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Study On Transgenic Synechocystis .sp PCC6803 Harboring Δ5 Desaturase Gene From Bacillus Subtilis

Posted on:2007-11-24Degree:MasterType:Thesis
Country:ChinaCandidate:K ZhangFull Text:PDF
GTID:2120360185475429Subject:Pharmacognosy
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Unsaturated fatty acid (UFA), which mainly come from the sea fish, have the extremely important nutritional and medicinal value. With the environmental deterioration, the resource of fishes is dried up day by day. Furthermore, the process to purify UFAs from fish fat are rather complicated. Algae are primary producer of UFAs, microalgae may become a new source of the edible UFAs, but under normal temperature the output is low. The reconstruction of blue-green alga with molecular biological methods may improve the content of UFAs, or synthesize new UFA.Synechocystis sp.PCC6803 is a mesophilic cyanobacterium, with its optimal growth temperature at 30°C and the favorable temperature from 25°C-40°C. In its genome, it has△6 (desA, sll0262) ,△9(desC, sll0541), A\2(desD, sIr1350)和ω-3(desB, sll1441) desaterase and elongation gene sll2024, which participate the synthesis of UFAs. DesB gene, encodingω-3 desaturase, is inducded at low temperature, so it synthesizes only trace polyunsaturated fatty acid at 30°C. In Bacillus Subtilis, there is des gene encoding A5Des, a inducible gene by low-temperature too. To obtain a transgenic Synechocystis strain producing UFAs, the author transferred A5des into wide-type PCC6803 strain.The experimentation are described in four parts:1. Selection and identification of transformants DR157 by transferring A5Des. The whole encoding sequence of△5des in Bacillus subtilis(natto) was amplified by PCR and recombined with promoter PglnA from Synechocystis sp.PCC6803, after inserting the recombining gene into the integration plasmid, a recombinant plasmid pJS157 was obtained, and transfered into Synechocystis sp.PCC6803. The double-crossover recombinants were selected and identified,2. Construction of single-crossover recombinants SR169 and transgenic strain SR169DR157. To obtain SR169, the promoter of desB induced by low temperature was replaced with promoter PglnA by single recombination. After transferring pJS157 into SR169, transgenic strain SR169DR157 was selected and identified.3. The physiological characteristics of transgenic Synechocystis. In order to optimize the cultivating condition and improve cultivation density, the growth velocity,cytochromes oftransgenic strains were analyzed.4. Analysis of the composition and content of UFAS in transgenic strains. Comparing to control sample, the composition of UFAs in DR157 and SR169DR157 didn't significantly varied, but the content of UFAs in DR157 and SR169DR157 was different from DR1188 at 30℃. However both composition and content of UFAs in DR157 and SR169DR157 varied from control samples at 20℃The main conclusions of this study are as follows:1. The expression product of△5 des in Synechocystis sp. PCC6803 can improve cell photosynthetic pigments directly or indirectly and cell growth, but to full play the physiological functions, carbon source is the restrictive nutrition factor.2. DesB gene controlled by glnA promoter can improve the content of an unidentified kind of UFAs in single cross-over recombinant and Delta5 des can synthesize a new kind of UFAs in Synechocystis sp. PCC6803, but low temperature is still the inducing factor for the synthesis of new UFA or improvement of the content of UFAS.
Keywords/Search Tags:Synechocystis sp. PCC6803, UFAs, A5 des, transgenic strain
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