Stress Tolerance Analysis Of PsGSTU1, PsCSase1 And PsnsLTP Gene From Polygonum Sibiricum Laxm

This paper will research transgenic (single-gene: PsGSTUl and PsCSasel; dual-gene: PsGST+PsCSase) tobacco in resistance to abiotic stress, at the same time observe whether it has synergy and stacking effect between double price genes; PsnsLTP gene which got from Polygonum sibiricum Laxm, constructed plant expression vector and yeast expression vector and successfully got transgenic tobacco and transgenic yeast. Appraisalling them using biology methods and analyzing the function of the transgenic tobacco resistant to biotic and abiotic stress, transgenic yeast resistance to abiotic stress. The results are as follows:1,Doing the following several different abiotic stress experiments about transgenic tobacco (PsGST, PsCSase, GC(PsGST+PsCSase)):Seed germination rate experiment (0.9%,1.2% NaCl; 10 mM,20 mM NaHCO3; 20% PEG6000; 200μM CdCl2; UV-B:90μW/cm2 ultraviolet); Tissue seedlings stress experiment (0.6%,0.9%,1.2% NaCl; 400μM CdCl2); Testing physiological indexes (SOD, POD, MDA and soluble protein) about greenhouse cultivated tobacco plants under pressure (NaCl, CdCl2, PEG6000 and UV). Whether the comparison of seed germination, tissue seedlings phenotype after stress or tobacco physiological indexes after stress, the results are all shown that: transgenic tobacco (PsGST, PsCSase, GC(PsGST+PsCSase)) resistant PEG6000, NaCl, NaHCO3, CdCl2 and ultraviolet ability higher than the control, but did not showed the synergistic effect about dual price gene, instead that tolerance adversity stress ability of transfering PsCSase tobacco is highest.2,Using agrobacterium-mediated technique to transfer the exogenous gene PsnsLTP into tobacco, by PCR and RT-PCR detection, gene has been successfully transferred into tobacco genome and normal transcription. Doing the following experiments about transgenic tobacco:Leaf discs resistant to salt and heavy metal experiment (3 mM,5 mM CuSO4; 200μM,400μM NCI; 100μM,200μM ZnCl2; 3 mM,8 mM CdSO4; 400μM, 800μM CdCl2; 100μM,200μM AlCl3; 1.2% NaCl; 15 mM NaHCO3); Seed germination experiment (0.9%,1.2% NaCl; 10 mM,20 mM NaHCO3; 200μM CdCl2; UV-B:90μW/cm2 ultraviolet); Tissue seedlings stress experiment (0.6%,1.2%,1.5% NaCl; 100μM, 200μM CdCl2; 7.5% PEG6000); Testing physiological indexes (SOD, POD, MDA and soluble protein)about greenhouse cultivated tobacco plants under pressure (NaCl, CdCl2, PEG6000 and UV). All test results showed that the resistance of transgenic tobacco for salinity-alkalinity, heavy metal and ultraviolet are obviously higher than the control.3,Transgenic tobacco (PsnsLTP) biological stress experiments:Placed Colletotrichum nicotianae and Pythium aphanidermatum which got from culturing in vitro respectively on transgenic tobacco and control tobacco leaves. The symptoms of control tobacco leaf is apparent, and symptom of transgenic tobacco is lighter. Results show that the exogenous gene PsnsLTP improve the tolerance of tobacco to the anthracnose and damping-off. Primarily validated the gene in the function of these pathogens.4,Construct the exogenous gene PsnsLTP yeast expression vector pYES2-nsLTP and transfer it into Saccharomyces cerevisiae INVScl, PCR and RT-PCR proved that the exogenous gene has been successfully transferred into INVScl and normal transcription. Under 5 M NaCl; 1 M NaHCO3; 1 M Na2CO3; 400μM CdCl2; 3 mM,5 mM,8 mM CdSO4 and 1 mM,3 mM,5 mM CUSO4 stress, with stress time and stress solution concentration increases, transgenic yeast survival rate is always higher than the control, and the difference became more and more obvious. Indicating that under the inducing of galactose, exogenous gene nsLTP can improve transgenic yeast INVScl-pYES2-nsLTP resistance to alkali, cadmium and copper. The experimental results and transgenic tobacco (nsLTP) experimental results are consistent, all show that the nsLTP genes can give plants certain resilience.