Effects Analysis Of Tobacco γ-tubulin Gene Silencing

γ-Tubulin is a conservative gene and an important component of microtubule organization center (MTOC). It plays an important role in microtubule nucleation, cell cycle and the formation of spindle. In animal and fungal cells, microtubule nucleation takes place at conspicuous MTOCs. Such as the centrosome or the spindle pole body (SPBs). Although there were not discrete MTOCs in higher plant cells, the highly ordered arrays of microtubules which coordinate cell division were assembled. However, the cell division mechanism mediated by microtubules was still unclear in higher plants. Recently, more and more researches focused on the function ofγ-tubulin. For example, Martine et al. used T-DNA inserted mutants to investigate the role of two Arabidopsisγ-tubulin genes in plant development and in microtubule arrays assemble in 2006. Pavla et al. also downregulatedγ-tubulin by inducible expression of RNAi constructs in Arabidopsis in 2006. They found thatγ-tubulin was essential for microtububle nucleation in plant development including stomatal patterning, polar tip growth and so on. We had already constructed a recombinant potato virus X (PVX) vector by inserting aγ-tubulin partial cDNA fragment in an antisense orientation. The recombinant vector was used to silence theγ-tubulin gene in tobacco (Nicotiana tabacum var. Samsun NN) by virus induced gene silencing (VIGS). We found thatγ-tubulin is essential for tobacco cell size and shape, leaf width and plant polar growth.In this study, we tried to get theγ-tubulin silenced stable mutants by Agrobacterium -mediated method. Results showed thatγ-tubulin downregulating in tobacco cells caused multiple branches, trumpet-shaped leaves and the aberrant polarity transportation of endogenous IAA. It indicated thatγ-tubulin is essential for plants devolpment, such as polar tip growth, shape of leaves, polar growth of roots and polarity transportation of endogenous IAA.In addition, paraffin sectioning was employed to observe the microsporogenesis and male gametophyte development in Nicotiana tabacum whichγ-tubulin was silenced by inserting a fragment (522 bp) on PVX. The results showed that there were five stamens in each anther and each stamen had four microsporangiums. The primary sporogenous cell formed microspore mother cells. Then following the meiosis of microspore mother cells, the tetrahedral tetrads were formed. The morphology character of positive control tobacco microsporogenesis was normal completely. But the microsporogenesis ofγ-tubulin silenced tobacco had changed. Some buds were normal in the period of primary sporogenous cell, but the faliments expand, then the bud fell off, which was in the period of microspore mother cells. Some buds had abnormal tapetum and microspore in the period of tetrahedral tetrads. These results manifested that PVX vector with 522 bp inserted fragment inducedγ-tubulin of tobacco silencing, which influenced microsporogenesis and male gametophyte development of tobacco.