Cloning Of Genes Related To Amlylose Synthsis In Potato Tubers And Studies On Potato Genetic Transformation

High-amylose-contained starch is the main material of producing bio-degradation plastic because of its special structure, fine physical and chemical characters. One of the main supplies of starch is potato. However, by far there is few potato cultivars that is high-amylase-contained in China. Therefore, our study aims at breeding high-amylose-contained potato cultivar by transforming encode sequence of Granule Binding Starch Synthase (GBSS) that controls amylose synthesis in potato tuber. Our research has drawn three conclusions as following: Firstly, potato genome DNA is extracted and used as template, via Polymerase Chain Reaction the 5'end flanking sequence of GBSS gene is cloned .Sequence analysis indicates the cloned fragment has 599 base pairs, and contains the main element TATA box and CAAT box .Though it shares 97.41% homology with the reported sequence, there is much difference between them: 136 base pairs deletion and 14 base pairs insert are found in our clone compared with the reported sequence. As the cloned fragment is a new promoter sequence, it is necessary to identify it's function. The plant expression vector pBIGGFP constructed by fusing the cloned sequence with GFP was transferred into steam segment, leaf and in vivo micro-tuber slices via the partical bombardment. The transient expression of GFP proved the cloned sequence was functional. Secondly, Tuber-specific-promoter Patatin is sub-cloned from plasmid pGEMpa, and sequence analysis indicates it has 968 base pairs. Thirdly, potato RNA is extracted from fresh tuber, by using RT-PCR GBSS gene is cloned. And the sequence analysis indicates the cloned fragment has 1824 base pairs and shares 99.29% homology with the reported sequence, shares 99.18 % comparability with amino acid sequence of the reported GBSS according to the deduced amino acid sequence translated by cloned GBSS. Constructing pBIGG , the plant expression vector of GBSS gene driven by GBSS gene 5'flanking sequence, and pBIPG ,the plant expression vector of GBSS gene driven by Patatin promoter, then transform the two plant expression vectors into potato .