Cloning And Characterization Of Neutral/alkaline Invertase Genes From The Latex Of Hevea Brasiliensis

Sucrose can be irreversibly decomposed into glucose and fructose by invertase, which is known as one of the key enzymes controlling sucrose metabolism and partitioning of photosynthetic carbon. In Hevea trees, rubber biosynthesis occurs in a specialized cell-laticifer, with sucrose as the primary substrate. It is verified in Hevea by physio-biochemical evidence that latex invertase is located in cytosol, with pH optima at the range of 7.25 to 7.40, and belongs to the category of neutral/alkaline invertases, and acts as one of the key enzymes regulating the sucrose metabolism and determining the yield of latex. To date, the literature regarding latex invertase is still scarce at the molecular level. Therefore, cloning and characterization of neutral/alkaline invertases from the latex of Hevea not only help elucidating the regulation of sucrose metabolism, but provide solid data for the understanding of its role in latex regeneration.Here, for the first time the genes encoding invertases are cloned and expressionally characterized in Hevea brasiliensis, and the major results are as follows.1. Degenerate primer pairs were designed according to the conserved peptides of available plant invertases, and the full length cDNAs of two invertases from the latex of Hevea were finally cloned using the technologies of RT-PCR and RACE. The two invertase genes were named HbNIN1 and HbNIN2, and their cDNA sequences were deposited in GenBank with accession nos. of GU573728 and GU573727. Both HbNIN1 and HbNIN2 predicted a pepetide of 577 amino acids. Bioinformatics analysis showed that the proteins coded by the two genes are putative neutral/alkaline invertase, possessing 12 structural domains typical of plant neutral/alkaline invertases. Online subcellular location analysis revealed that HbNIN1 was probably located in the cytoplasm, whereas HbNIN2 had a cellular localization of cytoplasm or chloroplast.2. Genomic DNA sequence cloning and analysis showed that both HbNIN1 and HbNIN2 had a transcription unit containing 4 exons and 3 introns. Both invertase genes were heterogenously expressed in E. coli with high levels, and the recombinant HbNIN2 demonstrated an apparent neutral invertase activity with pH optimum of about 7.0, but recombinant HbNIN1 had no detectable activities.3. The expression characters of two HbNIN genes were studied in planta by real time fluorescent RT-PCR analysis. Among the three Hevea tissues of latex, bark and leaf tested, in the Normal trees, both genes showed a predominance expression in latex. In the tapping Hevea trees, Both wounding and tapping up-regulated the expression of HbNIN2, but decreasing that of HbNIN1. in particular, HbNINl was more predominantly expressed than HbNIN2 in the first Tapping, then decreasing of HbNIN1 and increasing of HbNIN2. And abundancing expression of two genes in the normal tapping tree which the yield stability. Among the 7 plant hormones tested, the expression of HbNIN2 is significantly induced by JA, ABA and SA. By comparison, HbNIN1 is suppressed by JA and also stimulated by SA, although its effect is less obvious than that on HbNIN2. Ethylene could induce the expression of both genes, but its effect was less conspiculous than the three above-mentioned hormones. The expression of two invertase genes differed among different rubber clones, with the highest expression in PR107, the intermediate in Reyan8-79, and the lowest in Reyan7-33-97. Interestingly, the expression of both invertase genes correlated positively with the yielding levels of PR 107 trees.4. The 5'regulatory region of HbNIN2 from Hevea brasiliensis was isolated using Genome Walker. There are two core configurations in promoter. Several sequences of eukaryotic cis-regulatory element were found in the promoter, which function were proved by real time fluorescent RT-PCR analysis of HbNIN2.Brief, for the first time we cloned and characterized two invertase genes from the latex of Hevea brasiliensis. Our research showed that:both of two genes are coding for Neutral/Alkaline invertase; HbNINl gene is likely to be a significant role in controlling the sucrose metabolism in latex of the Tapping tree; while in the Normal tree, both of HbNIN1 and 2 may together to participate in regulating the sucrose metabolism of latex. Take together, our results contribute substantially to further identification of the determining invertase member corresponding sucrose metabolism in the latex, and understanding its role in the process of latex regeneration and rubber productivity.