Physiological Function Analysis Of Vacuolar ATPase B Subunit In Arabidopsis Thaliana

pH homeostasis is an necessary condition for plant growth and development.It is a main contributor to the regulation a variety of signal transduction as an important signal of plant cells.In eukaryotic,V-ATPase as proton pump has an important role in the maintenance of intracellular pH balance.V-ATPase of the plant is localted in the membrane of tonoplast and other secretion systems,such as endoplasmic reticulum(ER),Golgi,and some small vesicles membrane.The main function of V-ATPase is to cause vacuole acidification by catalyzing the hydrolysis of ATP,which can provid necessary energy for transportion of ions and metabolites.V-ATPase B as a catalytic subunit of V-ATPase can take part in the process of the hydrolysis of ATP.Meanwhile,our laboratory has proved that V-ATPase B subunit as an actin-binding protein plays an important role in regulating actin dynamics process.However,there is no report about that V-ATPase B subunit can regulate plant growth and development process.Thus,it is important to study the physiological functions of V-ATPase B,which can reveal the growth and development of plant.In this paper,Arabidopsis thaliana as a material was researched and the physiological functions of three subtypes of genes V-ATPase B subunit(AtVAB1,AtVAB2,AtVAB3)were studied,the main results are as follows:1.The RT-PCR technique was used to identify gene expression of vab1,vab2 and vab3 mutants inserted by T-DNA at the transcriptional level,but there was no significantly inhibitory ability.The growth and development of mutants has no significant phenotypic difference as comparted to the growth and development of wild type.2.AtVAB1,AtVAB2 and AtVAB3 CDS gene sequence was fused to green fluorescent protein 5 'end(GFP)sequence and gene was expressed by its own promoter.Homozygous lines were screened by tranfering vector into wild type.The subcellular localization of AtVAB1,AtVAB2 and AtVAB3 was observed by confocal microscopy and they were located in the vacuolar membrane.3.Construction of cDNA-RNAi vector was transfected into Arabidopsis and disturbed the target gene.The expression of AtVAB1 AtVAB2 and AtVAB3 identified by RT-PCR was not significantly inhibited.4.The vitro experiments suggest that the rate of pollen germination and pollen tube length of eDNA-RNAi transgenic plants were significantly descended as compared with wild type's.There was positively correlated with the time in respects of pollen germination and pollen tube length of wild-type.After two hours,pollen germination and pollen tube length of the cDNA-RNAi transgenic lines were not significantly changed.5.The rate of pollen germination except pollen tube length of cDNA-RNAi transgenic lines which were treated by 1 nM Lat B was more sensitive as comparted to wild type.6.Alexander staining experiments show pollen activity of cDNA-RNAi transgenic plants was not changed.The results of Aniline blue staining suggest that the rate of pollen germination was not significantly different between cDNA-RNAi transgenic plants and wild type,but pollen tube length of cDNA-RNAi transgenic plant becomes shorter as compared with wild type.7.By transfecting overexpression vector(OE.VAB1)into Arabidopsis,the results suggest that overexpression homozygous lines were characterized by larger seed,the defect of seedling growth,plants lose apical dominance and sensitive to salt stress.In summary,molecular biology,genetics and cell biology methods were used to prove that V-ATPase B subunit plays an important role in the respects of plant growth,pollen tube growth and response to salt stress signaling pathways.