| The origin of new genes was the important force driving the evolutionary innovations of life.Horizontal gene transfer（HGT）and de novo origination are the important mechanism for the origin of novel genes.HGT refers to the exchange of genetic material across species boundaries.De novo evolved genes are novel genes that arise from previously noncoding DNA.Although HGT was believed to play important roles in adaptive innovation of green plants,its role in plant detoxification ability remains to be explored.In addition,de novo genes are important for the innovation of multiple eukaryotic genomes,whereas the origin,evolution and biological function of de novo genes are poorly understood.In this study,we uncovered the origin and evolution of plant SagB,OsJDG9 and OsDNE8 genes based on the exhaustive sequence analyses,and the roles of the horizontally transferred gene SagB and two rice de novo genes OsJDG9 and OsDNE8 in detoxification of nitroaromatics,the divergence between indica and japonica,and regulation of yield-related traits,respectively.The following includes the main findings:Part Ⅰ The origin and evolution of SagB gene and its roles in the tolerance of nitroaromatic stresses.Nitroaromatic substances are not only the widespread naturally-occurring products,but the important chemical feedstock that are commonly used for the synthesis of pesticides,pharmaceuticals,explosives,and other manufactured chemicals.These substances are persistent and recalcitrant to degrade in the environment,and have exhibited high toxicity and mutagenic activity to virtually all living organism.Thus,the clean-up of nitroaromaticcontaminated environments has long been a major international concern.Phytoremediation is proposed as a cost-effective and sustainable alternative to conventional remediation technology.Although some transgenic plants have been developed for phytoremediation of nitroaromatic contaminants through expressing some bacterial genes,nitroaromatic detoxification by plant endogenous gene remains to be demonstrated.The streptolysin S（SLS）-associated gene B（SagB）belongs to the nitroreductase superfamily,and is widespread in plants and bacteria.However,the investigations on the origin and evolution of plant SagB gene and its biological functions are still lacking.In this study,we revealed the origin and evolutionary pattern of plant SagB gene based on the sequence and phylogenetic analyses,and its roles in the resistance of nitroaromatic stresses through the molecular biology of rice and Arabidopsis.The following includes the main results:1.Sequence similarity analysis showed that SagB gene was widespread in nearly all sequenced land plants,including bryophytes,ferns,and seed plants.Land plant SagB genes shared the highest sequence identities with those of bacteria,and the phylogenetic analysis revealed that the ancestor of land plant SagB gene might be acquired from bacteria through a single HGT event.Further analysis showed that land plant SagB genes underwent positive selection during their early evolution.2.qRT-PCR and GUS assays showed that rice OsSagB and Arabidopsis AtSagB genes were constitutively expressed in all detected rice and Arabidopsis tissues,and had the highest transcripts in the roots.The subcellular localization analyses revealed that both Arabidopsis and rice SagBs are localized to the chloroplasts.In addition,the expression of both AtSagB and OsSagB genes was induced by multiple nitroaromatics,including the explosive 2,4,6trinitrotoluene（TNT）,natural compound 1-nitropyrene（1-NP）and herbicide pendimethalin（Pen）,suggestive of their functions in nitroaromatic tolerance.3.In vitro assays showed that plant SagB protein had the ability to degrade multiple nitroaromatics,including TNT,1-NP and Pen.In addition,the nitrite-oxidizing bacterial SagB protein enzymatically degrade multiple nitroaromatic substances,suggesting that the SagB enzyme activity was relatively conserved between land plants and bacteria.4.We further constructed the overexpression and knockout/knockdown lines of rice and Arabidopsis SagB genes,and performed their phenotypic observations under nitroaromatic treatments.The results showed that the SagB overexpressing plants exhibited longer roots than those of wild-type（WT）plants,while the roots of SagB knockout or knockdown lines were shorter than those of WT plants,suggesting SagB genes significantly increased plant tolerance to nitroaromatics.Further evidence showed that SagB genes increased tolerance and detoxification of TNT through facilitating its transformation to the amino derivative（ADNT）.5.Compared with WT,overexpression of SagBs in Arabidopsis and rice increased their ability of TNT uptake,and removed more TNT from the growth culture,whereas knockout or knockdown of SagB genes repressed this ability,suggestive of its potential application in phytoremediation of nitroaromatics.Our findings not only revealed that land plant SagB gene was acquired from bacteria through HGT,and that HGT plays essential roles in the origin of plant detoxification ability,but provided a new strategy and