| Tobacco, annual or perennial herbs, is an important economic crop. Because of its abundant glands, it has a large number of green tissue, making it an ideal model for the study of the genetic engineering of the plant species.α-Farnesene is a volatile sesquiterpene that is synthesized by the mevalonate(MVA)pathway through the action of α-farnesene synthase. So far, the research of apple α-farnesene synthase focus on the relationship of α-farnesene and oxidation of α-farnesene with apple superficial scald. However, limiting research about apple α-farnesene synthase effect on the synthesis of terpenoids and the characteristic of transgenic plants. In this study, Introduced apple α-farnesene synthase gene into tobacco and the third generation transgenic plants were used for the experimental material. The analysis of signaling pathway and metabolite, then,explain the reason and mechanism of transgenic plants with early senescence characteristic.The main results as follow;(1) The synthesis and release of plant terpenes with spatial specificity(organs). Expression analysis of Md AFS in different parts(roots, stems, leaves, flowers) of transgenic plants, the lowest expression in stems, following roots and leaves, flowers have the highest expression level, 3.6-fold compared to stems. GC/MS analysis of tobacco volatile terpene components,we can not detect the release of farnesene in the different parts of transgenic plants. Treatment with Potassium, farnesene can be detected in the flower of transgenic plants.(2) Transcript level of Md AFS was induced by 4°C, 42°C, wound, methyl jasmonate(Me JA), gibberellic acid(GA), salicylic acid(SA), ethephon(ETH), polyethylene glycol-6000(PEG-6000) and hydrogen peroxide(H2O2). Treatment of 4°C at the higher level all the time; wound show increasing and then decline; Me JA, GA, SA, ETH, PEG-6000, H2O2,and 42°C treatment showed the burst up-regulated at a certain time point. In the experimental condition, transcript level of Md AFS was induced by light in apple seedling.(3) At present, there are not available genome databases for tobacco(Nicotiana tabacum NC89). De nove transcriptome assembly of leaves and apical buds of tobacco, got a total of249185 unigenes. Different expression analysis showed that antioxidant acivity had 26down-regulated unigene including APX and CAT. In addition to, carotenoids metabolism had34 down-regulated unigenes, plastoquinone metabolism had 14 down-regulated unigenes,ascorbate metabolism had 10 down-regulated unigenes and nitrogen metabolism had 62down-regulated unigenes.(4) The transgenic plants accumulated more reactive oxygen species(ROS). The antioxidant defense system was compromised in transgenic plants, decreased the activity of antioxidant enzyme(APX, CAT, and SOD), lower level anitoxidant substance(carotenoid,polyphenol, and flavonoid), and enhanced the sensitivity to oxidative stresses.(5) For hormonal regulation, the results indicated that JA, SA, and ET have lower levels and reduction the release rate of ET, therefor, BR has a higher level, CTK has not significant change in transgenic plants.(6) Photosynthesis is a main reducing power and carbon source of terpenoids synthesis pathway, The results showed that transgenic plants had decreased net photosynthetic rate(Pn),starch and soluble sugar content dropped significantly. The analysis of photosynthesis,photosynthetic proteins and carbon fixed signal pathway found 87, 127, and 195down-regulated unigenes, respectively.(7) The synthesis and release of terpenes are expressed in a time specific manner(developmental stages). The expression of Md AFS gene increased with the development of transgenic tobacco continuously, reaches its peak at flowering stage. The correlation analysis of key enzyme genes in terpenoids synthesis pathways, the results showed that Md AFS is positive relation to HMGR, other genes(DXS, DXR, IDI, FPPS, and GPPS) are negatively correlated.(8) The results of q RT-PCR showed that HMGR1, HMGR2 and HMGS are up-regulated and other key genes(DXS, DXR, IDI, FPPS, and GPPS) are down-regulated in transgenic plants. The treatment of HMGR specific inhibitor lovastain(MEV) results implied that HMGR had a higher transcript level in transgenic plants. But the transgenic plants are not sensitive to the fosmidomycin(FOS) treatment, which is DXR specific inhibitor.(9) In transgenic plants, the content of chlorophyll and carotenoids had a lower level, and the value of Fv/Fm was significant decreased; in transgenic leaves, the release ofmonoterpenes, limonene and linalool, are not sensitive to Me JA treatment. The results of q RT-PCR showed that chlorophyll synthase gene was down-regulated, chlorophyllase gene was up-regulated. Pyruvic acid is the raw material of terpenoids synthesis. The content of chlorophyll and carotenoids can be restored by pyruvic acid treatment, indicating that reducing the synthesis is the main reason for reduction of chlorophyll and carotenoid.Key words: tobacco, MdAFS, sesquiterpene, senescence, transcriptome... |
PDF Full Text Request