| The origin of plants is one of the most important events in the history of life,which greatly promotes the change of global climate and formation of ecosystems.From the ocean to fresh water to land,plants continue to break through the boundaries of their own survival.In the long process of evolution,plants have obtained many new genes and traits in order to adapt to the complex and changeable environment.Previous studies have shown that horizontal gene transfer,as an important new gene acquisition mechanism,plays a key role in plant adaptive evolution.Drought,as an abiotic stress,is a serious threat to agriculture and global food security.The gene which codes guanine deaminase was found among the differentially expressed genes in rice under drought stress.As a key enzyme in purine metabolism pathway,GDA has the catalytic activity of deamination of guanine to xanthine and promotes plant growth and development by maintaining nitrogen homeostasis.Although there has been some research of the physiological and biochemical activity of this gene in plants,there is no detailed research on its origin and evolution and its function in rice.Therefore,in this study,the origin and evolution model of plant GDA gene were clarified by sequence analysis,phylogenetic analysis and adaptive evolution analysis,and the function of OsGDA gene in rice was preliminarily identified by bioinformatics and molecular biology.The main results of this study include:1.Based on the homology search of GDA gene in plants,it was found that GDA gene existed widely in plants,but homologous gene was not found in cruciferous plants.The results of phylogenetic analysis showed that the plant GDA homologous sequence was divided into three branches,in which all streptophytes were in same branch,and green algae were divided into two branches.Gene structure analysis showed that the number of introns was conserved in most plants,and there was a great difference in intron phase between streptophytes and green algae.Then,we predicted conserved motifs of plants GDA protein sequences,and it was found that the motifs within different branches of the phylogenetic tree were consistent,but there were obvious differences between branches.Sequence consistency analysis showed that the sequences within different branches were more consistent,but the sequence consistency among populations was poor.These results suggest that different plant branches may be the result of independent origin.The result of further homology search of plant GDA sequence in all cellular organisms showed that the protein existed in archaea,bacteria,fungi and other eukaryotes.Sequence consistency analysis found that streptophytes and green algae branch I had high homology with bacteria,while green algae branch Ⅱ showed more similar with fungi.The result of phylogenetic analysis revealed that the gene in ancestor of streptophytes originated from chloroflexi through an HGT event,while the GDA gene in the two green algae branches originated from chloroflexi and mucoromycota,respectively.2.Adaptive evolutionary analysis found that plants experienced positive selection in a short time after horizontal transfer of GDA gene,while purified selection dominated in the subsequent evolution,but the positive selection effect was still significant in streptophytes and green algae branch Ⅱ.Through the analysis of significant positive sites,we found they distributed outside the aminohydrolase domain,indicating that functional innovation may have taken place in GDA genes in the evolutionary process of these two populations.3.qRT-PCR analysis was performed to analyze the expression pattern of rice OsGDA gene in different growth periods,we found that it was expressed in all parts of rice at different growth stages,and the expression was extremely up-regulated in leaves and sheaths at filling stage.It was suggested that the gene may play an important role in yield formation.The results of subcellular localization of OsGDA protein showed that OsGDA was located in cell membrane,cytoplasm and nucleus.Based on the analysis of the expression under different hormone and abiotic stress,it was found that the gene was induced by high temperature,low temperature,drought and salt stress.At the same time,it showed a positive response to IAA,GA,ABA and 6-BA.This result suggested that OsGDA gene may enhance the resistance of plants to stress by participating in the hormone regulatory network.4.Through the analysis of OsGDA promoter sequence,it was found that there were a variety of response elements in this sequence,including light response,hormone response such as auxin,salicylic acid,methyl jasmonate and abscisic acid,drought and hypoxia induced response and so on.The existence of these acting elements establishes the basis for the transcriptional regulation of the gene.Further construction of the co-expression gene network and GO enrichment analysis showed that the co-expression genes were widely involved in the membrane system,Golgi apparatus and apoplast region,and had physiological and biochemical activities such as redox,glycosylation and transmembrane transport,and participated in related biological processes.In addition,KEGG pathway analysis also found that these co-expression genes were related to the synthesis and metabolism of secondary metabolites and signal transduction pathways.5.In this study knockout lines and overexpression lines were constructed with Nipponbare as the background,and the major agronomic traits of each line were investigated.Compared with the wild type,the overexpression lines showed the characteristic of dwarfing,and produced more tillers,and finally increased the yield per plant.Furthermore,in order to simulate drought condition,the materials of different lines were treated with PEG-6000 and soil culture dehydration.The results showed that the survival rate of overexpression lines was higher than that of wild type,while the survival rate of knockout lines decreased.The expression analysis of wild type showed that the expression of OsGDA increased significantly after treatment,and decreased on the 9th day of treatment.In addition,expression analysis of the samples stopped irrigation for 6 days showed that overexpression could significantly increase the expression of genes related to abscisic acid synthesis and transduction,and the negative regulatory gene of gibberellic acid signal was also significantly induced.Taken together,our results not only clarified that plant GDA gene was obtained from bacteria and fungi through three independent HGT,but also further indicate that the gene is widely involved in hormone response to improve stress resistance in rice,which provides new resources for rice resistance breeding,but also provides a reference for the application prospect of this gene in cruciferous plants. |
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