HbNIN2’s activity.This study first systematacially screened the HbNIN2 interacting proteins,and examined the regulatory modes of the resulting interaction proteins on the E. coli or yeast expressed HbNIN2 recombinant proteins. The results obtained will forma foundation for futher exploring the molecular regulation of the latex invertase, and help elucidate the molecular mechnisms underlying the regulation of sucrose metabolism and latex regeneration in Hevea laticifers. In addtion, the results will provide theoretical basis for molecular breeding for higher rubber yield.