Functional Study Of A Novel Gibberellin 2-oxidases Gene,PpGA2ox,from Kentucky Bluegrass(Poa Pratensis L.)

Kentucky bluegrass（Poa pratensis L.）,a typical cool season grass,is widely used in temperate regions because of its good quality.However,the fast-growing speed and frequent mowing requirement leading to the high maintenance cost prevents its further application.Therefore,breeding semi-dwarf or dwarf P.paratensis is becoming an increasingly important goal for breeding.GA2ox is a key enzyme involved in gibberellin metabolism process,and it is closely associated with of plant architecture regulation.Although several GA2ox genes have been identified and functionally studied in plants,the gene in P.pratensis has not been reported and its function remained largely unknown.The objectives of this study were to isolate a gibberellin 2-oxidase gene from P.pratensis,to determine its promoter activity,and to systematically reveal its function using bioinformatics analysis,transgenic approches,RNA-sequencing and yeast two-hybrid screening.The main findings of this study are:1. A gibberellin 2-oxidases gene,PpGA2ox（NCBI accession No.KX254272）,was cloned from Kentucky bluegrass cv.‘Baron’using RACE method.Its open reading frame was 1047 bp in length,corresponding to 348 amino acids.Conserved domain prediction showed PpGA2ox belongs to the GA2ox superfamily.Phylogenetic analysis exhibited that the PpGA2ox protein was most closely related to Ta GA2ox.The expression character analysis revealed that the transcripts of PpGA2ox were found in all investigated tissues including leaf,stem,and root,but the highest transcription was observed in the young leaves,indicating that PpGA2ox function in the tissues with higher bioactive gibberellin contents.Its expression could be regulated by exogenous GA₃,Me JA,IAA or drought stress.Subcellular localization result demonstrated that PpGA2ox was localized in both the nucleus and cytoplasm.2. The up-stream 1109 bp sequence in front of the ATG start codon was obtained using genome walking method.Plant CARE online prediction proved that the fragment not only contained promoter basic elements including TATA-box,CAAT-box,but also contained ten cis-elements related to Me JA,four cis-elements related to drought stress and one cis-element related to meristem expression.The promoter sequence was infused in front of the GUS reporter gene and transformed in Arabidopsis.Histochemical staining results of the transgenic lines showed that stronger GUS activity was identified in the both leaf apex and shoot apical region of seedlings and in the raised position of leaf margin of mature plants.Moreover,the expression of GUS could be induced by exogenous GA₃ but suppressed by PAC.3. Transgenic Arabidopsis plants overexpressing PpGA2ox displayed delayed seed germination and decreased germination rate.PpGA2ox-overexpressing lines showed dwarf phenotype,and the cells were smaller than the control.Hormone contents analysis revealed that the content of endogenous active GAs,ZR,and IAA in transgenic lines were lower than the control.The expression level of GA metabolism-related genes in transgenic Arabidopsis was increased.The dwarf phenotype of the PpGA2ox-overexpressing lines could be rescued by exogenous GA₃.The results proved that the overexpression of PpGA2ox was responsible for the decrease of gibberellin content and the dwarf phenotype.Interestingly,PpGA2ox-overexpressing lines exhibited later flowering than the control.Further investigation revealed the expression of the genes promoting flowering were down-regulated but the expression of the genes delaying flowering were up-regulated.The leaf color of the transgenic lines was greener than that of WT and the chlorophyll content in the transgenic lines was higher than the control.The mature transgenic plants remained obvious dwarfing phenotype,but with smaller siliques and more rosette leaves.The YPH500 yeast cells transformed with PpGA2ox showed enhanced resistance to drought stress but showed no difference under salt stress,suggesting the potential role of PpGA2ox in drought stress.Transgenic Arabidopsis lines were further utilized to verify the function of PpGA2ox in drought stress.The results of morphological observation,physiological measurement and drought related genes determination proved the function of PpGA2ox in drought stress.We hypothesized that PpGA2ox contributed to the improved water retention,cell membrane integrity,and osmotic adjustment which in turn enhanced drought stress resistance of transgenic plants.4. RNA-sequencing analysis of PpGA2ox-overexpressing Arabidopsis lines was adopted to uncover the underlying transcriptional mechanism.GO and KEGG enrichment analysis showed PpGA2ox participated in gibberellin signaling,photosynthesis,plant developmental processes and environmental stress.This was consistent with the results of the dwarf phenotype,hormone contents decrement,chlorophyll content accumulation and improved drought stress resistance in PpGA2ox-overexpressing Arabidopsis lines.These results verified the function of PpGA2ox in regulating plant architecture and the resistance to environmental stress.5. Using agrobacterium tumefaciens mediated transformation of callus,stable transformed rice（Oryza sativa）plants overexpressing PpGA2ox were generated.GUS staining results showed the target plastmids was successfully transformed in rice.The overexpression of PpGA2ox in rice caused dwarf phenotype,decreased root length,and shortened flag and the second leaves length than control.Whereas,leaf blades width was not influenced.The expression level of GA metabolism-related genes in transgenic rice was decreased.The leaf color of PpGA2ox-overexpressing lines was greener than that of WT and the chlorophyll content in the transgenic lines was improved.The expression level of the genes related to chlorophyll synthesis was increased,while the expression level of the genes related to chlorophyll degradation was reduced.The photosynthetic efficiency of transgenic plants was raised,and the expression level of the genes that play essential roles in the photosynthetic system was upregulated.Moreover,PpGA2ox-overexpressing lines showed shorter inflorescence than the control,and the length of first internode were shorter than control.6. A high quality normalized full length c DNA library of Kentucky bluegrass was constructed using SMRT c DNA synthesis method to further explore the regulating mechanism of PpGA2ox.Its titer was 2.5×10⁶ cfu·ml^-1,and the average length of insert segments was longer than 1000bp,and the recombination rate was 100%.The transcriptional auto-activation assay demonstrated that PpGA2ox could not activate itself.Two candidate proteins were obtained by yeast two-hybrid screening.We assumed that PpGA2ox could interact with these proteins to participant in gibberellin metabolism,osmotic adjustment and developmental processes.Further investigation is still needed to verify the assumption.This study proved that PpGA2ox could play important roles in regulating plant architecture and improving plant resistance to drought stress,providing theoretical basis for breeding dwarf and stress tolerant Kentucky bluegrass cultivars.