RESUMO
In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.
Assuntos
Genes de Plantas , Hevea/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Mitocondriais/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Sequência de Bases , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hevea/metabolismo , Látex/biossíntese , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Estresse Oxidativo , Casca de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Tapping panel dryness (TPD) occurrence in high latex yielding rubber tree (Hevea brasiliensis) is characterized by the partial or complete cessation of latex flow upon tapping leading to severe loss in natural rubber production around the world. The goal of this study was to identify genes whose mRNA transcript levels are differentially regulated in rubber tree during the onset of TPD. To isolate TPD responsive genes, two cDNA libraries (forward and reverse) from total RNA isolated from latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. In total, 1,079 EST clones were obtained from two cDNA libraries and screened by reverse Northern blot analysis. Screening results revealed that about 352 clones were differentially regulated and they were selected for sequencing. Based on the nucleotide sequence data, the putative functions of cDNA clones were predicted by BLASTX/BLASTN analysis. Among these, 64 were genes whose function had been previously identified while the remaining clones were genes with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. RT-PCR analysis was carried out to validate the up-regulated genes from both the libraries. Among them, two genes were strongly down-regulated in TPD trees. The level of mRNA transcripts of these two genes was further examined by conventional Northern and RT-PCR analysis. Results indicated that the expression level of two genes was significantly lower in TPD trees compared to healthy trees. Many TPD associated genes were also up-regulated in TPD trees suggesting that they may be involved in triggering programmed cell death (PCD) during the onset of TPD syndrome. The results presented here demonstrate that SSH technique provides a powerful complementary approach for the identification of TPD related genes from rubber tree.
Assuntos
Hevea/genética , Látex/metabolismo , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes de Plantas/fisiologia , Hevea/metabolismo , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop for natural rubber production. At present, more than 9.5 million hectares in about 40 countries are devoted to rubber tree cultivation with a production about 6.5 million tons of dry rubber each year. The world supply of natural rubber is barely keeping up with a global demand for 12 million tons of natural rubber in 2020. Tapping panel dryness (TPD) is a complex physiological syndrome widely found in rubber tree plantations, which causes severe yield and crop losses in natural rubber producing countries. Currently, there is no effective prevention or treatment for this serious malady. As it is a perennial tree crop, the integration of specific desired traits through conventional breeding is both time-consuming and labour-intensive. Genetic transformation with conventional breeding is certainly a more promising tool for incorporation of agronomically important genes that could improve existing Hevea genotype. This chapter provides an Agrobacterium-mediated transformation protocol for rubber tree using immature anther-derived calli as initial explants. We have applied this protocol to generate genetically engineered plants from a high yielding Indian clone RRII 105 of Hevea brasiliensis (Hb). Calli were co-cultured with Agrobacterium tumefaciens harboring a plasmid vector containing the Hb superoxide dismutase (SOD) gene and the reporter gene used was beta-glucuronidase (GUS) gene (uidA). The selectable marker gene used was neomycin phosphotransferase (nptII) and kanamycin was used as selection agent. We found that a suitable transformation protocol for Hevea consists of a 3-d co-cultivation with Agrobacterium in the presence of 20 mM acetosyringone, 15 mM betaine HCl, and 11.55 mM proline followed by selection on medium containing 300 mg/L kanamycin. Transformed calli surviving on medium containing 300 mg/L kanamycin showed a strong GUS-positive reaction. Upon subsequent subculture into fresh media, we obtained somatic embryogenesis and germinated plantlets, which were found to be GUS positive. The integration of uidA, nptII, and HbSOD transgenes into Hevea genome was confirmed by polymerase chain reaction (PCR) as well as Southern blot analysis.