Cloning And Identification Of5’ Flanking Sequences Of Terminase Genes Of Potato SGAs Biosynthesis

Potato disease is a main factor limiting potato production all the time, As the rapidvariation of disease and the emerge of patience of insect pests, the resistance of varietiesbred soon be lost. So, how to breeding varieties having widely, durable resistance to diseasesand pests has become the important content of potato breeding research. Steroidalglycoalkaloids（SGAs）existing in the body of potato plant is a kind of steroid alkaloids haswidely biological activities, peculiar smell and toxicity, It affects the edible flavor of tuberand Its processing quality, but it is closely related to the potato’s own ability of resistence todiseases and pests. In recent years breeders hope to regulate the content and spatialdistribution of SGAs in potato plant to make it has a high content in the ground part but verylow in the underground part, then to make the plant have a high resistance to plant diseasesand insect pests and at the same time not cause the potato food safety problems. Themetabolism of SGAs is a complex network system, many factors can affect Its synthesis,Environmental factors such as temperature, light, moisture and mineral all can influence Itsformation and accumulation. Researches have shown that solanidine glycosyltransferase isthe key enzyme of SGAs biosynthesis at the end of SGAs anabolic branch. So by studyingthe upstream regulation sequences of the downstream key enzyme genes, importantpromoters can be found, It provides theoretical basis for regulating SGAs synthetic at themolecular level and carrying out the space and time control of Its synthetic. The main resultsof this research are as follows:1. By using the genome walking, three2183bp、2098bp and2392bp promoter fragmentsseparately upstream from the translation initiation site of the sgt1、sgt2and sgt3gene wereobtained. Bioinformation analysis of these sequences showed that all the three promoters hadthe core promoter element TATA box、CAAT box and cis acting elements. The promotersequences have been submitted to the GenBank（sgt1promoter：KC759163；sgt2promoter：KC331038；sgt3promoter：KC331037and sgt1promoter and sgt3promoter sequences werereleased for the first time.2. By subcloning of the sgt2promoter, seven5’ flanking promoter region deletionsequences were obtained, and the seven sequences were fused with gfp::gus reporter genereplacing the CaMV35S promoter originated from pCAMBIA1304to construct seven5’flanking deletion binary vectors in which the gfp::gus reporter gene was driven by the5 ’flanking promoter region deletion sequence.3. With the transient expression analysis of tobacco leaves through Arobacterium–mediated transformation, we preliminary analyzed the function of sgt2promoter usingGUS histochemical staining. The result showed that each promoter fragment had the activityof promoter. The1078bp promoter fragment upstream from the translation initiation site hadthe highest expression level and the bp promoter fragment upstream from the translationinitiation site still could drive the high expression of gus; With the gfp transient expressionanalysis of the onion epidermis through Arobacterium-mediated transformation,we analysedthe subcellular localization of sgt2full-length promoter, the result showed that the gfp wasexpressed in cell nucleus and cell wall place of onion epidermal; The seven5’flankingdeletion binary vectors and pCAMBIA1304vector were introduced into wild type tobaccothrough Arobacterium-mediated transformation, generating more than eighty plants resistentto hygromycin（have not yet identified）.