| The synthesis and accumulation of nicotine is known to be controlled by various developmental, environmental, and chemical factors. The commercial practice of topping can result in nicotine increase in the leaves of Nicotiana tabacum. The factors controlling the topping-induced nicotine biosynthesis increase likely involve a complex physiological response in the plant as a result of altered phytohormones and wound induced signaling. Although the number of cloned genes involved in nicotine biosynthesis has been accumulating recent years, the regulation mechanism of nicotine biosynthesis is far from understood. In the present study, we used suppression subtractive hybridization and differential screening strategy to identify differentially expressed genes in the roots of tobacco plants (Nicotiana tabacum L. K 326) 24 h after topping. After differential screening, 560 significantly differently expressed clones among 1950 positive ones were acquired, and 273 high quality expressed sequence tags (ESTs) were acquired after sequncing. The expression of selected genes was analyzed by reverse transcription polymerase chain reaction and RNA gel blot hybridization, and the result indicated that their transcription amount increased after tobacco topping. A full-length cDNA sequence of NtNAC-R1 was cloned and Phylogenetic analysis showed that NtNAC-R1 belonged to special NAC family in Solanaceae. Expression analysis indicated that NtNAC-R1 had a highest expression level in root. The transgenic tobacco plants with reverse NtNAC-R1 gene were got through Agrobacterium mediated transformation and the expression of nicotine biosynthesis key enzymes encoded genes were analyzed in transgenic and contol plants. This study highlighted the complex molecular changes occurring in tobacco plants after topping. Identification of these decapitation-responsive genes will provide further insights into molecular mechanism of nicotine biosynthesis and regulation.The results were as follows:1. The positive relationship between nicotine biosynthesis ability and expression level of key enzyme related to nicotine biosynthesis in the roots of tobacco was confirmed. The tobacco (Nicotiana tabacum) plants were topped after water cultured for 65 d, total RNA was extracted respectively from the roots of topped and untopped tobacco plants 24h after topping. Expression analysis of putrescine N-methyl transferase gene (PMT) was carried out by semi-quantitative RT-PCR with the tobacco actin as control and Northern blotting with the digoxin-labelled PMT RT-PCR product as probe showed that PMT expression level increased after topping. The top leaf nicotine contents of the topped plants also increased. This suggested that 24h after topping was the proper time for SSH library construction.2. A tobacco tip roots suppression subtractive hybridization (SSH) library was constructed using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester. The insert size of positive clones was 200-1 000 bp confirmed by PCR. After differential screening, 560 significantly differently expressed clones among 1950 positive ones were acquired, and 273 high quality expressed sequence tags (ESTs) were acquired after sequencing. 3. The results of bioinformatics analysis indicated that these 273 high quality ESTs mainly involved in alkaloid biosynthesis (4%), plant hormone metabolism (3%), signaling / transcription (18%), stress / defense (32%), protein metabolism (9%), carbon metabolism (6%), other metabolism (15%) and function unknown (13%). The expression of selected genes was analyzed by reverse transcription polymerase chain reaction and RNA gel blot hybridization, and the result indicated that their transcription amount increased after tobacco topping. The tobacco tip roots suppression subtractive hybridization library will contribute new data to the list of possible candidate genes involved in nicotine biosynthesis regulation.4. NAC transcription factor screened from the SSH library was cloned and functionally analyzed. A full-length cDNA sequence of a putative tobacco NAC transcription factor was cloned using in silico cloning approach from the SSH library and was confirmed by RT-PCR. After Bioinformatics analysis, the full sequence named NtNAC-R1. NtNAC-R1 had a 936bp open reading frame in length, encoding 311 amino acids, with a typical conserved domain of NAC transcription factor family. Phylogenetic analysis showed that NtNAC-R1 had a highest homology with the NAC domain in petunia and belonged to special NAC family in Solanaceae. The expression of NtNAC-R1 in E.coli (Escherichia coli) BL21 showed activity in prokaryotic cells. The expression pattern of NtNAC-R1 in root before and after tobacco topping was analyzed by RT-PCR and Northern blotting, and the result indicated that NtNAC-R1 with a decreased expression level at 2h and 4h after topping, then rise. The data indicate that NtNAC-R1 gene maybe has a response for signal transduction caused by tobacco topping. The cloned tobacco root NAC transcription factor gene will provide a foundation for further study on root development and regulation of nicotine biosynthesis after tobacco topping.5. The transgenic tobacco plants with reverse NtNAC-R1 gene were got through Agrobacterium mediated transformation. The expression analysis of nicotine biosynthesis related key genes in the transgenic and wild type tobacco plants showed that the expression level of PMT and ODC decreased, which indicated NtNAC-R1 gene may play an important role in the nicotine biosynthesis regulation in some degree.The innovation points of this research were as follows:1. The direct molecular proof for nicotine biosynthesis was provided.2. A tobacco tip roots suppression subtractive hybridization (SSH) library was constructed and the differentially expressed genes before and after tobacco topping were analyzed.3. The NtNAC-R1 transcription factor gene from the SSH library was cloned and its expression profile was analyzed.4. The transgenic tobacco plants with reverse NtNAC-R1 gene were aquired, and the expression of nicotine biosynthesis related key genes was analyzed. |
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