Study On Expression Profiles Of Sucrose Transporter Hbsut5 From Hevea Brasiliensis

In para rubber tree (Hevea brasiliensis), rubber biosynthesis occurs in a specialized cell-laticifer, with the sucrose as the primary substrate. Through a phloem-mediated long-distance transportation, sucrose reaches the laticifers in the phloem of trunk bark, which is the major site for rubber harvesting. Due to lacking plasmodesmata connections between laticifers and their neighboring cells, sucrose imports into the aticifers by a transmembrane pathway. In higher plants, it has been made clear that sucrose tranporters (SUTs) mediate transmembrane transportation of sucrose, and play a critical role in photosynthetic carbon partitioning between different sinks. In Hevea tree, electrophysiological evidence has demonstrated that sucrose co-transport with protons into the laticifers, and there exists proton-sucrose co-transporters in the plasmamembrane of laticifers. Therefore, the studies regarding the physiological roles of SUT in Hevea tree, espetially its regulation in sucrose loading to laticifers, not only help unravel the mechanisms underlying the mechanisms of carbon partitioning between sinks, but contribute to the elucidation of the regulation of sucrose loading to laticifers.In our preliminary studies, HbSUT5 was revealed to be down-regulated by tapping and wounding, but not affected by Ethrel treatment in latex. This expression mode was distinct to that of HbSUT3, which proved to be the key SUT member determining sucrose loading to laticifers and related to rubber productivity. Therefore, this article started on researching the expression regulation profiles of HbSUT5 and its physiological function. The major results are as follows:Using the yeast expression system we found that HbSUT5 coding protein is a functional sucrose transporter, the rusults showed that HbSUT5 is a sucrose transporter of high affinity, low transport capacity.Expression levels of HbSUT5 in latex, bark and leaves was fully displayed higher or the highest in HbSUT family members, and with the development of leaves it drivened into decline. Tapping or wounding treatment can regulated latex yield, in virgin trees, with the frequency increased of tapping and the processing time extended of wounding, the expression of HbSUT5 was decrease, this was contrary with the results of HbSUT3. And we found JA and SA can regulate HbSUT5 expression.HbSUT5 was presumably the single or lower copy gene in rubber genome. Its 5' regulatory region was obtained using GenomeWalker, consists of 959bp, containing the typical eukaryotic core promoter region and a number of important cis-acting element. Several sequences were found:a jasmonic acid response related components CGTCA-motif, an auxin response related components TGA-element, a salicylic acid response related components TCA-element, and a TC repeats which may be related to resistance and stress response, as well as a meristem tissue expression related components CAT-box, Protoplast transient expression studies show that promoter sequences can activate the expression of the reporter gene GFP in tobacco leaves.Using fluorescence quantitative RT-PCR technique, the author through screening 23 candidate internal control genes of rubber tree, identified the use of internal control genes in 8 different experimental conditions when analysis the gene expression. Study of the gene expression in normal latex of tapping trees can use eIF-1 and MPD1 as internal control genes; ethrel treated sample can use UBC2 and MPD2 as internal control genes; JA treated sample can use UBC2 and DRH2 as internal control genes; SA treated sample can use Ef-2 and MPD2 as internal control genes; and UBC2 and PP can be used in the study Reyan 7-33-97 gene expression in individual plants; the sample of different cultivar clones and wild germplasm materials can use UBC2 and UBC3 as internal control genes; This study found gene expression were variety in different tissues materials, need more than five internal control gene to correcte; in the study of gene in the leaves at different developmental stages, 18S-rRNA and Act4 are suitable use as internal control genes.Comprehensive analysis on tapping, wounding and some hormone treatment to the effect of HbSUT5 expression, and comparing different tissues and different developmental stages on HbSUT family expression, as well analyzing the promoter elements of HbSUT5, we preliminary hold that HbSUT5 may participate in manipulation of laticifer sucrose supply, and it possibly function on the carbon delivery in many kinds of tissue especially young development tissues.The results of this study do great help to the elucidation of the regulation of HbSUT5 and its physiological role. For the first time, the internal control genes suitable for gene expression analysis in Hevea tree were systematically screened under different conditions, which contributes a great deal to Hevea molecular biology.