Cloning, Expression And Functional Characterization Of Trehalose-6-phosphate Synthase Genes In Hevea Brasiliensis

Trehalose is synthesised from UDP-glucose and glucose-6-phosphate through two enzymatic steps:first by trehalose-6-phosphate synthase(TPS) to produce trehalose-6-phosphate(T6P), and then by trehalose-6-phosphate phosphatase (TPP) to dephosphorylate T6P and produce trehalose and inorganic phosphate (Pi). Trehalose is a very stably non-reducing disaccharide, and it can stabilize the structure of membranes and protein in harsh environmental conditions of heat, cold, high osmotic pressure and drought. As a result, trehalose acts as an important protective molecule in maintaining biological functions and life processes in living organisms. However, studies of the enzymes involved in trehalose synthesis is still lacking in rubber trees. Therefore, cloning and characterization of trehalose-6-phosphate synthase in Hevea trees not only help elucidating the regulation of trehalose metabolism, but also provide solid data for the understanding of its roles in resistance to abiotic stresses.Here, the genes encoding trehalose-6-phosphate synthases were cloned for the first time and expressionally characterized in Hevea brasiliensis. The major results are as follows.1. By searching the EST sequences available in rubber trees, we obtained assembled contigs of putative trehalose-6-phosphate synthase genes. And then the full length cDNAs of two trehalose-6-phosphate synthase genes of Hevea trees were cloned by using the technologies of RACE and PCR. The two trehalose-6-phosphate synthase genes were named HbTPS1 and HbTPS2. HbTPS1 predicted a peptide of 928 amino acids, and HbTPS2 predicted a peptide of 940 amino acids. Online subcellular location analysis revealed that HbTPS1 was probably located in the endoplasmic reticulum, whereas HbTPS2 was probably located in mitochondria or nucleus. Hydrophobicity analysis revealed that both HbTPS 1 and HbTPS2 were hydrophilic proteins.2. The expression characters of the two HbTPS genes were studied by real-time quantitative RT-PCR analysis. Among the eight Hevea tissues (leaf, bud, female flower, male flower, seed, branch bark, old leather and latex) collected in the regularly tapped rubber trees, both HbTPS genes showed a predominance expression in bark. In the latex of virgin Hevea trees, both wounding and tapping up-regulated the expression of the two HbTPS genes. HbTPS2 was more abundantly expressed than HbTPS1 in the latex of regularly tapped trees, whereas in the latex of wounding treated rubber trees HbTPSl was more abundantly expressed than HbTPS2. Among the treatments of six plant hormones, the expression of HbTPS1 was conspicuously up-regulated by ET and 2,4-D. After GA treatment, the expression levels of the two genes first decreased and then increased.In rubber seedlings, HbTPS1 and HbTPS2 showed differential expressions in response to low temperature (4℃), high temperature (40℃) and drought (20% PEG6000) stresses. After the low temperature treatment, the expression level of HbTPSl significantly increased in the bark and xylem, and first increased and then decreased in the root. And HbTPS2 expression significantly increased in the bark, and first increased and then decreased in the xylem and root. After the high temperature treatment, the expression of HbTPS1 first increased and then decreased in the leaf, and markedly increased in the root. HbTPS2 expression first increased and then decreased in the xylem, and markedly increased in the root. After the treatment of drought stress, HbTPS1 expression level conspicuously increased in the root while HbTPS2 expression first decreased and then increased substantially in the bark, xylem and root.3. The 5’regulatory regions of HbTPSl and 2 genes were isolated using Genome Walking technology, with 1596-bp and 1509-bp in length, respectively. A typical eukaryotic core promoter sequence was found in the 5’regulatory region of HbTPS2. A number of eukaryotic cis-acting regulatory elements were found in these two promoters, some of which have been proved in the above expressional analysis.4. The tissue specificity of HbTPS expression was analysed by mRNA in situ hybridization in young stem and midrib of Hevea brasiliensis. The results showed the mRNA transcripts of the two TPS genes were located mainly in phloem. HbTPS1 mRNA levels were relatively higher in the midrib than in young stems, whereas the expression of HbTPS2 was significantly higher in young stem than in midrib.5. Yeast complementation assays showed that both HbTPS genes can complement the functional deficiency of the TPS-deficient yeast mutant strains, suggesting the functionality of the two HbTPS genes.6. Transgenic Arabidopsis plants overexpressing HbTPS1 displayed improved tolerance to freezing, high temperature, drought and salinity stresses compared with wild type Arabidopsis plants. Transgenic Arabidopsis overexpressing HbTPS2 growred much more faster than the wild-type Arabidopsis plants, and showed characters of early flowering.In this paper, two TPS genes were cloned and characterized for the first time in Hevea brasiliensis. The results suggest that the two HbTPS genes have multiple functions, including stress tolerance, growth and flowering promotion.Take together, our results do great help to the further elucidation of the biological functions of the two HbTPS genes, and also provide two reliable target genes for future transgentic rubber breeding.