gene target for the phytoremediation of nitroaromatic-polluted environments.Part Ⅱ The origin and evolution of rice OsJDG9 gene,and its roles in contributing to the divergence of grain shape between indica and japonica subspecies.Rice（Oryza sativa L.）is one of the most important crops in the world.During the long process of rice domestication,it produced abundant genetic diversity,and obvious genetic divergence.Among them,the divergence between O.sativa L.ssp.indica and O.sativa L.ssp.japonica subspecies was one of the most important genetic divergence.There were obvious differences in morphological and physiological characteristics between indica and japonica subspecies,and their most significant morphological difference was the grain shape.In general,indica had slender grains,while japonica had short and round grains.Although multiple grain shape-related genes and their regulation pathways have been uncovered,mining the key gene controlling the divergence of grain shape between indica and japonica subspecies,and illustrating its roles in contributing the divergence of indica and japonica subspecies remain to be explored.De novo evolved genes are novel genes that arise from previously noncoding DNA.Despite several reports showed that de novo genes are important for the genomic innovation of eukaryotes,the comprehensive investigations on the origin,evolution and biological functions of rice de novo genes are still lacking,and the roles of these genes in the divergence between indica and japonica are poorly understood.In this study,we revealed the origin and evolution of a rice de novo gene OsJDG9 based on the comparative genome,transcriptome and proteome analyses,and its roles in the divergence of grain shape between indica and japonica subspecies based on rice molecular biology and population evolutionary genetics.The following includes the main findings:1.Sequence similarity analysis showed that a japonica gene located on chromosome 9 of rice shared high sequence similarities with indica,O.rufipogon,O.barthii,O.glumaepatula,and O.longistaminata.Further comparative analysis of various Oryza species revealed that the full coding sequence of this gene was only present in the vast majority ofjaponica cultivars and several O.rufipogon accessions,while the other Oryza species do not contain the feature due to a single nucleotide A to G substitution at the start codon site of OsJDG9.These findings suggested that OsJDG9 evolved from the previously noncoding DNA sequences through de novo origination,and was inherited by most japonica cultivars.Thus,this gene was named as OsJDG9（japonica-dominate gene on Chromosome 9）.2.Transcriptomic data and qRT-PCR analyses of OsJDG9 gene in various Oryza populations showed the transcripts of this gene only in japonica and O.rufipogon.but no transcripts in indica and the other Oryza species,suggesting that this gene has the transcription ability.The specific peptide of OsJDG9 protein was identified from proteomic database,suggesting that this gene was expressed at the protein level.Protein sequence and structure analyses showed an alpha helix at the C-terminus of OsJDG9 protein with large intrinsic disordered regions.Further analyses revealed that OsJDG9 was expressed in vitro.Subcellular localization analyses suggested that OsJDG9 was localized to cell membrane.3.qRT-PCR and GUS analyses of OsJDG9 gene showed that this gene was expressed in various rice tissues,and the expression of OsJDG9 is relatively stronger in young panicles of 2-9 cm in length,and the transcript levels of this gene decreased as the panicle matures,suggestive of its roles in young panicle development of rice.4.We further constructed the knockout and overexpression lines of OsJDG9 gene in two japonica backgrounds（Zhonghua 11 and Nipponbare）.Phenotypic analyses showed that OsJDG9 knockout mutants exhibited slender grains than WT plants,while overexpression of this gene increased the grain length and width,suggestive of its roles in regulating rice grain shape.5.Cross-section and scanning electron microscopy analysis of spikelet hulls before heading stage showed that OsJDG9 gene is an inhibition of cell size,but a positive regulator of cell number.Further RNA-seq analyses revealed that OsJDG9 controls grain shape by coordinately modulating cell number and cell size of the spikelet hull through regulating the transcript levels of cell expansion and cell cycle-related genes.6.We further analyzed the genomic sequences of OsJDG9 gene in 2315 cultivated accessions from the 3000 rice genomes project,and the results showed that the vast majority of japonica cultivars（99.01%）contained the full coding sequence,while most of indica cultivars（97.58%）do not contain the feature of coding sequence due to a single nucleotide A to G substitution at the start codon site of OsJDG9,suggestive of the divergence of this gene between indica and japonica subspecies.Thus,we defined the rice cultivars with or without the start codon as OsJDG9Jap and OsJDG9Ind types,respectively.7.The geographic distribution of 2315 cultivated accessions