Gene Cloning And Expression Analysis Of The Rop Gene Family In Hevea Brasiliensis

Nature rubber from Hevea brasiliensis is a raw material of industrial and strategic importance, and the self-sufficient rate of our country is very low and most (3/4) depends on imports. Tapping is a key factor in the production of natural rubber, which can significantly promote the natural rubber biosynthesis and induction of secondary laticifers differentiation. Latex regeneration is a typical isoprenoid secondary metabolism that represents the main metabolic activity in the laticifers cell, and constitutes one of the limiting factors in determining rubber productivity in regularly tapped Hevea trees. Therefore, to clone the key genes and clarify the mechanism underlying latex regeneration, and will provide important target genes and theoretical guidance for directional molecular breeding in Hevea tree.ROP/RAC is the unique plant subfamily of the conserved Rho family of small GTPases. Accumulating evidence has shown that ROP/RAC act as predominant switches in signal transduction in plants, are involved in the control of many development processes, such as polarized cell growth, cell morphogenesis, regulation signal transduction events and defense and stress responses. And also involved in the metabolic regulation of plant secondary substances. In the rubber tree, we have found one by tapping regulated expression of Rab gene. But the ROP family genes in rubber tree latex regeneration process role have been no reports at home and abroad.Here, for the first time the genes encoding ROP small GTPases are cloned and expressionally characterized in Hevea brasiliensis, and the major results are as follows:1. Using the method of PCR and RACE, five ROP small GTPases genes were cloned from Hevea brasiliensis. The five genes were named HbRopl, HbRop2, HbRop3, HbRop4 and HbRop5, and their cDNA sequences were deposited in GenBank with accession nos. of JX316015, JX316016, JX316017, JX316018 and JX316019. All genes predicted to encode proteins of 22 KD; Bio informatics analysis showed that the proteins coded by the five genes contains seven domains typical of plant ROP GTPases.2. Genomic sequence cloning and analysis showed that HbRopl-4 had a transcription unit containing 6 exons and 7 introns belonging to group Ⅱ, but the HbRop5 belongs to group Ⅳ containing 7 exons and 8 introns. The prokaryotic expression vectors of all five genes were constructed, of which HbRop5 was expressed with high levels in E. coli.3. The expression characters of five HbRop genes were studied by semiquantitative RT-PCR and real-time quantitative RT-PCR analysis. The expression patterns of the five HbRop genes were investigated from different aspects, including the tissue-specific expression, expression in response to tapping, wounding, and seven hormone treatments, and the expression between the healthy and TPD trees. The five HbRops showed differential but redundant tissue-expression patterns, and were predicted to be involved in stress and defense response in rubber tree supported by the results of phylogenetic analysis and expressional response to wounding. The transcripts of HbRop3 and HbRop5 were conspicuously regulated by tapping in virgin trees, suggesting the roles in latex regeneration. HbRop2,3 and 5 showed synergistic expression with the induction and formations of secondary laticifers after mechanical wounding in the bark of one-year-old branch. Of the five HbRops, HbRop5 was the only one being markedly upregulated by cytokinin. The transcripts of HbRop3 increased gradually with the increase in TPD severity, suggesting a role in the occurrence and/or development of TPD in Hevea brasiliensis.4. The 5’regulatory regions of the five HbRops were isolated and analyzed by bio informatics. A number of ecis-acting regulatory elements were predicted in the promoter, some of which have been preliminarily proved expressional analysis performed on the five HbRops.5. The tissue specificity expression of HbRop3 and HbRop5 were analysed by mRNA in situ hybridization. The results showed both Rop genes were mainly expressed in phloem tissues, suggesting a relation with the formation of secondary laticifers from cambium.Take together, our results contribute substantially to further identification of the determining member of ROP small GTPases in the latex, and facilitate its role in the regulation of latex regeneration in Hevea brasiliensis, and are therefore theoretically and practically important.