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Cloning and functional analysis study of proline metabolism related gene in Solanaceae plants
virus induced gene silencing
Tobacco mosaic virus
|摘要:||植物於乾旱、寒害、鹽害和重金屬等逆境下，普遍以調節脯胺酸（proline）含量來回應環境變化。要瞭解菸草（Nicotiana benthamiana）調節脯胺酸含量的機制，須就其脯胺酸生成代謝相關基因進行功能分析。由於菸草脯胺酸生成代謝相關的基因尚未被選殖與定序，因此本論文以番茄（Lycopersicon esculentum cv. TA204）的基因組為材料建構脯胺酸生成代謝相關基因的片段。共有四個脯胺酸生成代謝相關的基因片段(約450 bp)被建構，其中包括三種脯胺酸生成酵素基因（pyrroline-5-carboxylate synthetase, P5CS; pyrroline-5- carboxylate reductase, P5CR; ornithine aminotransferase, OAT）與一種脯胺酸代謝酵素基因（proline dehydrogenase, PDH）成功的被選殖與定序。將這些基因序列比對已登錄於基因庫的番茄基因，發現至少有99% 的相似性。本論文遂以這些基因片段為探針，進行菸草脯胺酸生成代謝相關基因的功能分析。菸草的處理包括乾旱、過量銅與病毒誘導基因消寂（virus induced gene silencing, VIGS）等三項。試驗結果顯示，乾旱處理誘導菸草脯胺酸累積，同時也升高P5CS與OAT基因的表現，而PDH基因的表現則被抑制。此結果顯示，乾旱誘導菸草脯胺酸累積的機制，包括升高脯胺酸合成與降低脯胺酸代謝。另一方面，過量銅處理降低菸草脯胺酸含量，同時也降低P5CS與OAT基因的表現，PDH基因的表現亦被抑制。此顯示過量銅處理降低菸草脯胺酸含量的機制，是降低脯胺酸合成。雖然脯胺酸的代謝也被降低，但降低脯胺酸合成的效果，大於降低脯胺酸代謝的效果。試驗結果也顯示P5CR基因的表現與脯胺酸含量變化無關，推測該基因並未參與脯胺酸合成或該基因片段可能是假的。本論文進一步利用病毒誘導基因消寂系統，以確認乾旱處理下，菸草脯胺酸生成代謝相關基因的調節機制。三種脯胺酸生成代謝相關基因（P5CS、P5CR及PDH）被植入菸草鑲嵌病毒（Tobacco mosaic virus, TMV）的質體，並命名為TMV-P5CS、TMV-P5CR及TMV-PDH。試驗結果顯示，以TMV-P5CS感染菸草，可降低乾旱誘導脯胺酸累積的量。如果以TMV-PDH 感染菸草，則可提升乾旱誘導脯胺酸累積的量。以TMV-P5CR感染菸草不影響乾旱誘導脯胺酸累積的量。本論文的結論是：由番茄所選殖脯胺酸生成代謝相關的基因片段，可用來瞭解菸草脯胺酸含量調節的機制。其次，利用病毒誘導基因消寂系統，可用來偵測所選殖基因片段的真偽。以利進一步分析基因的功能。|
Fluctuation of proline level is a widespread episode among plants in response to environmental stresses, such as drought, chilling, salinity and heavy-metals. To reveal the proline episode in tobacco (Nicotiana benthamiana), the functional analysis of proline metabolism related genes (PMRGs) is vital. Because these genes are not yet cloned in tobacco, this thesis work begins with the construction of the fragment of tomato (Lycopersicon esculentum cv. TA204) PMRGs. Four PMRGs fragments (around 450 bp) that includes 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) were cloned successfully with 99% or more sequence identity to those genes of tomato available in Genbank. Taken these genes fragments as the probes, the functional analysis of PMRGs in tobacco were subjected to the treatments of drought, excess copper and virus-induced gene silencing. Our results demonstrate that drought increased proline contents in tobacco leaves along with the increase of P5CS and OAT gene expression, and with the suppression of PDH gene expression, suggesting that drought induced proline accumulation in tobacco was through the up regulation of P5CS and OAT gene expression and down regulation of PDH gene expression. On the other hand, excess copper decreased proline contents in tobacco leaf disks along with the suppression of P5CS and OAT, indicating that excess copper reducing proline content was via down regulation of P5CS and OAT gene expression. It was noticed that PDH gene was suppressed under excess copper treatment which is supposed benefit to proline accumulation, but that was unlikely able to overcome the action of P5CS and OAT genes. It was also noticed that, the gene expression of P5CR was not affected under both drought and excess copper stresses, suggesting that P5CR gene does not play a role in proline episode or another possibility could be that the fragment we cloned was a fake. The regulations of tobacco PMRGs in response to drought stress were confirmed by using the system of virus induced gene silencing (VIGS). Three PMRGs, P5CS, P5CR and PDH were constructed into Tobacco mosaic virus (TMV) plasmid, and named as TMV-P5CS, TMV-P5CR and TMV-PDH, respectively. The infection of TMV-P5CS on tobacco suppressed proline accumulation under drought stress. On the other hand, the infection of TMV-PDH on tobacco enhanced proline accumulation. The infection of TMV-P5CR on tobacco did not affect proline accumulation under drought stress. Based on these results we conclude that, among the PMRGs fragments we cloned from tomato, the clones of P5CS, OAT and PDH were closely related to the proline episode in tobacco. In addition, it is suggesting that VIGS can be used to confirm the function of PMRGs and for further functional analysis.
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