showed that the OsJDG9Ind type is mainly present in low latitude,while the proportion of OsJDG9Jap type has a trend of increasing along with the increased latitude,suggesting a regional differentiation in OsJDG9.In addition,we analyzed the parameter of genetic divergence for OsJDG9 and its flanking regions between indica and japonica subspecies,and the result showed that the estimated values of OsJDG9 locus are relatively higher than its flanking genomic regions,suggestive of the genetic differentiation in OsJDG9 between the two subspecies.8.The nucleotide diversity value（π）for OsJDG9 gene and its flanking regions were estimated in 2315 cultivated rice and 28 wild populations（O.rufipogon）,and the results showed that the π value for OsJDG9 gene is much lower than its flanking regions in both cultivated rice and O.rufipogon,suggesting that low nucleotide diversity in the locus of OsJDG9 might be the result of natural selection.Further analyses showed that none of the Tajima’s D values are statistically significant in all the three populations,suggesting that the locus of OsJDG9 did not escape from neutral evolution during rice domestication.9.The phylogenetic analysis of OsJDG9 from 2315 cultivated rice and 28 O.rufipogon suggested that the OsJDG9Ind type indica clade fell into the branches of O.rufipogon Ⅰ（Or-Ⅰ）,while the OsJDG9Jap type japonica clade fell into the branches of O.rufipogon Ⅲ（Or-Ⅲ）.Further haplotype network showed that the indica with OsJDG9Ind type and the japonica with type OsJDG9Jap have the closest relationship with those in Or-Ⅰ and Or-Ⅲ,respectively.These findings revealed that the two types OsJDG9Ind and OsJDG9Jap are independently originated from Or-Ⅰ and Or-Ⅲ of O.rufipogon.10.We re-sequenced the genomic sequences of OsJDG9 gene in 296 rice cultivars,and the results showed that 144 out of 145 japonica cultivars possess this gene,while 138 out of 151 indica cultivars do not contain this gene due to the loss of start codon,suggestive of the divergence of this gene between japonica and indica.Further phenotypic analyses of 151 indica cultivars showed that compared with the OsJDG9Ind type,the grain length was significantly reduced,but the grain width was significantly increased in the OsJDG9Jap type,suggesting that OsJDG9 also regulates grain shape in the indica background,and has contributed to the divergence of grain shape between the OsJDG9Ind and OsJDG9Jap type.Our investigations not only revealed the de novo origination of OsJDG9 from the previously noncoding regions,but uncovered the important roles of this de novo evolved gene in regulating the divergence of grain shape between indica and japonica,and provided a new gene target for the genetic improvement of grain shape in rice.Part Ⅲ The origin and evolution of OsDNE8 gene,and its roles in regulating rice yield traits.Rice is one of the most important crops,and the staple food for more than half of the populations in the world.As the major goal of breeding,rice yield is mainly determined by productive panicle number per plant,grain numbers per panicle and grain weight.Therefore,mining yield-related genes and illustrating their potential regulation mechanism are important for realizing high-yield rice breeding.De novo evolved new genes from previously noncoding sequences play key roles in driving eukaryotic evolutionary innovation.Although recentinvestigations revealed that de novo genes play important roles in the development and environmental adaptation in eukaryotes,the biological functions of these genes in rice are poorly understood.In this analysis,we revealed the origin and evolutionary pattern of a rice de novo gene OsDNE8 based on bioinformatics and population evolutionary genetics,and its roles in regulating rice development and yield traits through rice molecular biology.The following includes the main results:1.Sequence similarity analysis showed that a japonica gene located on chromosome 8 of rice shared high sequence similarities with indica,O.rufipogon,O.glumaepatula,and O.meridionalis.Further comparative analysis of these Oryza species revealed that the full coding sequence of this gene was only present in japonica and O.rufipogon,while the indica cultivars（Minghui 63 and Shuhui498）and the other Oryza species do not contain the coding feature of OsDNE8 due to the premature stop codon.These findings suggested that OsDNE8 evolved from the previously noncoding DNA sequences through de novo origination by removing the stop codon to obtain the coding feature.Thus,this gene was named as OsDNE8（de novo evolved gene on Chromosome 8）.2.Transcriptomic data and qRT-PCR analyses of OsDNE8 gene in various rice populations showed the transcripts of this gene only in japonica,but no transcripts in indica and the other Oryza species,suggesting that this gene has the transcription ability.Protein sequence and structure analyses showed that OsDNE8 is likely a stably folded protein with an alpha helix at its core and disordered termini.Further analyses showed that OsDNE8 was expressed in vitro.Subcellular localization analyses showed that OsDNE8 