| α-linolenic acid(ALA) is a vital ω-3 series polyunsaturated fatty acids, with enhanced intelligence, protected eyesight and prevented cardiovascular, regulation of blood pressure and blood lipids, etc, which can metabolize into DHA and EPA. At present, the major source of ALA is abyssal fishes, while vegetable oil with low ALA content, such as rapseed oil, soybean oil, sunflower oil et al, is the main edible oil of China national, so our citizens’ ADI(acceptable daily intake) of ALA can not meet the need of our body when we consume a normal diet. For improving the situation, it’s significant to develop the plant resources enriched ALA. Rap oil is one of the Chinese mainly edible oil, if you can increase the ALA content of rapeseed, you can improve the nutritional value of rapeseed oil and provide a source of high levels of ALA.ALA is mainly stored in plants’ seed oil, by ω-3 fatty acid desaturase(FAD3, FAD7, FAD8) catalyzing conversion from linoleic acid. ω-3 FAD gene is a key rate-limiting enzyme gene in ALA synthesis pathway. By regulating the expression of the genes, it can increase the ALA content of plants. Perilla frutescens is the oil crop which is richest in ALA. In this study, we cloned two high expression genes respectively named FAD3 and FAD7 from Perilla frutescens by PCR, constructed the seed-specific expression vector pC2301M1NPB-EFAD3 and pC2301M1NPB-EFAD7 and transformed American tobacco and zhongshuang 11 by agrobacterium-mediated genetic transformation. The main research results as follows:1. The full-length of the gene of FAD3 and FAD7 is respectly 1240 bp and 1370 bp by cloning from Perilla frutescens. The homologous of FAD3 and FAD7 are bouth up to 99% with the published FAD3 and FAD7 of Perilla frutescens by NCBI online alignment.2. Constructed the seed-specific expression vector pC2301M1NPB-EFAD3 、pC2301M1NPB-EFAD7, successfully. The vectors contain the seed-specific promoter that named nap A and screening marker nptⅡ.3. With the tobacco-leaf as explant, transgenic tobaccos were studied by agrobacterium-mediated genetic transformation. Batch resistant plants were obtained by Kan selective agent. Molecular identification of resistant plants showed that 24 strains containing the NAP+FAD3 gene fragment and 26 strains containing NAP+FAD7 gene fragment. It’s indicated that FAD3 and FAD7 of Perilla frutescens are integrated in tobaccos chromosome. qRT-PCR analysis showed that the expression of FAD3 of Perilla frutescens was instability in transgenic tobacco seeds, elevated also reduced. Fatty acid composition analysis of tobacco-seed shows that there was one transgenic tobacco with PfFAD3 had a high ALA content, it’s content of ALA about 2.4 times to the negative FAD3 transgenic tobacco, meanwhile the content of linoleic acid was increased and the content of oleic acid was reduced. The content of ALA in others FAD3 transgenic tobaccos had no difference with the negative FAD3 transgenic tobacco. There was no difference detected between positive FAD7 transgenic tobaccos and negative in ALA content.4. With the cotyledons of 4 days Brassica campestris as explant, transgenic Brassica campestris were studied by agrobacterium-mediated genetic transformation. The Kan resistance screening and molecular identification of transgenic plants showed that 1 strain containing the FAD3 gene of Perilla frutescens NO.8. It’s indicated that FAD3 of Perilla frutescens was integrated in Brassica campestris chromosome. RT-PCR analyses showed that PfFAD3 only expressed in the seeds of the NO.8 Brassica campestris not in leaves. It’s indicted that the seed-specific expression vector successfully restricted the expression of PfFAD3 gene in the seed. qRT-PCR analysis showed that the expression of FAD3 gene of Perilla frutescens was significantly higher than negative NO.5 transgenic rapeseed in transgenic rapeseed NO.8, it’s indicted that the FDA3 gene of Perilla frutescens was normal expressed in the NO.8 rapeseed. |
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