Analyses Of TUA2 Involved In Seed Germination Under ABA Stress In Arabidopsis Thaliana

Tubulin is a kind of protein which generally expresses in eukaryocyte , and it is made up of tubulin heterodimers composed byαtubulin andβtubulin.There are 6 different TUA subgroups and 9 different TUB subgroups in Arabidopsis . Many experimental results showed that different tubulin subgroups not only expressed effectively in specified developmental stage and tissue , but also change their expression level under environmental stresses. Thus , tubulin is a kind of housekeeping gene , and it participated in masses of singal respond reaction .Our research used the tua2 mutant of TUA2 subgroups as material , and we undertaked the research of germination under the TUA2-participated ABA stress by reverse genetics , genetic transformation , Tail PCR , semiquantitative-PCR and other technology.The major results showed as follow :1. We found an ABA-sensitive mutant from several T-DNA insertion mutants by phaenotype observation . Then we found the T-DNA insertion site ( located in the upstream promoter region of TUA2 ) with Tail PCR ,and the level of TUA2 genetic expression raised in this T-DNA insertion mutant. In addition we obtained the mutant inserted in the corresponding site form the SALK library ,and got three overexpression mutants in different strains by genetic transformation.2. We constructed TUA2 transgenic expression vector for subcellular localization , transiented expressed the fusion gene of TUA2 and GFP by particle gun transformation . The results showed that the TUA2 mainly distributed at cellular membrane.3. By observation of germinationrate under ABA stress , the TUA2 mutant is found , and the overexpressionmutant all emerged a phaenotype of germination delayed. We supposed that TUA2 may participate in the germination regulating process of ABA.4. We analyzed the expression level of ABA pathway related gene by RT-PCR, found that the expression level of mutant had been depressed compared to HAB and ABI in wild type under ABA stress. It implied that TUA2 may participated in ABA negative regulation process.