Expression Of Trehalose-6-Phosphat Synthase Gene In Prokaryote And Study On Its Function

Based on cDNA of tps 1 gene, pUC 18 plasmid and pET-30a(+) plasmid,we have completed to construct recombinant expression vector pHHSMOOl and pHHS-M002 by gene cloning technology, and made tpsl gene functionally expressed in Esche-richia coli. In addition, we have studied the resistance of recombinant strain to bad environment.1 .Transformated into E.coli DH5 a and induced by IPTG, the foreign gene tpsl in the expression vector pHHSMOOl has expressed. The result indicated that from three IPTG concentration the best induced dote is 0.7mmol/l, the expressed foreign protein account for about 6.2% of the total cell protein. The better is 1 mmol/1 lPTG,lhe target protein account for about 4%. By expression vector pHHSM002, tpsl gene also expressed efficiently in E.coli BL21(DE3)PlysS ,the target protein amount to 17.8% in cell.2.This study used high sensitive HPLC to quantitily detecte trehalose in viro.The result suggested that a certain amount trehalose is observed ,which indicated that not only tpsl gene can express in E.coli ,but also the expressed protein has its biology activity, tpsl gene gived the engineer strain ability to yield trehalose after being incorporated. At the same time, the experiment suggested that the trehalose-6-pho-sphate synthase is the critical enzyme for trehalose synthesis in yeast, which conforms with the previous experiment result.3.The resistances to bad environment of engineer strain have also been studied in this dissertation. Result from salt tolerance experiment indicated that the survivor ratio of BL21 and BL21(pET-30a(+)) quickly decreased in 0.4~0.6mol/l NaCl concentration ,while the recombinant strain BL21(pHHSM002) died slowly and show its salt resistance in wide range.Seen from each salt concentration, the recombinant strain also survived more than control group, when salt concen- tration amount to 0.9mol/l ,the latter was all disrupted to die while the former maintain 8.5% survivor.Experiment result from resistance to high temperature exhibited better ability to heat tolerance in recombinant strain. After heat shock at 50℃ for one hour ,the transformant has 15% survivor while all the control strain died ,but this experiment alsoshow that the transformant can't tolerate the higher temperature than 52 ℃.Cold tolerance experiment exhibited both the transgenic strain and the control strain can survive at low ratio in -20℃ and -196℃ environment for 24hrs ,but at the same temperature condition, the survivor ratio of recombinant strain has 3~4 times higher than the control group. In experiment, we also discovered higher survivor of strain in liquid nitrogen than that in -20℃ environment ,which indicates that quick freeze hurt strain less than slow freeze does, and the expressin of tpsl gene can help the body survive better in low temperature environment.