Cloning And Functional Analysis Of OsTUA2

Lateral roots are important for plants to uptake water and nutrient.The studies on lateral root development have been hightlighting.We isolated three rice mutants with defect in lateral root development from an EMS-generated rice mutant library (Kasalath,indica).Comparision of the number of lateral root primordial(LRP) of 3d-old seedlings with the number of LR of 7d-old seedlings indicates that the reduction of lateral roots of the mutants is caused by both reduction of LRP initiation and defect in LR emergence.Map-based cloning and complementation test confirmed that the mutation phynotypes in the three mutants are caused by different mutations in a gene forα-Tubulin protein 2 OsTUA2(LOC_Os11g0247300,7879771bp) located in Chromosom 11.The three alleles,therefore,designated as Ostua2-1,Ostua2-2 and Ostua2-3. The Ostua2-1 mutant caused by a point mutation exchanging of T with A at site of ORF 309bp,resulting in one amino acid substitution(F to L).The Ostua2-2 mutant changed bases from TCC at ORF 563-565bp to GAGAG,and the third mutant caused by 23 bases deletion between ORF 292bp and 314bp.Because of the different mutations,more severely mutated phenotypes of Ostua2-2 and Ostua2-3 were observed than Ostua2-1.RT-PCR analasis indicated that the OsTUA2 is expressed in whole tissues,including roots,shoots,leaves and panicles.Subcellular location of GFP fused OsTUA2 showed that it locates in whole cell.Tubulins are conserved in organisms,and the mutation of OsTUA2 influences the plant growth including reduction in plant growth rate,delay of heading date,and reduction in panicle size.In addition to the suppression of lateral root development,the elongation of primary roots and adventitions roots of mutants also reduced.Under Pi-starvation,the elongation of roots of Ostua2-2 and Ostua2-3 mutants are enhanced and the difference between WT and the mutants ranges from 60%under control to 15%under this condition after 35 days,indicating the mutants are more sensitive to Pi-starvation.To examine the effect of salt stress on mutants phenotype,we compared 10-day -old seedlings grown on 25mM NaCl with the control(on normal culture conditions). It showed that both shoots and roots of WT are repressed slightly,but the shoots of mutants are repressed severiously,the leaves withered and the first leaf scorched.The second leaf of Ostua2-1 huddled up and could not extend.The second leaf of Ostua2-2/-3 curled up and the tip region scorched.The Ostua2-1 root was a little shorter than the control,while the Ostua2-2/-3 root repressed heavily and became much shorter.It reveals that the mutants are more sensitive to salt stress than WT.The microtubule inhibitor Propyzamide remarkably reduced the primary root growth in mutants Ostua2-2 and Ostua2-3,and root tips showed swollen with proliferation of root hairs.It shows that the two mutants are more sensitive to Propyzamide than WT.