| The GCN(general control non-derepressible)system in yeast is a regulation mechanism of the initiation of protein translation under amino acid starvation.In the absence of amino acids,GCN1 interacts with GCN20,the complex GCN20-GCN1 regulates GCN2 activation,and GCN2 kinase in turn phosphorylates eIF2α to inhibit the translation initiation.AtGCN1 in Arabidopsis is a homology to GCN1.Previous experiments showed that AtGCNl is involved in the regulation of GCN2 activity and protein translation under cold stress.We suppose that a GCN system exists in Arabidopsis to regulate protein translation under endoplasmic reticulum stress(ER stress).The gcnl-1 mutants are here found to be more resistant to TM than the wild type;The expression of ER stress-related genes are up-regulated in gcn1-1 and the phosphoration level of eIF2α was down-regulated in gcnl-1 under ER stress conditions,compared with wild type.Taken together,these results show that AtGCNl may regulate protein translation by regulating the phosphorylation of eIF2α in response to ER stress.The translation initiation factor eIF2 was modified by site-directed mutagenesis to change the serine at position 51 to alanine or aspartic acid,and the transgenic plants were constructed to mimic the phosphorylation state and non-phosphorylation state of eIF2.The phenotype of the transgenic plants was tested and the results are not consitant with to the supposed phenotype.There were two eIF2 homologs in Arabidopsis.A homozygous mutant is screened out and the other homolog has no T-DNA mutation so the mutant eIF2α will be created by CRISPR and the double mutants will be obtained for further verification.During my postgraduate study,the optimization of corn pollen transformation technology was also carried out.In this experiment,Chang 72 and Zheng 58 corn pollen were used as materials.Ultrasonic treatment and electric shock treatment were used to transform ps1300+GFP and CRISPR vectors into corn pollen to detect the transformation efficiency.If CRISPR vectors are transformed to corn pollen,as long as the foreign gene is expressed in the corn pollen,Cas9 can edit the genome of corn pollen.In order to promote the efficiency,we improve the methods of corn pollen transformation.The projects aim to investigate whether AtGCN1-mediated eIF2α phosphorylation pathway is involved in ER stress,the regulation mechanism of AtGCNl in ER stress and the optimization of pollen-mediated transgenic methods in maize transformation. |
