| Stylo(Stylosanthes spp.)used as an important tropical legume and green manure crop,has highly adaptable to low phosphorus(P)strees in acid soils,and can effectively utilize soil organic P.At present,whole-genome map of stylo has not been released,which limits the further study on molecular mechanism of low P adaptation in stylo.Therefore,a detailed genome map of stylo was constructed for the first time,and by combining transcriptome,proteome and metabolome methods,the genes involved in efficient utilization P in stylo were excavated and their biological functions were analyzed.The main results were shown as follows:(1)A 1.173 Gb size genome of Stylosanthes guianensis(Reyan No.5)was produced by using three generation(Nanopore)and next generation(MGI)sequencing technology,combined with optical map(Bio Nano)and chromosome conformation capture(Hi-C mapping)technology.The length of contig N50 and scaffold N50 reached to 2.36 Mb and13.61 Mb,respectively.98.71%of genome sequence(1.158 Gb)was anchored to 10chromosomes of stylo.Totally,34,985 protein-encoding genes was predicted based on the de novo,homology and RNA dependent methods,the average length of genes was 2,650.17bp.(2)Comparative genomic studies revealed that Reyan No.5 stylo experienced a papilionoid whole-genome duplication(PWGD)event(～58 million years ago)shared by Leguminosae Papilionoideae plants,and had no recently species-specific whole-genome duplication(WGD)events.Genomic evolutionary analyses estimated that stylo had the closest relationship with the two subgenomes of Arachis hypogaea(A.duranensis and A.ipaensis),and their the most recent ancestor diverged 18.9(15.0～22.7)million years ago.(3)The utilization capacity of exogenous organic P adenosine diphosphate(ADP)was evaluated in 20 Stylosanthes guianensis.Results showed that 20 genotype stylo had the ability to utilize exogenous organic P(ADP),but the capacities had significant genotypic variations.Subsequent analysis revealed that the root secreted-APase(acid phosphatase)activity of stylo also has significant genotypic variations,and there was a significant positive correlation between the ability of utilization exogenous ADP and the activity of root secreted-APase in stylo.(4)To identify the APase involved in stylo organic P utilization,the gene family members of purple acid phosphatase(PAP)were identified at whole-genome level in this study.Results showed that stylo genome contained 25 menbers of SgPAPs gene.Subsequently,transcriptome and proteomic analysis showed that 13 and 8 SgPAPs members at transcriptome and protein levels were significantly up-regulated by low phosphorus stress in roots of P efficient stylo T277.In addition,metabolome analysis showed that the relative concentrations of 9 phosphate-containing nucleotides and 7 phospho-lipid in stylo roots were significantly decreased under low phosphorus stress,with the up-regulation of numerous phosphatases.(5)In order to clarify the potential relationship between the relative expression level of SgPAPs gene and the capacity of utilization exogenous organic P in stylo,the 8 SgPAPs up-regulated by low P stress at transcription and protein levels,was detected the relative expression under adding exogenous ADP treatment(+ADP)by q RT-PCR.The results indicated that the relative expression level fo 8 SgPAPs in 20 genotype stylo roots had significant genotypic variations.Among them,the relative expression levels of SgPAP7 and SgPAP10 in different genotype stylo roots were significantly positively correlated with the utilization ability of exogenous ADP,and SgPAP7 had the higer correlation,the correlation coefficient(R~2)was 0.3558(P<0.01).(6)The recombinant protein of SgPAP7 was expressed and purified by E.coli.The analysis of biochemical enzymatic characteristics in vitro suggested that SgPAP7 had a broad spectrum of hydrolytic activity to a series of phosphorylated substrates,and the enzyme activity to ADP was the highest.The optimum p H value of SgPAP7 was 6.0 and the optimum temperature was 40°C.Subsequently,the potential biological functions of SgPAP7in plants were analyzed by transgenic Arabidopsis thaliana.Results indicated that overexpression of SgPAP7 could significantly improve the acitives of endogenous and root secreted APase in transgenic Arabidopsis thaliana,and the utilization capacity of exogenous ADP in overexpressed strains was significantly enhanced.In addition,under low phosphorus stress in soil,compared with wild-type Arabidopsis thaliana(WT),the biomass of transgenic Arabidopsis thaliana with overexpression of SgPAP7 was significantly higher,and the soluble P concentration of overexpression strains in young leaves was also significantly evaluated,but old leaves soluble P concentration was significantly decreased.The above results indicated that SgPAP7 had the dual functions of root secreted-APase and intracellular APase,which was not only involved in the utilization of exogenous ADP,but also reactivation utilization of intracellular P.Taken together,the study firstly constructed a genome map of stylo,and analyzed the responses of stylo SgPAPs family gene to low P stress by combining transcriptome,proteomics and metabolomics techniques.Through biochemical enzyme properties analysis and transgenic function verification,the biological function of SgPAP7 involved in P efficiency utilization was elucidated.The study not only lay a foundation for further revealing the mechanism of low P adaptation in stylo,but also provide candidate genes for developing stylo cultivars with high P efficiency. |
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