| Hevea brasiliensis is the only source of commercial natural rubber. Laticifer is the main place of biosynthesis and accumulation of latex in rubber tree. Differentiation and amount of laticifer are the key factors which affect the production of rubber. Mechanical damage can induce differentiation and formation of laticifer. Jasmonic acid (JA) is a key signalling molecule of mechanical damage. Treatment with exogenous JA can induce diffferentiation and formation laticifer, however, the molecular mechanism underlying remains unclear. To clone the laticifer-differentiation-related genes in Hevea brasiliensis, we obtained several ESTs related laticifer different throught differentially expressing analysis of a suppression substactive hybridization (SSH) cDNA library of barks. These ESTs include two dehydrins and one NAC transctiptional factor (TF) gene, all of which seem to relate with dehydration. The full length cDNA of NAC gene was obtained by Rapid Amplification of cDNA Ends (RACE). It was 1116 bp in length containing a 870 bp open reading frame (ORF) that encoding a protein of 287 amino acids with a predicted molecular mass of 32.8KDa. The the sequence, containing a DNA binding dormain on N terminal and putative transcription active dormain on C terminal, indicating a typical structure of NAC TF, was therefore termed as HbNAC1. In comparison to several NAC genes of other plants, HbNAC1 protein shows sequence identity ranging from 31% to 67%. To reveal the physiological function of the gene, further investigations were conducted as following:1. The expression patterns of HbNAC1 were identified by using semi-quantitative RT-PCR: Analysis showed that the expression of HbNAC1 in different tissues was various, the expression was strongest in seeds, and stronger in leaves, latex and bark, but lowest in roots. Additionally, mechanical wounding had no obvious effect on the expression of HbNAC1. Furthermore, when treated with six different plant hormones, HbNAC1 was obviously up-regulated after treatment with ABA and ET, slightly increased by JA, but down-regulated by GA and CKT.2. Determination of transcriptional activity:five fragments of HbNAC1 with different length were inserted into pBD-GAL4-cam and transferred into yeast, the result of transcription activity assay showed that both full-length sequence and C terminal of HbNAC1 indicated strong transcriptional activity, whereas DNA binging domain in N terminal alone did not. Analysis of truncated sequence of C terminal showed that transcriptional activity lost when 75 aa were truncated from C terminal, and truncation of 15 aa did effect, which indicating that the sequence is essential for transcriptional activity. 3. Cellular location of HbNACl:GFP fusion expression vector of GFP-HbNAC1 were constructed and transformed into tobacco. HbNAC1 were verified to transferred into TO plants by PCR. Further investigation would be carried out in near future.4. Determination of physiological role of HbNAC1 throught genetic transgene:plants over-expression vector and RSDX fusion transcriptional repression vector of HbNAC1 gene were constructed and transformed it into Arabidopsis using Floral dipping method. TO transgenenic seed have been harvested. Hygromycin selection is ongoing. Drought and dehydration experiments would be carried out in near future.NAC transcription factors that are unique to plants are the new type transcription regulatory factors that have multiple biological functions. They play a key role in regulation of plant growth and development, hormone level and responses to various kind stresses. Some transcription factors of the NAC familly play a key role in the JA pathway, thus characterization of HbNAC1 function is important to reveal the mechanism of rubber biosynthesis. This work lay a good foundation for further study of the signal pathway during jasmonic-induced laticifer differentiation. |
PDF Full Text Request