Functional Study Of Horizontal Transfer Originating Vascular Bundle Related Gene OsBSP1 In Rice

Vascular bundles can transport water and nutrients and provide mechanical support for plants,which is of great significance for adaptive evolution of plants.Bark Storage Protein(BSP)plays an important role in nitrogen cycling in perennial plants.In our previous study,we found that BSP gene was acquired by vascular plants from bacteria through a horizontal gene transfer event,and undergo gene replication and functional differentiation in vascular plants.However,the biological function of BSP gene has not been elucidated in the monocot crop rice.In this study,the biological functions of BSP gene in rice were preliminarily analyzed through expression pattern analysis,transgenic material creation,phenotypic observation,agronomic traits identification and cytological observation.The main results are as follows.(1)Expression pattern analysis showed that the expression level of OsBSPl gene was higher in root,stem and leaf sheath,but lower in leaf and floret,and the expression level of OsBSP1 gene was significantly higher in vegetative growth stage than in reproductive growth stage.In leaves,OsBSP1 was mainly expressed in vascular bundles.In stem,leaf sheath and root,OsBSP1 was expressed in vascular bundle tissue and parenchyma cells.In addition,the results of tobacco subcellular localization experiments showed that OsBSP1-GFP fusion protein was localized in the cell membrane and cytoplasm.(2)OsBSP1 overexpression and CRISPR-Cas9 transgenic materials were constructed and their phenotypes were identified at different developmental stages.The results showed that the plant height,root length and root number of OsBSP1 overexpression lines were significantly higher than those of wild type at seedling and tillering stages,while the plant height and root length of CRISPR-Cas9 mutant were significantly lower than those of wild type.At maturity,the plant height,stem length and aboveground biomass of OsBSPl overexpression lines were significantly higher than those of wild type,while the above indexes of CRISPR-Cas9 mutant were significantly lower than those of wild type.These results indicate that OsBSP1 has significant positive effect on rice growth and development.(3)By observing the tissue sections of rice stems at heading stage,it was found that the large vascular bundle area of OsBSP1-Cas9 plants was significantly lower than that of the wild type,while the large vascular bundle area of OsBSP1 overexpression plants was significantly higher than that of the wild type.In addition,the number of vascular bundles and internode diameter of OsBSP1 overexpression plants were significantly higher than those of wild type The results showed that OsBSP1 gene may be involved in vascular bundle development.(4)The investigation on yield traits of transgenic rice materials showed that the effective tiller number,main panicle length and grain number per panicle of OsBSP1 overexpression lines were significantly increased,while significantly decreased in OsBSP1-Cas9 plants compared with wild type.The yield per plant of OsBSP1-OE increased by 23%on average compared with that of wild type,while the yield per plant of mutant decreased by 28%.These results indicate that OsBSP1 positively regulated rice yield by regulating effective tiller number and grain number per panicle.(5)The expression levels of vascular bundle developing-related genes in rice were detected.Results showed that the expression levels of TAL and VEP1 were decreased in OsBSP 1-Cas9 plants and increased in OsBSP1 overexpression lines.These results suggest that OsBSP1 may cooperate with TAL and VEP1 in regulating vascular development in rice.(6)Measurement of yield traits revealed that the effective tiller number and main panicle length of OsBSPl overexpression lines were significantly increased compared with wild type,and significantly decreased in OsBSP 1-Cas9 plants.The yield per plant of OsBSP 1-OE increased by 23%on average compared with that of wild type,while the yield per plant of mutant decreased by 28%,indicating that OsBSP1 had a positive effect on rice growth and yield.In summary,the results of this study showed that the horizontally acquired BSP gene positively affected the plant height,root length,biomass,plant height and effective tiller number at the vegetative stage,and then had a positive effect on rice yield through promoting vascular development in rice.The results provide important information for revealing the role of horizontal gene transfer in the evolution of vascular plants,and provide reference for genetic improvement of rice yield.