Knockout And Prokaryotic Expression Of Vascular Bundle Development Related Genes Sm-Nvas In Tobacco

Vascular bundles of plants are the most important conducting tissues of plants.The formation and development of vascular bundles is a biochemical process that is affected by multiple genes and regulatory pathways.We have cloned a novel gene sm-Nvas expressed specifically in the vascular bundles in tobacco.To analyze its function,we have constructed successfully the expression vector with CaMV35S::sm-Nvas::GUS and transformed into tobacco.It showed that there were dwarf plants,thick stems,thick veins,the more number of vascular bundles and the shorter length of parenchyma cell of vascular bundle in the transgenic plants.Therefore,it is believed preliminarily that sm-Nvas is related to the development of vascular bundle.Based on these study results,the main contents of this study are as follows:(1)In order to further analyze the function of the gene,the knockout vector pc1300-Cas9-gRNA-sm-Nvas containing two sequences of sm-Nvas was constructed successfully and transferred into Agrobacterium by electrotransformation.Then,the knockout vector transferred into tobacco by Agrobacterium tumefaciens mediated leaf disc transformation method.The positive transgenic plants were identified and the edit of the gene was analyzed.Based on the comparative observation between transgenic plants and wild plants,it is further revealed that the gene plays an important role in the growth and development of vascular bundle.(2)Meanwhile,the prokaryotic expression vector pMAL-p5x-sm-Nvas was constructed.The water-solubility of sm-Nvas protein was enhanced by optimizing the induction conditions.The soluble purified protein lays the foundation for further elucidating the enzymatic properties of the gene product and the molecular mechanism of the gene in the development of tobacco vascular bundles.