Screening And Phenotype Identification Of T-DNA Insertional Mutants Related To Transmembrane Protein In Arabidopsis Genes

The transporter proteins with transmembrane domains(TMDs)in plants are the indispensable functional molecules during the plant growth and development.They participate in the life process of plants,such as absorpting and transporting various ions,chemical molecules and some phytohormones in plants.The mechanism research of transporting substances across the membrane in Arabidopsis thaliana as the model plant,have become a hotspot in the field of molecular biology.There are about 13%of genes encoding transporter proteins in Arabidopsis genome,while part of these genes function are still unknown.In this study,we screened and confirmed some T-DNA insertion mutants of some genes which encode putative transporter proteins,furthermore,we explored some features of these mutants,and provided the research materials and some cues for deeply researching those putative transporters genes.First we ordered 62 T-DNA insertion mutants from the Arabidopsis biological resource center(ABRC),which belong to 33 genes respectively.These genes all have at least 3 transmembrane domains(TMDs)predicted by TMHMM.Using the "double primers" PCR method,we confirmed most of those T-DNA insertion mutants,and screened 50 homozygous mutants.These seeds provided important material for the following experiments.We selected two homozygous single mutant of At2g03330 and At3g11810 genes respectively,and constructed 4 heterozygous double mutant lines using artificial cross-pollination method,and then successfully screened those 4 homozygous double mutants lines.These homozygous double mutants also provide important experimental materials for following study of mechanism of those two genes with functional redundancy.During researching function of those genes,we found some individual plants of 5 mutants survived after treatment of high temperature stress while the wild type plants all died,but the survival rate of those 5 mutants is differert.Furthermore,according to one of those mutants which have the highest survival rate after high temperature treatment,we cloned full length genomic DNA of that gene from wildtype genome,then inserted it into the binary vector of pCAMBIA1300,and provided important experimental basis for confirming the gene function by transforming the whole gene into that mutant through A.tumefacien mediated gene transferring by floral dipping.In summary,the research carried out preliminary exploration of some genes encoding putative transporters with several transmembrane domains.First we confirmed most of these T-DNA mutants and screened 50 homozygous single mutants.Besides that,we successfully got 4 homozygous double-mutants of At3911810 and At2g03330 with functional redundancy,through artificial cross pollination.In addition,we also treated some mutants under high temperature stress,and speculate these genes'function being related to Arabidopsis temperature susceptibility.These works can provide materials and some cues for the following research.