Clone And Expression Analysis Of The Key Enzymes Of Ammonia Assimilation From Hevea Brasiliensis

Rubber(cis-1,4-polyisoprene),an isoprenoid polymer with no known physiological function to the plant, is produced in about 2000 plant species with varying degrees of quality and quantity. Rubber is the raw material of choice for heavy duty tires and other industrial uses requiring elasticity, flexibility, and resilience,such as sars and planes et al. Hevea brasiliensis has been the only commercial source of natural rubber mainly because of its abundance in the tree, its quality, and the ease of harvesting. Glutamate dehydrogenase (GDH) is present mainly in mitochondria in higher plants and catalyses both the amination ofα-oxoglutamate,with NADH as the electron donorand the deamination of glutamate to ammonia andα-oxoglutamate ,with NAD+ as the electron recepter. The enzyme seems to function in assimilation of ammonia under stress conditions such as high temperature , in senescence and other abnormalities. It also functions in higher plants to direct carbon skeletons into the citric acid cycle under conditions of carbon stress. It has important academic meaning and applied value to study potential function of cysteine protease in Hevea brasiliensis.In this study, first strand cDNA synthesis was performed by reverse transcribing total latex RNA using a modified oligo-dT primer anda PCR product of 830bp was obtained from the cDNA , using specific primers for GDH genes designed based on the reported sequence of Arabidopsis thaliana Glutamate dehydrogenase (AtGDH,gi:12321666)gene. The product was ligated to pGEM-T vector and sequenced. Length of the coding region was 830bp.A comparison of composition nucleic acid sequence and deduced amino acid sequence of GDH among the different plants show the sequences are highly homologous , suggesting its potential involvement in dehydrogenase. HbGDH has high sequence homology to Arabidopsis thaliana NADP-specific glutatamate dehydrogenase that reported in the data base. The phylogenetic tree of amino acid sequence of GDHs among the different plants show HbGDH and AtGDH together. The predicted amino acid sequence of the HbGDH has high similarity to those of HbGDH (82%), OsGDH(79%). Sequence alignment indicate that there is about two conserved regions GLFV-dehydrog-N and GLFV-dehydrog .HbGDH may have the activity of dehydrogenase and have relate to nitrogen metabolism in plants. RT-PCR showed that HbGDHgene expression has organization difference: highest in the latex, secondly in the leaf, lowest in the bark.The exact physiological roles of GDH in plant carbon and nitrogen metabolism and remobilization in plants remain largely speculative.