Expression Of Wheat Fatty Acid α-Dioxygenase In Vitro And Analysis Its Transcript Pattern In Wheat

The living creatures have been creating special physiological and biochemical procedure to adjust themselves to the stressed environment during the long evolutional process, such as stress signals binding acceptor, signals transduction and magnification, changing of genes'transcript and translation pattern and returning to normal post stressing.Ploy unsaturated fatty acid (PUFA) and their derivatives play important roles in cellular signals transduction and controlling genes'expression pattern. For example, in mammals, the products from arachidonic acid pathways, prostanoid and leukotriene have a great correlation with pathological tissues, cancers and body inflammatory reactions. In plants, JA is one of the products fromα-linolenic acid pathways。Besides its important roles in growth and develop, JA also takes part in plant hypersensitive reaction and cell death as its counterpart in mammals. The first biological reaction of PUFA is catalyzed by fatty acid oxygenases, whose products are called as oxylipins. The oxygenase existing in living creatures include cyclooxygeanse for prostanoid biosynthesis, lipoxygenase for leukotriene and jasmonate biosynthesis in mammals and plants respectively. The newer one oxygenase,α-dioxygeanse, which catalyses the first step of the fatty acid alpha carbon atom oxidation, is reported in many plants recently and its products involve in plant HR and develop.We first homologically cloned the wheat fatty acid alpha dioxygenase sequence (DOX) using special primers obtained from wheat EST databases, and we expressed DOX in vitro in different vectors and E.coli hosts. The results of SDS-PAGE demonstrated that the vectors pEHISTEV-DOX,pET32a-DOX in E.coli host BL21(DE3)codon plus have an efficient yield as inclusion body. At the same time, we got positive clones of Pichia pastoris containing multicopies of pPIC9k-DOX, which have the ability to resist 2mg/mL G418.Then we analyse the transcript pattern of wheat DOX gene by radioactively labelled probes and statistic its homology EST in genbank databases. The results imply that the wheat DOX may have the same expression pattern as its counterpart in rice, which involves in both develop and resistant in stress. It is different from that of Arabidopsis, which have two copies of DOX, DOX1 and DOX2, taking part in HR during uncompatible pathogenic infection and senescence respectively.Finaly, we used special primers cloning the wheat NAD+-dependent aldehyde dehydrogenases (ALDH) sequence cDNA from wheat leaf infected by Blumeria graminis, which have a putative role on catalyzing the Cn-1 fatty acid aldehyde into Cn-1 fatty acid to complete the alpha oxidation cycles. Wheat ALDH EST statistical analysis showed that its homology EST sequences come from the same cDNA library as that of wheat DOX, which means that the ALDH subunit and the DOX subunit may have great possibilities to combine enzymic complex having fatty acid alpha oxidation functions.