| Caragana intermedia is widespread and is very important for ecological environment which can improve the soil and hold water at arid region of China northwest. In order to make more production,oleic acid desaturase (FAD2) gene which code the key enzyme of polyunsaturated fatty acid synthesis were cloned from young seed of C. intermedia by RACE. And they were expressed and analyzed in yeast. These studies found the rationale for C. intermedia genetics improvement and breeding the new fuchsins with good quality oil or cold resistance. The main results are as follows:1. Three full-lengthΔ12 fatty acid desaturases genes were cloned from pedo-seeds of C. intermedia., which named FAD2-2A(GenBank No. EF503621), FAD2-1A(GenBank No. EU433557) and FAD2-1B(GenBank No. EU433558 ), respectively. The sequence analysis of these three genes and fad2-2B gene(CaFAD2, GeneBank accession AY957394) which was cloned from caragana intermedia leaves by Wang Y.D. have done. The deduced first 283 amino acid sequences of Fad2-1A and fad2-1B showed high identity which was 98.9%, and the FAD2-1A protein is 97aa longer than FAD2-1B protein. The identity of FAD2-2A and FAD2-2B deduced amino acid sequences was 81.6%, and that of FAD2-2B and FAD2-1A was lowest 64.7%.2. The four FAD2 proteins structure and features were analyzed by bioinformatics methods, such as DNAMAN, DNAStar software, and http://www.us.expasy.Org, http://www.ch.embnet.org internet means and so on. The results indicated that FAD2-2B had signal peptide and the others had not signal peptide cleaved sites. The secondary structure of FAD2-2A and FAD2-1A were similar and coil was main with little strand and helix. FAD2-2B and FAD2-1B secondary structure are coil and strand mainly. The four proteins were membrane-bound proteins and have five or six transmembrane domains, respectively. The half life of FAD2-2A protein was 220 h, and that of the others were 30 h or 31 h. FAD2-2B relative translation effectiveness was 1 and that of the other proteins were 5. The palmitoylation sites of FAD2-1A, FAD2-1B, FAD2-2A and FAD2-2B were 5, 4, 8, 9, respectively. 3. Southern-blotting results indicated there are four copies of FAD2 gene in C. intermedia. genome at least, and these are coincident with the four FAD2 genes were cloned. The real-time PCR was performed to detect the relative expression levels of FAD2-2A, FAD2-1A and FAD2-1B mRNA. FAD2-2A transcript showed a low level in the root, middle-stage seeds and pre-mature seeds, and FAD2-2A was expressed more in the first-stage seeds and tender leaves than in the other tissues. FAD2-1A transcripts showed the lowest level in the root and the highest level in the first-stage and middle-stage seeds, and it was expressed abundantly in the tender leaves too. FAD2-1B was expressed abundantly in the middle-stage seeds and its transcript was very low in the other tissues. In all tissues the FAD2-1A transcript level changed most, and that of FAD2-2A changed lest. According to all of these, we deduced that FAD2-2A takes responsibility of desaturating oleic acid in the membrane lipid mainly; FAD2-1A is in charge of desaturating oleic acid in the store lipid of seeds and leaves; and FAD2-1B is in charge of desaturating oleic acid in the store lipid of seeds, too.4. The Expression vectors pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were constructed by double enzymes digesting and transformed yeast. The yeasts with pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were inducted to express the exogenous genes at 20℃and were treated by cold(15℃) and high temperature(30℃). FAD2-2A transcription increased quickly at first stage and then keep the high level with induction medium at 20℃. The change tendencies of FAD2-2A protein and fatty acid productions are similar with that of transcription. The transcription, protein and fatty acid productions of FAD2-1A increased gradually at the whole process. And that of FAD2-1B kept low level at the first stage and increased at the middle stage.5. When the yeasts were treated by low temperature, the expression of FAD2-2A and FAD2-1A increased obviously, and the FAD2-1B transcription increased a little.But the FAD2-1B protein and fatty acid production did not change. If the yeasts were treated by high temperature, the expression of FAD2-2A and FAD2-1A increased firstly and then decreased, but FAD2-1B protein did not change.6. The analysis results of real-time PCR, SDS-PAGE and fatty acid content suggested that FAD2-2A and FAD2-1A expression were regulated in transcript level with all three kinds of treating methods; FAD2-1B were regulated in transcript level with 20℃, were regulated at transcript and translation stages with cold, and at transcript, translation and after translation stages with high temperature treatment.7. FAD2-2A change was fastest to response to temperature and FAD2-1B was slowest. In this study, the FAD2 genes were researched from transcripts, translation and productions, the inherent regulating mechanism of the three genes were provided. All of these give the conditions and the theory guidance for Caragana intermedia gene engineering breeding to improve the seed oil quality. |
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