Genetic Difference Of A Dwarf Mutant And Characterization Of The DXS1 Gene In Poa Pratensis L.

Kentucky bluegrass(Poa pratensis L.),as one of the turfgrasses widely used in temperate regions,has the characteristics of good cold resistance,drought tolerance,long green time,high turf quality and etc,but needs more frequently mowing to maintain a higher turf quality.Space environment can induce non-directional variation in plants and seeds,which may lead to good traits in line with breeding direction.In the progeny of Kentucky bluegrass seeds returned to the ground after space flight in this study,we found some mutant with reduced plant height and darkened leaves.Based on M4 generation of dwarf mutant(A 16)and wild type(WT)as the research material,the study of comparative analysis in cell morphology,genetic variation,plant phenotype,physiological phenotypes,and molecular level,and functional research on 1-deoxy-D-xylulose-5-phosphate synthase 1(DXS1)gene which regulate plant growth and development and its promoter,are carried out on A16 and WT.These data will benefit to understand the variation of space mutation in dwarf mutants and the effect of space mutation on related genes,and then has guiding significance for breeding dwarf varieties of Kentucky bluegrass and also contributes to better use of space mutation for plant breeding.The results are as follows:(1)The difference between A 16 and WT at the cellular morphysiological and genetic level:Space mutagenesis has significant effect on the morphology and anatomical structure of Kentucky bluegrass leaves.Epidermal cells on WT leaves were long and thin,and tended to extend laterally.However,the upper epidermal cells of A]6 were obviously spindle-shaped,much shorter and wider,and tended to extend radially.And the distribution of stomata in the leaves of A16 was denser and smaller.In addition,transcriptome sequencing was performed on both of them:InDel polymorphism analysis showed that there were a large number of random insertion/deletion mutations between the genome/transcriptome of A16 and WT,indicating that the space environment had a mutation effect on the Kentucky bluegrass.There were 4,203 differentially expressed genes between A16 and WT.(2)The difference of phenotype and physiology between A16 and WT was showed as follow:in terms of plant height,the decreased plant height in A16 may be related to the down-regulation of DXS1 gene,GA3ox4 gene in gibberellin synthesis pathway,FMO gene and PINS efflux protein in auxin biosynthetic pathway,and even the content of gibberellin and auxin in A16 are both lower than those in WT.In terms of leaf color,the chlorophyll content of dwarf mutant is significantly higher than WT,and the ratio of chlorophyll a/b is lower than WT.The difference in leaf color may be caused by up-regulated expression of UroS(chlorophyll synthesis gene)and down-regulation expression of CLH1(chlorophyll degradation gene).In terms of drought and salt tolerance,based on the endogenous ABA content,physiological index and differential expression of stress resistant genes,the drought resistance of A16 was stronger than that of WT,and there was no significant difference in salt tolerance.In terms of disease resistance,the expression level of PR1L and NPR1L in A16 was at a low level before pathogen induction,but the transcription levels were significantly improved after pathogen infection,and was significantly greater than that of WT,indicating that WT and A16 had differences in induced disease resistance.(3)The functional analysis of DXS1 gene in Kentucky bluegrass shows that the DXS1 gene of Poa pratensis(PpDXS1)contains an ORF frame of 2139 bp,which is similar to DXS1 protein of Aegilops tauschii and Brachypodium distachyon,and belongs to the first clade of DXS gene family.The PpDXSl gene is expressed in leaves,leaf sheaths and roots in Kentucky bluegrass,and the expression level in leaves is highest.The treatment of GA3,ABA,JA and pathogen infection can improve the expression of PpDXS1 gene.In addition,antisense expression of PpDXSl in Kentucky bluegrass resulted in significant reduction of plant height,GAs and IAA contents in transgenic lines,while ABA and chlorophyll contents were higher in one of the transgenic lines(antiDXS 1-102).Subsequently,transcriptome analysis of antiDXS1-102 transgenic lines and control plants(CK)showed that these genes involved in IPP/GGPP synthesis,GA and IAA biosynthesis and signal transmission,and chlorophyll degradation are down-regulated in antiDXS1-102 plants,while the expression of genes related to chlorophyll and ABA biosynthesis is up-regulated,which is consistent with the result hormone content.(4)Comparative analysis of the DXS1 gene promoter sequences from A16 and WT revealed that there was a 715bp insertion fragment and a 500bp deletion fragment in the promoter region of the PpDXSl gene from A16,suggesting that space environment could induce insertion/deletion mutations in the promoter region of the DXS1 gene.The analysis of promoter activity in dual luciferase reporting system and transgenic Kentucky bluegrass showed that the relative activity of PpDXS1 promoter sequence from A16 was lower than WT,and the decrease of activity was caused by the deletion fragment in A16.Moreover,the G-box binding protein(GBF1)which binds to the g-box element in the deletion fragment,has transcriptional activation on promoter activity,and PpDXS1 gene is significantly improved in overexpressed GBF1 transgenic Kentucky bluegrass.Therefore,we speculate that the deletion fragment contained G-box element of the PpDXSl promoter region from A16 and the transcriptionally activated function of the GBF1 protein may be the reason for the decrease in the activity of the DXS1 gene promoter of A16 and the decrease in the expression level of DXSI gene regulated by the promoter.In conclusion,space mutagenesis had a great influence on Kentucky bluegrass in terms of cell morphology,plant phenotype,physiological performance and molecular expression,including plant height,leaf color,physiological resistance and gene expression.In addition,the space environment influences the gene expression of PpDXS1 gene promoter by inducing insertion/deletion mutations,which regulating the growth and development of plants.