Origin And Evolution Of The Land Plant NRPSL Gene And Preliminary Functional Characterization Of The Rice OsNRPSL Gene

The origin of the land plant is one of the most critical events in the evolution of terrestrial life,which played a crucial role in the establishment of modern terrestrial ecosystems.There is evidence that the land plant emerged from the streptophyte algae,which have the ability of transforming from aquatic to terrestrial.In order to adapt to the changing environment,the ancestor of the land plant acquired new phenotypes and metabolic pathways in the process of transition to land.Non-ribosomal peptide synthetase is a large-scale assembled biocatalyst.Its products play an important role in the interaction between plants and pathogens.The NRPSL gene encodes a homologue of non-ribosomal peptide synthetase,but its function in rice is completely unknown.In this study,phylogenetic and positive selection analyses were performed to investigate the origin and evolution of the NRPSL gene in land plants.Based on the method of molecular biology,the pattern of spatiotemporal expression and the expression under abiotic stress and hormone of the NRPSL gene in rice was explained by real-time qPCR.Based on the method of reverse genetics,CRISPR/Cas9 mutants,and overexpression lines of the rice OsNRPSL gene were constructed to reveal the function of the rice OsNRPSL gene.The main results are as follows:1.NRPSL gene is widespread in land plants,but there is no homologous in chlorophyte green algae and streptophyte algae.Phylogenetic analysis showed that there were homologous genes in fungi and most bacteria.In the phylogenetic tree,the land plant NRPSL genes formed a single branch,which fell into the branches of fungi,and the plant NRPSL gene had the highest sequence similarity with its homologous gene in fungi.The result showed that the NRPSL gene in land plants was obtained from the fungi through the horizontal gene transfer event.The analysis of adaptive evolution indicated that the land plant NRPSL gene had undergone positive selection in the early stage of evolution,and the dominant force contributing to the evolution in land plants was purifying selection.These findings indicated that the NRPSL gene was fixed by positive selection in the early stage of land plant evolution.2.The analysis of temporal and spatial expression of the OsNRPSL gene showed that the gene had higher expression in the root through the period of vegetative growth.The analysis of the expression patterns under the abiotic and hormone treatment of the OsNRPSL gene revealed that the expression of this gene was induced by oxidation,high temperature,drought and salt stresses,and GA and IAA treatments.The increased expression of the gene under induction indicated that the gene may be related to the response of stresses.3.The phenotypes of the OsNRPSL gene mutants and overexpression lines were identified at maturity.Induced plant height,increased tillers with the narrowed and shortened leaves all hinted the inhibited development of the mutants,while the leaves of overexpression lines became wider and longer.Moreover,the analysis of the phenotypes of transgenic plants and the wild type under the treatment of PEG showed that the root length of both mutants and overexpression lines was shorter than that of wild-type before and after the treatment,and the shoot length of mutants was shorter than that of wild-type,while the shoot length of overexpression lines was significantly longer than that of wild-type.It was found that O2-accumulation in leaves of mutant lines was more than that of wild-type,while that of overexpression lines was less than that in wild-type.Moreover,the water content of mutants was lower than that in wild-type before and after the treatment,while the overexpression lines was higher than that in wild-type.All of these above demonstrated that the OsNRPSL gene may be involved in the regulation of drought response.These results not only provided crucial information for revealing the origin and evolution of the land plant NRPSL gene but also provided a reference for further revealing the biological function of the OsNRPSL gene,especially the function in response to drought stress.