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Silencing And Functional Analysis Of NtFAD2 Gene From Tobacco

Posted on:2006-03-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:M F YangFull Text:PDF
GTID:1103360182472489Subject:Botany
Abstract/Summary:PDF Full Text Request
One of the most important desaturases in plants is microsomal oleoyl-phosphatidylcholine desaturase (FAD2), located in endoplasmic reticulum. It catalyzes the desaturation of oleic acid into linoleic acid. Plant oil with high level of linoleic acid and other polyunsaturated fatty acid is associated with a lower oxidative stability, and its oxidation products are harmful to human health. Therefore, one of the most important aims of improving the quality of plant oil is to cultivate crops with seed oil containing high level of oleic acid. Furthermore, the structure, function and regulation of FAD2 enzyme are far from being elucidated. RNAi technology developed recently can be used to silence genes efficiently. In our work, we intend to use this technology to silence FAD2 desaturase, and then investigate the changes of lipid compostion in membranes and seed oil, and get further insight into the function of FAD2 enzyme and its gene expression patterns. In this paper, the FAD2 gene of tobacco (Nicotiana tabaccum), herein designated NtFAD2, was cloned by PCR-based method, and a partial coding sequence of the putative NtFAD2 gene was used to create intron-containing construct expressing hairpin RNA for silencing endogenous NtFAD2 gene. After the transformation of tobacco mediated by Agrobacterium tumefaciens, some NtFAD2-silenced mutants were established in the population of the primary transformants. The analyses of fatty acid profile of the transgenic line S61 showed that, besides a drastic increase of oleic acid level in total leaf lipid and in phosphatidylcholine and phosphatidylethanolamine, the silencing of NtFAD2 also resulted in pleiotropic effects on glycerolipids of leaves, i.e. a significant increase of oleic acid level in plastidial lipids and a palpable decrease of palmitic acid levels in some individual polar lipids. The significant increase of oleic acid found only in leaf and seed lipids of transgenic lines proved the diversity of the silencing effects in different organs. The silencing construct showed severer effect on NtFAD2 enzyme in organs with high NtFAD2 activities than in organs with low NtFAD2 activities. The similar decrease of NtFAD2 gene transcripts in most tissues of transformant suggested that oleic acid contents in some organs had no direct relation to the abundance of NtFAD2 gene transcripts. It is necessary to investigate the stability of high-oleic-acid feature of NtFAD2-silenced tobacco at different temperatures, since temperatures affect the desaturation degree of plant fatty acid, and such an investigation will facilitate the application of RNAi technology to cultivate oil plant of good quality. Results showed clearly that the desaturation of leaf lipids of NtFAD2-silenced tobacco was also regulated by temperatures, and the level of oleic acid decreased significantly at low temperatures, and the level of polyunsaturated fatty acid increased accordingly. The fatty acids in leaves of wild-type tobacco fluctuated in the same way, but more slightly. The analyses of individual lipids from NtFAD2-silenced tobacco suggested that the resuscitation of the activity of silenced NtFAD2 enzyme was at least a factor in the decrease of oleic acid levels in transgenic plant at low temperatures. However, temperatures had no obvious impact on the degradation of NtFAD2 mRNA in the mutant. We assumed that the rebound of oleic acid level in NtFAD2-silenced plant were adaptive activities of plant grown at low temperatures. The oleic acid content of total lipids in seeds of transgenic tobacco decreased at low temperatures. Thus, such transgenic plants must be grown at suitable temperatures, or in suitable seasons and areas to escape the affect of low temperatures, especially when they are in seed formation stages.
Keywords/Search Tags:tobacco, FAD2, oleic acid, gene silencing, temperature
PDF Full Text Request
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