Cloning And Mutation Of GlgC Gene And Construction Of Plant Expression Vector For Increasing Starch Content Of Potato

Potato (Solanum tuberosum L.) is the only crop that can be used as grain and vegetables in the world. It plays very important role in daily life and national economy. Because of its fine processing characteristics, potato starch has extensive range of application, and some application can't be substituted with starch of other source. Under the historical opportunity of the development of western regions, potato processing will become the major industry of western rural economy. But starch content of potato cultivars presently used have a few variety can up to 20%. For meeting the rapid development of potato process industry, the work of breeding high starch content potato is extremely urgent. Because the potato breeding material of the high starch content is extremely deficient and potato is the homology tetraploid, by routine breeding method to cultivate high content starch potato variety will not only take quite a long time and energy but also meet many difficulties. Gene engineering can break species limits and transform plant with the gene from other species. The further investigation of relevant enzymes of starch biosynthesis and improvement of molecular biotechnology in recent years, makes by gene engineering technology to control the activity of relevant enzyme of starch biosynthesis and increase of starch content of potato tuber possible.In this study, potato genome DNA is extracted from the in vitro of Taihong . According to known sequences of potato tuber-specific expression promoter Class I Patatin , we cloned a 1030bp distinctive fragment by polymerase chain reaction (PCR) using artificial synthetic special primers and combined it with plasmid pUCm-T easy vector and transform E. coli DH5 a with it. Digestion by enzyme and sequence analysis indicated that the homology between this fragment and that reported sequence is 94% . It includes control elements of TATA box (situated upstream of transcription initiation -21 ~-13bp) and CAAT box(situated upstream of transcription initiation site -59~-48bp); There is core enhancer homology sequence TGGTTATG in upstream of CAAT box. The glgC gene was amplified using a PCR carried out on chromosomal DNA from E.coli BL21 with the special primers. The 1296bp fragment representing the glgC gene was cloned in pUCm-T easy vector and transform E.coli DH5 a with it. The nucleotide sequence of the clone was confirmed by sequencing. The homology between this fragment and that reported sequence is 99%. In order to make ADPase lessdependent on the activator F6P and less sensitive to inhibition by the inhibitor AMP, cloned mutation gene glgC336 using splicing by overlap extension of PCR.The glgC and glgC336 genes were subcloned into pET-28a-c(+) expression vector respectively, the N-terminal His-taq was introduced in the recombinant enzyme. The recombinant protein was produced after induced by IPTG. The total proteins of positive and control bacteria were analyzed on a 15% polyacrylamide gel. This indicates the molecular weight of expressed protein fit in theory value well.By introducing Patatin and glgC (or glgC336) into plasmid pBI101, we constructed the plant expression vector pBIPG (or pBIPGT) which puts glgC gene under the control of Patatin promoter. This work was a good foundation for breeding potato varieties with high starch content by transformation.