| Summary: | 碩士 === 國立中興大學 === 農藝學系 === 93 === Many plants synthesize and accumulate proline in response to osmotic stress. Four major proline metabolism related genes (PMRGs) that include three proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR) and ornithine aminotransferase (OAT), and one proline catabolism gene, proline dehydrogenase (PDH) regulate proline biosynthetic pathways in plants. We previously demonstrated that drought stress increased the proline content in tobacco (Nicotiana benthamian) leaves along with the increase of P5CS and OAT gene expression, and with the decrease of PDH gene expression. In this study, to characterize transcriptional regulation of the key proline enzymes in tobacco N. benthamiana, four PMRGs DNA fragments (around 400 bps each) were identified and cloned. The sequences of those gene fragments from tobacco shared high homologies with more than 82.1% nucleotide sequence identity to those of tomato available in Genbank. To assess the relative important role of PMRGs involved in proline metabolism during drought stress in the tobacco plants, those gene fragments were constructed into Tobacco mosaic virus (TMV) viral vector to establish a system that can suppress multiple genes expression simultaneously via virus-induced gene silencing (VIGS). The P5CS activity increased in the primary stage (24 hour treatment) of drought stress, but the OAT activity increased in the later stage (36 hour drought treatment). Suppression of the P5CS gene expression via VIGS increases proline accumulation more efficient than that of suppression of OAT gene expression. In addition, the gene expression of P5CR did not affect proline accumulation under both drought and VIGS, suggesting that P5CR gene does not play important role in drought-induced proline accumulation. On the other hand, the PDH gene activity increased in rewater and infection of TMV containing antisense RNA of P5CS, OAT and PDH on tobacco. The fact that PDH gene was suppressed under drought treatment is possibly benefit to proline accumulation, but is unlikely able to overcome the action of P5CS and OAT genes. Based on these results, we suggest that drought-induced proline accumulation was via regulating the increase of P5CS and OAT gene expression. Thus both ornithine and glutamate biosynthesis pathways contribute key roles to the drought stress-induced proline accumulation in the N. benthamiana plants. P5CS especially plays a major role for the proline accumulation in the early stage of drought stress. In addition, the VIGS system for suppressing multiple genes simultaneously developed in this study can not only be used to confirm the function of PMRGs but also study other metabolic pathway in the further.
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