Studies On Transcriptional Activation Of ABI5 Subfamily

Abscisic acid(ABA),a plant hormone,widely exists in bacteria,algae and plants.It can regulate the growth and development of plants and respond to a variety of biological and abiotic stress responses such as bacteria,low temperature,drought and high salt.The ABI5 family is a basic leucine zipper(bZIP)transcription factor,which is involved in the gene expression and regulation of plant seeds in the late embryonic development and ABA signal transduction.Early studies in our laboratory have shown that there are two regions in ABI5 that can activate reporter genes,one is the non conservative region at the carboxyl end,and the other is the conservative region II(region II).In general,yeast one hybrid or yeast two hybrid can be used to study the transcriptional activation of transcription factors.Does the conserved region II of ABI5 family also have transcriptional activation in plant cells? Moreover,can SnRk2.6 kinase activate the transcription activation of ABI5 and interact with ABI5 in yeast cells? In order to answer these questions,we have studied the transcriptional activation of ABI5 family members in yeast cells and plant cells.The main results of this paper are as follows:(1)The activity of transcriptional activation of ABI5 family member conservative region ? was analyzed by yeast single hybrid method in Saccharomyces cerevisiae AH109,and the activity of ?-galactosidase was measured to quantitatively analyze its transcriptional activation activity.The results showed that DPBF4CR?had relatively high transcriptional activation activity,while DPBF5CR?,ABF2CR?,DPBF2CR?,ABF1CR?,ABF4CR?,the transcriptional activation activities of DPBF3CR?,ABI5CR? and At5G42910CR? decreased in turn.(2)The activity of transcription activation of SnRk2.6 kinase and ABI5 was analyzed by yeast two-hybrid method.The experimental results showed that pGADT7 did not interact with ABI5,that is,ABI5 itself had no activity of transcription activation,but SnRk2.6 kinase could interact with ABI5 in yeast cells,that is,SnRk2.6 might activate the activity of transcription activation of ABI5,and the results showed that ABI5 had no activity of transcription activation Serine 42,Serine 145,Threonine 156 and Threonine 201 may be involved in the activation of ABI5 by SnRk2.6.(3)The results showed that the GUS / LUC ratio of ABF1CR?+pHQRep(6x)+pLUC in experimental group 1 was 0.111,the GUS / LUC ratio of ABF2CR?+pHQRep(6x)+pLUC in experimental group 2 was 0.055,and the GUS / LUC ratio of ABF4CR?+pHQRep(6x)+pLUC in experimental group 3 was0.108,The GUS / LUC ratio of ABI5CR?+pHQRep(6x)+pLUC in experimental group 4 was 0.843,and that of DPBF2CR?+pHQRep(6x)+pLUC in experimental group 5 was 0.376,The GUS / LUC ratio of DPBF3CR?+pHQRep(6x)+pLUC in experimental group 6 was 0.096,that of DPBF4CR?+pHQRep(6x)+pLUC in experimental group 7 was 2.557,that of DPBF5CR?+pHQRep(6x)+pLUC inexperimental group 8 was 1.371,that of At5G42910CR?+pHQRep(6x)+pLUC in experimental group 9 was 0.353.It can be seen from the above data that in the transient expression analysis system of plant,with the extension of culture time,the activity of GUS enzyme increases steadily,and there is endogenous activity of GUS enzyme in Arabidopsis protoplast,which indicates that the conservative region ?(CR ?)of ABI5 family members has transcriptional activation activity in plant cells,and there are ABI5 families in plant protoplast cells The transcriptional activity of the conserved region ?(CR ?)of DPBF4 in the family members is relatively high,which is similar to the result obtained in yeast single hybridization.(4)In order to further study the transcriptional activation activity of conserved region II of ABI5 family members in Saccharomyces cerevisiae AH109 and plant protoplast cells,the vector pGADT7-SnRK2.6 and pYLUC vector were constructed in this study,which were used in subsequent yeast two hybrid experiments and transient expression analysis experiments.(5)In order to investigate whether the repeated sequence of the GAL4 binding site UASGal1 added in the reporter gene can increase the expression of the downstream reporter gene,this experiment mainly used the internal reference vector pLUC,and re-conducted the DPBF4 member conserved region ? in plant protoplast(CRII)transcription activation activity study,the results showed that the GUS / LUC ratio of pHQEff-6+pHQRep(3×)+pLUC in experimental group 1 was 195.876,the GUS / LUC ratio of pHQEff-6+pHQRep(6×)+pLUC in experimental group 2 was275.814,and the GUS / LUC ratio of pHQRep(3×)+pLUC in experimental group 3was 123.703,and the GUS / LUC ratio of pHQRep(6×)+pLUC in experimental group 4 was 159.013,The data showed that the GUS / LUC ratio of pHQEff-6+pHQRep(6×)+pLUC in experimental group 2 was 1.41 times of that of pHQEff-6+pHQRep(3×)+pLUC in experimental group 1,and the GUS / LUC ratio of pHQRep(6×)+pLUC in experimental group 4 was 1.29 times of that of pHQRep(3×)+pLUC in experimental group 3.However,pHQEff-6 contains DPBF4 conserved region ? gene coding fragment,pHQRep(3×)and pHQRep(6×)contain the repeat sequence of UASGal1,the upstream activator of GAL4 DBD,so the results can show that DPBF4 conserved region ? can activate the transcription of GUS in Arabidopsis protoplast cells.Moreover,the increase of the repeat of the binding site UASGal1 of GAL4 in the reporter gene can increase the expression of the downstream reporter gene.However,the increasing trend of this activity is not linear with the number of repeat of UASGal1.