Poplar Salt Stress Response Genes PtRabA2f Function

Small GTP-binding protein take part in signal transduction, cell proliferation,cytoskeletal reorganization,cell membrane transport and so on in eukaryotic cells.These proteins function as molecular switches periodically regulated activation andinactivation state, the cycle was linked to the GTP binding and hydrolysis. In woodyplants, sequencing of small G protein family had114members in populus trichocarpa, itwas divided into four subfamilies, Rab reached66, accounting for57%of thetotal members. Moreover, populus trichocarpa small G protein family members aremore than21of arabidopsis, this difference can suggest that small G proteinfamily function specificity in populus trichocarpa.This experiment is in-depth study on the PtRabA2f gene function of populustrichocarpa. The test amplified PtRabA2f gene from populus trichocarpa cDNA thourghRT-PCR method, ORF fragment size is651bp. When building a plant expression vector,the experiment chose to use GateWay technology.First the experiment connectthe target gene RabA2f and intermediate vector pENTR-SD-D-TOPO,finally, theexperiment obtains intermediate vector pENTR-RabA2f, the RabA2f connects to theplasmid pGWB2formation of pGWB2-RabA2f through the replacement method, Byusing agrobacterium mediated method respectively topopulus trichocarpa andarabidopsis, obtains overexpression system populus trichocarpa and arabidopsis.By contrast WT, pGWB2and T2-PtRabA2f-5arabidopsis growth conditions. TheT2-P-5group is significantly better in the normal flat plate. the result shows fresh heavy,plant height, leaf bright and large, root length long etc, the growth, root length and freshweight are better than those of pGWB2group and WT group in stress conditions, haveless affected under stress conditions.In order to detect the populus trichocarpa RabA2f gene expression characteristics, the Actin1gene is used as the internal standard gene, using semi-quantitative PCRmethod. Under200mmol/L NaCl,8%PEG,200mmol/L mannitol and10mmol/L H2O2stress, compared with the control group, the PtRabA2f gene expression levels showed asignificant increase in state and every time is higher than WT group and pB2group, thehighest expression level in24h. When in200mmol/L NaCl treatment of24h,T0-PtRabA2f-3populus trichocarpa expression is3.54times of WT type, when in200mmol/L mannitol treatment of24h, T0-P-3populus trichocarpa expression is1.36times of WT type, when in10mmol/L H2O2treatment of24h, T0-P-3populustrichocarpa expression is2.81times of WT type, when in8%PEG treatment of24h,T0-P-3populus trichocarpa expression is2.37times of WT type, when in droughtstress treatment of24h, T0-P-3populus trichocarpa expression is2.5times of WT type.For WT, pB2and T2-PtRabA2f-5group arabidopsis, the each of the reaction timethe arabidopsis GTP hydrolysis ability changes is measured in the non-stress conditions,experiments showed GTP hydrolysis ability maximum in90min. The test selects0h、2h、12h、24h to measure GTP hydrolysis ability， in60mmol/L NaCl,8%PEG,200mmol/L mannitol,10mmol/L H2O2stress. The results show that the GTPhydrolysis ability of WT group are lower than T2-PtRabA2f-5over expression system ofarabidopsis GTP hydrolysis ability.In the80mmol/L NaCl: the group of T2-P-5arabidopsis chlorophyll contentdecreases at24h to144h, and reachs a minimum value at the time of144h, in144h,chlorophyll content began to increase, compared with the control group，chlorophyllcontent improves15.35%in144h, chlorophyll content improves17.34%in168h, theresults change little. In the8％PEG, compared with the control group, the arabidopsischlorophyll content improved25.31%in144h, the arabidopsis chlorophyll contentimproves26.64%in168h, the results change little In the200mmol/L mannitol:compared with the control group, the arabidopsis chlorophyll content improves24.43%in144h, the arabidopsis chlorophyll content improves25.99%in168h, the results change little. In the10mmol/L H2O2: the Arabidopsis chlorophyll contentimproves36.33%in168h, the results change little. These results show that the transgenic ofPtRabA2f arabidopsis system chlorophyll content is more stable and less affected understress conditions.In the80mmol/L NaCl: the group of T2-PtRabA2f-5conductivity value is smallerthan the group of WT and pB2conductivity, conductivity changes slowly and with theprolongation of time this trend changes more obvious, compared with the control group,the group of T2-P-5electrical conductivity decreases by6.01%in24h, the group ofT2-P-5electrical conductivity decreases by29.81%in168h, the results showthat significant differences. In the200mmol/L mannitol: compared with the controlgroup, the group of T2-P-5electrical conductivity decreases by6.34%in24h, the groupof T2-P-5electrical conductivity decreases by25.21%in168h, the results change little.In the8％PEG: compared with the control group, the group of T2-P-5electricalconductivity decreases by6.25%in24h, the group of T2-P-5electricalconductivity decreases by27.54%in168h, the results change little. In the10mmol/LH2O2: compared with the control group, the group of T2-P-5electricalconductivity decreases by4.62%in48h, the group of T2-P-5electricalconductivity decreases by28.67%in168h, the results change little.These results show that the transgenic of PtRabA2f arabidopsis system electricalconductivity is more stable and less affected under stress conditions.