Molecular Characterization Of Tobacco (Nicotiana Tabacum L.) Expansin Genes

The plant cell wall is a very important feature to both plants and man.As such,its manipulation has the potential to bring about some significant breakthrough in the field of agriculture and its downstream industries.Besides providing the strength that is needed by the plants,cell walls define cell shape,size and function.As such,there is constant assembly,remodeling and disassembly of the cell wall during the plant’s lifetime.These processes of cell wall degradation,synthesis and modification are facilitated by a variety of plant proteins and enzymes.Expansins are an example of such cell wall proteins and they are involved in cell wall modifications underlying many plant developmental and physiological processes.Decades after their initial discovery in cucumber,it is now clear that these small proteins have diverse biological roles in plants.Through their ability to enable the local sliding of wall polymers by reducing adhesion between adjacent wall polysaccharides and the part they play in cell wall remodeling after cytokinesis,it is now clear that expansins are required in almost all plant physiological development aspects from germination to fruiting.Among other possible mechanisms,expansins are believed to achieve these many roles through their p H dependent non-enzymatic wall loosening ability.Although a lot of work on expansins has been done thus far,only a few tobacco expansins have actually been studied in tobacco.However,from the so many expansin experiments which involved either overexpression,suppression or knockout of these genes,it has been demonstrated that these genes can bring significant changes to plants in many ways.This research work therefore aimed at identifying and categorizing the expansin genes in tobacco as well as generating and characterizing transgenic tobacco plants overexpressing expansin genes.This thesis presents two related studies.The first study concerns genome-wide analysis of tobacco expansin genes and their global expression in tobacco.In this study,we identified a total of 52 tobacco expansin genes.These 52 expansins were grouped into their respective subfamilies: 36 Nt EXPAs,6 Nt EXPBs,3 Nt EXLAs and 7 Nt EXLBs.Phylogenetic analysis showed that these 52 tobacco expansins were further divided into 13 subgroups.It was also observed that although the α-expansin subfamily of this species was the largest subfamily,just like most other species,the size of the Nt EXLB subfamily was relatively larger than what has been reported in other species.Gene structure analysis revealed that genes within the same subfamilies/subgroups exhibited similar characteristics such as gene structure and protein motif arrangement.In this study,cis-acting element analysis revealed that each expansin gene was regulated or several expansin genes were co-regulated by both internal and environmental factors.35 of these genes were identified as being expressed according to a microarray analysis.In contrast to most Nt EXPAs which had higher expression levels in young organs,Nt EXLAs and Nt EXLBs were preferentially expressed in mature or senescing tissues,suggesting that they might play different roles in different organs or at different developmental stages.Also,microarray analysis and q PCR studies revealed a spatial-temporal expression pattern of these expansin genes in tobacco.Nonetheless,the results also showed that the tobacco expansin genes are expressed throughout the entire life cycle of tobacco,with some genes having high expression levels at different developmental stages,suggesting their probable involvement in a certain developmental period directly or indirectly.As the first step towards genome-wide analysis of the tobacco expansin gene family,this work provides solid background information related to structure and expression as well as regulatory cis-acting elements of the tobacco expansins.The second study is concerned with the generation and characterization of transgenic tobacco plants overexpressing selected expansin genes.In this study,we begun by assessing the spatial-temporal expression pattern of the tobacco expansin genes.Since tobacco is mainly grown for its leaf,our target genes were those which are predominantly expressed in the leaves.Basing on the reads per kilo base per million(RPKM)values,we selected 8 expansin genes which were shown to be predominantly expressed in the leaves.We cloned,generated and characterized transgenic tobacco plants overexpressing these genes.All but Nt EXPA11 did not result in significant phenotypic changes.We therefore focused our attention on Nt EXPA11 and investigated the effect of overexpressing it on tobacco growth and development.Expression analysis of this gene using q PCR showed that although it was present in several tissues,its transcript levels were highest in the stems,flowers and leaves and lowest in the roots.After overexpressing it in tobacco,it modulated plant growth and development.The transgenic tobacco plants exhibited enhanced growth characteristics under in-vitro,greenhouse and field conditions.Compared to wild-type tobacco plants,Nt EXPA11-ox plants demonstrated an increased growth rate which was characterized by a vigorous root system as well as significantly bigger leaves and internode lengths.In comparison with the wild-type plants,transgenic tobacco plants showed a 0.75-fold and 0.5-fold increase in plant height and leaf length respectively under in-vitro conditions.Most interestingly,the transgenic plants had significantly improved root system architecture.A closer look at the cells of these plants revealed that transgenic plants had bigger cells compared to their wild type counterparts.Furthermore,these transgenic plants exhibited an improved tolerance to salt and drought stress.Under salt stress,transgenic plants accumulated relatively less levels of H2O2 compared to the wild type plants.However,when allowed to complete their life cycle under normal growing conditions,transgenic plants developed at least three leaves less than the wild type plants even though they had significantly bigger internodes.Collectively,these results demonstrated that combined with other tools,expansin genes could be an effective strategy to improve crop production.