Construction Of Vector With GST And CS Dual-gene From Polygonum Sibiricum And Its Transformation To Yeast And Tabacco

This research obtained GST and CS from cDNA library of Polygonum sibiricum under salt stress and were constructed as single and dual yeast expression vector as well as single and dual plant expression vector.The expression vectors were transformed into yeast and tobacco and successfully gained the transformants containing single or dual genes.These transformants were detected through molecular examination and analysed the tolerance of these transgenic yeast and tobacoo,furthermore,the relationship between two genes in cocperation with tolarating to adverse surroundings was compared and analysed.The primary results were as follows:1,This article has analysed the GST and CS gene by virtue of bioinformatics.The preliminary results indicated that GST belonged to Tau family,presumeably assuming that it played a huge part in getting rid of the toxic arising from heavy metal and salt stress.CS,guided by signal peptide,located in the cell matrix,acting as cell cytoplasmic protein.The analysis of fluorescent RT-PCR showed that after two days under 3%NaHCO₃ stress,the expression of GST and CS in leaf and stem reached the peak,with the largest expression of CS in stem occurring at the second day in comparision with the greatest expression of GST coming up at the third day.During the process of stress,the expression of GST and CS in underground stem both rose upward while the expression in leaf and stem showed a downward trend.2,After constructing the prokaryotic expression vector pET28a-GST containing GST,the GST gene was transformed into E.coli and the specifi protein(26 kDa) successfully expressed. Under 3%NaHCO₃ stress,the BL21-pET28a-GST,expressing the GST,show a obviously rapid growth than control(BL21-pET28a).Above all illustrated that the protein induced by GST under 3%NaHCO₃ stress,could promote the growth of bateria.The result showed that GST gene had the ability to resist to salt stress.3,After constructing the eucaryotic expression vector pYES2-CS,the gene was transformed into yeast.The experiments illustrated that under NaCl or NaHCO₃ stress,the transformants had an apparent survival rate that control and the amount of GSH and Cys in transformants got boosted.The results showed that over-expression of CS gene in yeast really enhanced the yeast's tolerance to salt,which poved that CS gene was capable of resisting to salt.4,Completely constructing the eucaryotic expression vector(pYES2-GST)embracing GST or CS or dual-vector containing GST with CS,they were transformed into yeast,named as ty-gst,ty-cs and ty-gc.Under 1 mol·L^-1 NaHCO₃ or 5 mol·L^-1 NaCl stress,the transformants showed a stronger tolerance to salt than wild yeast,but there was no remarkable difference among the transgenic yeasts.After treated with heavy metal,the transformants(ty-gst,ty- cs and ty-gc) showed a far greater ability for resisting to the toxic of CdSO₄ and CuSO₄ than wild yeast,especilly the strongest tolerance to CdSO₄ of ty-gc.Among these transgenic yeasts,the tolerance of ty-cs and ty-gc for CuSO₄ were higher than that of ty-gst. The results indicated that GST and CS gene born the tolerance for CuSO₄ while in transgenic yeasts CS may coordinate with GST to raise the tolerance of yeast for CuSO₄.5,After constructing the single expression vector(pROKâ…¡-GST,pROKâ…¡-CS) and the dualvector (pROKâ…¡-GC),they were transformed into tobacco,named as TY-GST,TY-CS and TY-GC.The tolerance for salt in this expriment manifested that the activity of SOD and POD in TY-GST,TY-CS and TY-GC all higher than that in wild control.There was notable difference among the activity of POD in these transformants,which meant that the activity of POD in TY-GC was greater than any other transgenic tobacoo containing only one gene. The experiment of tolerance for heavy metal confirmed that wild tobacoo's leaves became old and yellow while the syndrome of transgenic plants assumed far later,but there was no prominent difference among the transformants.