was localized to cell membrane,cell cytoplasm and cell nucleus.These findings suggested that OsDNE8 might be a protein-coding functional gene.3.We further analyzed the genomic sequences of OsDNE8 gene in 2310 cultivated accessions from the 3000 rice genomes project,and the results showed that 99.72%japonica cultivars and 54.12%indica cultivars contained the full coding sequence,while 45.82%indica cultivars do not contain the feature of coding sequence due to the premature stop codon of OsDNE8.Thus,we defined the rice cultivars with the full coding feature as OsDNE8,and those with the premature stop codon as osdne8,respectively.The geographic distribution of OsDNE8 and osdne8 types from 2310 cultivated accessions showed that the OsDNE8 and osden8 types are mainly present in high and low latitudes,respectively,suggestive of a regional differentiation in OsDNE8.4.The genetic diversity of OsDNE8 gene and its flanking regions were estimated in 2310 cultivated rice and 28 O.rufipogon populations,and the results showed that the nucleotide diversity（π）and population mutation rate（θW）values for OsDNE8 gene is relatively higher than its flanking regions in both cultivated rice and O.rufipogon,suggesting that no obvious selection occurred in the locus of OsDNE8 during domestication.Further analyses showed that none of the Tajima’s D values are statistically significant in all the three populations,suggesting that the locus of OsDNE8 did not escape from neutral evolution during rice domestication.5.The phylogenetic analysis of OsDNE8 from 2310 cultivated rice or 354 re-sequenced populations and 28 O.rufipogon revealed that OsDNE8 originated from O.rufipogon,and was inherited by most japonica varieties,while this gene in indica might be the result of introgression.Further haplotype network showed that the japonica with OsDNE8 type have the closest relationship with those in O.rufipogon,suggesting that OsDNE8 originated from O.rufipogon,and then was inherited by japonica varieties.6.qRT-PCR and GUS analyses of OsDNE8 gene showed that this gene was expressed in various tissues of rice at the different growth stages,and the expression of OsDNE8 is relatively higher in stem and young panicle at the heading stage,suggestive of its roles in stem and young panicle development of rice.7.We further constructed the overexpression and knockout lines of OsDNE8 gene in the japonica Zhonghua 11 background.Phenotypic analyses showed that compared with the wildtype（WT）,the plant height and internode length was significantly increased in OsDNE8 knockout mutants,but decreased in the overexpression lines.Cross-section of the third internode of stem showed that the cell length was significantly increased in OsDNE8 knockout mutants than that in WT,but decreased in the overexpression lines.These findings showed that OsDNE8 was an inhibition of plant height by negatively regulating cell length to control internode length.8.Compared with WT,the plant lodging index was significantly decreased in OsDNE8 overexpressing plants,but increased in the knockout mutants,suggesting that this gene was a positive regulator of plant lodging resistance in rice.Further analyses suggested that OsDNE8 regulates plant lodging resistance through influencing plant height and internode length.9.Phenotypic observations on panicle-related traits of OsDNE8 transgenic plants showed that compared with WT.OsDNE8 knockout mutants exhibited increased panicle length,secondary branches per panicle,and grain numbers per panicle,whereas OsDNE8 overexpressing plants showed the opposite traits,suggesting that OsDNE8 has large effects on panicle architecture and negatively regulates grain numbers.10.Phenotypic observations on grain shape of OsDNE8 transgenic plants showed that compared with WT,the grain length was significantly increased in OsDNE8 knockout mutants,but decreased in OsDNE8 overexpression lines.By contrast,the grain width was decreased in OsDNE8 knockout mutants,but increased in OsDNE8 overexpression lines.These findings showed that OsDNE8 gene regulates rice grain shape.11.Compared with WT,the productive panicle numbers per plant was significantly increased in OsDNE8 knockout mutants,but decreased in OsDNE8 overexpressing plants,suggesting that this gene was an inhibitor of productive panicle numbers.By contrast,overexpression or knockdown of OsDNE8 has no effect on 1,000-grain weight of rice.Further analyses showed that the grain yield per plant was increased by 20.49%and 20.83%in two OsDNE8 knockout mutants,but decreased by 20.42%and 19.35%in two OsDNE8 overexpression lines,respectively.These findings suggested that OsDNE8 was an inhibitor of grain yield by negatively regulating productive panicle numbers and grain numbers.Our investigations not only revealed the de novo origination and population evolutionary patterns of rice OsDNE8 gene,but uncovered the important roles of this gene in rice development,and provided a novel gene target for rice high-yield breeding and the genetic improvement of multiple traits. |
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