| As the main compoments of fish oil, EPA and DHA are necessary for the normal development of retina and cerebral cortex of the baby and children , and plays an important role in prevention of cardiovascular disease. Their sanitarian function has been widely known. Usually the EPA and DHA synthesis of human can meet the normal physiological activities , but pregnant, infant, baby and the elderly and disease patients need supplementary EPA and DHA.Currently,the mai sources for these LCPUFAs are oils from fish and microbes. However, shrinking fish populations is making the output has not meet the growing needs of people, and this kind of fish is not suitable for pregnant women and infant consumption because of pollution, and the high cost of microbial oil extraction .through the single-celled microalgae refined fish fermented way high cost. Therefore, search the Sustainable,safe and economic alternative sources becoming the urgent problem to be solved. Recently, genes encoding desaturases and elongases from microbes have been identified and successfully expressed in oilseed plants. However, the levels of LCPUFAs produced in transgenic plants expressing these genes are still much lower than those found in native microbes.To solve the problem of low levels of LCPUFAs produced in transgenic plants, the cloning over-expression activity is obviously one of the most effective ways besides the method of optimization of gene expression and select the reasonable metabolic pathways. However, the very low homology of desaturases and elongses involved in the anabolic processes of LCPUFAs makes it difficult to clone these new genes. Currently, this new genes are mainly cloned throngh hybrid screening of cDNA library and scale of sequencing, is time-consuming, and cost highly.In recent years, the explosive growth of public to biological information regarding the various database of nucleic acid, protein sequences of biological information , offers a powerful support of information resources to analysis and identification for certain genes. Our research use the protein sequences of known desaturases and elongases as the targets using the PSI-BLAST program, on one hand, to repertoire the known sequences systematically, on the other hand, to search for some unknown homologous sequences, and preliminary screening and identification the putative desaturases/elongases by means of bioinformatics. On this basis, we calculated the phylogenetic trees of the entire desaturases and elongases respectively and rendered the detailed distribution table of these genes, provide reference guide for cloning and identification these new genes comprehensivly and systematically.The main results are as follows:1. We selected 267 known and 107 putative desaturases, 243 known and 389 putative elongases from NCBI datebase, and identified 400 new genes encoding desaturases and elongases.2. We calculated the phylogenetic tree of the 532 desaturases with the neighbor-joining methord, there are 6 individual branches in this phylogenetic. We defined the 4 large branches as the following subfamilies: (1) First Desaturase,(2) Omega Desaturase,(3) Front-End Desaturase,(4) Sphingolipid Desaturase. This result verified the existing classification standards of dasaturases to be correct. The other 2 small branches may be the unique phenomenon in the process of evolution.3. We calculated the phylogenetic tree of the 871 elongases as above, and there are two individual branches in this phylogenetic. We defined the 2 large branches as the following subfamilies: (1) S/MUFA elongases, (2) PUFA elongase. There is a converd amino acids motif in two subfamilies, means the possibility of the same origin of all the elongases, and the differentiation of functions may be the result of adapted to environmental changes in evolutionary process.4. Our phylogenetic and motif analysises indicate that the functions of desaturases and elongases have diverged via two phases. In the first phase, desaturases and elongases diverged into four and two subfamilies, which had different ranges of substrate specificities. In the second phase, we believe that a variety of substrate specificities or regioselectivities diverged within the individual subfamilies. In other words, the first phase restricted the range of functions and then, in the second phase, functions diverged within their ranges.We analied the phylogenetic tree of desaturases and elongases.We noticed there was a unique small branche in the phylogenetic tree of desaturases whose sequences belongs to Phytophthora infestans, which had low genetic relationship with daseturases from other organisms. The genome sequencing of Phytophthora infestans has been completed in September 2009, and we searched 3 putative daseturases and 4 putative elongases from the genome of Phytophthora infestans. On one hand, to test our bioinformatics analysis results and revealed the unique position of Phytophthora infestans in the evolution of desaturases. On the other hand, to clone the over-expression genes and improve the levels of LCPUFAs produced in transgenic plants. So we cloned the 3 putative daseturases and 4 putative elongases from Phytophthora infestans, and their functions were verified by heterologous expression in yeast. The main results are as follows:1. Homology cloned a fragment of 911bp length, and sequencing results indicated it was PinE4 - a new gene of elongases. Generated the yeast expression vector pYSE2.0- PinE4 and the function was verified by heterologous expression in yeast, the results showed the PinE4 encoded a elongase the enzyme could convert LA to EDA.2. Homology cloned a fragment of 1kb length, and the sequencing results indicated it had 2 introns. We got PinE3 of 741bp length after the deletion of 2 introns,which is new elongase. We cloned PinD1 of 1347bp and PinE1 of 1022bp and PinE2 of 1044bp successively, a new desaturase and two elongases. We have verified their functions by heterologous expression in yeast repeatedly, but the results all were incredible, we should research their functions furtherly.
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