Gene Cloning And Allele Mutants Analysis Of Epidermal Cell Mutant Tub4^S95Fin Arabidopsis

Cytoskeletons are closely associated with many life activities,including the maintenance of cell shape,cell division,cell movement and material transportation.The distribution and arrangement of microtubules are very important for the maintenance of cell shape.In Arabidopsis,the rapid growth tissues and organs will display helical growth and abnormal cell division phenotypes when the tubulin mutated or some mutated microtubule bound protein(MAP)binding to microtubules,resulting in disordered microtubule arrangement.In this study,we screened an epidermal cell mutant tub4S95F from an EMS-mutagenized pool,which shows some abnormal phenotypes,such as right-handed rosette leaves,swelling pavement cells,shorter and thicker hypocotyls in light condition and shorter and twisted hypocotyls in dark condition.We tested cell ploidy of pavement cells by flow cytometry and found the cell numbers of 4C,8C and 16C ploidy are higher in tub4S95F than in wild type,indicating that increased endoreduplication leads to pavement cell swelling.We mapped the mutant gene to a 200 Kb region through map-based cloning and found a candidate gene TUB4.Through DNA sequencing,we found there is a C to T substitution at 284 bp in the first exon of TUB4,resulting in a serine to phenylalanine substitution at the 95 amino acids.The genetic complementation experiment shows that the hypocotyl and pavement cell defects have been restored.This result strongly suggests that the TUB4S95F is responsible for the tub4S95F growth defect phenotypes.In order to have a further understanding of the TUB4's function on plant development,we analyzed the T-DNA mutants(tub4-1,tub4-2)of TUBA.The T-DNA insertion site is on promoter region and 3'UTR region,respectively.The qRT-PCR result shows that the expression levels of TUB4 gene in these two lines are decreased differently compared with wild type,but far from knock out,so no obvious phenotypes are seen.Through studying different tubulin point mutation seedlings,the rosette leaves exhibit right or left handed patterns,the pavement cells show strong or medium swelling phenotypes and the hypocotyls of some lines are twisted under dark condition.These results suggest that the impact of point mutation is more severe than TUB4 gene knock down leading by T-DNA insertion.Besides,we crossed the left-handed mutant tub4S351F with right-handed mutant tub4S95F,the epidermal cells and hypocotyls of F1 generation seedlings show normal phenotypes.