| Tiller is a special branching structure that is not dependent on the main stem during the growth and development of grass species used as turf or forage.It directly affects plant density and productivity.Each tiller can form its own leaves and roots,thereby facilatating photosynthesis and efficient use of nutrients and water in the soil,as well as plant tolerance to environmental stresses.Tiller development involves two stages:axillary bud initiation and subsequent outgrowth.Whether tillering buds can outgrowth into tillers after formation is mainly affected by external environment,endogenous hormones,heredity.The initiation and outgrowth of tillers are very sensitive to drought stress.Drought stress significantly inhibits the development of tillering,which seriously affects plant density.At the same time,some key genes related to tiller development also play a vital role in the genetic regulation of tillering.Therefore,it is of great significance to study how drought inhibits tiller development process and the genetic regulation mechanism of key genes of tillering development in grass spcies for developing high-density grass germplasm.In this study,we first simulated the drought stress by adding polyethylene glycol 6000(PEG6000)to the hydroponic environment,and studied the effect of drought on the initiation or outgrowth of tall fescue,which is a widely used turf and forage grass species,and whether drought inhibition of tillering is mediated through the strigolactone signaling pathway.Secondly,from the above experimental results,we can obtain two key factors for tillering development,FaMAX2(more axillary growth 2)and FaTBl(teosinte branchedl),which are induced by drought.The function and potential mechanism of FaMAX2 affecting tillering development were studied through heterogenous expression of FaMAX2 in rice.Finally,the function and molecular regulation mechanisms of FaTB1 were studied by overexpression of FaTBl in tall fescue and yeast one-hybrid and yeast two-hybrid system.The main research content and results as follows:1.The objective of this study was to investigate whether drought-inhibition of tiller growth is mainly due to the suppression of axillary bud initiation or subsequent outgrowth in perennial grass species and to determine whether drought-inhibition of tiller development and growth in grass species are associated with strigolactone(strigolactone,SL)accumulation and signaling.Seedlings without axillary buds and two axillary buds were grown in 20%polyethylene glycol 6000(PEG6000)solution to induce drought stress.Plant height,number of leaves,axillary buds,tillers and length of axillary buds were monitored during of stress.Both axillary bud initiation and outgrowth were inhibited by drought stress,with outgrowth being more sensitive to drought stress.qRT-PCR analysis showed that expression level of genes involved in axillary bud activity was down-regulated at 14 d of drought stress while genes involved in axillary bud dormancy was up-regulated(FaTB1 up-regulated expression).Strigolactone(SL)content was elevated under drought stress in crowns.qRT-PCR analysis showed that expression level of genes involved in SL biosynthesis and signaling transduction(FaMAX2)were up-regulated during drought stress.Combined with the previous transcriptome data,the expressions of FaMAX2 and FaTBl were significantly up-regulated in crown under drought stress,suggesting that FaMAX2 and FaTB1 plays a key role in drought inhibition of axillary bud outgrowth.2.In order to study the function of FaMAX2 in the regulation of tillering development of tall fescue,the FaMAX2 gene sequence was cloned from tall fescue,and the open reading frame(ORF)frame was 2160 nucleotides,encoding 720 amino acids.Cluster analysis showed that FaMAX2 and LpMAX2 had the closest evolutionary relationship,followed by OsD3 and BdMAX2.Amino acid sequence structure analysis revealed that the amino acid sequence encoded by this gene has a specific F-box domain(F-box)and a leucine-rich repeat(leucine-rich repeat,LRR).The Arabidopsis protoplasts were transiently transformed with a green fluorescent protein fusion vector for subcellular localization,and FaMAX2 was found to be localized in the nucleus.The function of FaMAX2 in rice was verified by heterologous overexpression,and it was found that,compared with the wild type,the development of tillers in transgenic rice was inhibited,which was caused by inhibiting the outgrowth of rice axillary buds.The bud activation and dormancy,the synthesis and transduction of strigolactone and the expression pattern of the downstream gene of MAX2 were analyzed in transgenic rice.It was found that the bud activation genes OsCDKB2-1 and OsCycD2 in transgenic rice showed down-regulated expression in some lines.The bud dormant genes OsDRM1 and OsARP showed up-regulated expression,indicating that the axillary buds initiation in the overexpressing lines may remain dormant and unable to outgrowth and cause a reduced tillers phenotype,while the Strigolactones downstream genes OsD53,OsIPAl and OsTBl is up-regulated,indicated that the inhibition of tiller development by transgenic lines was also related to the key factors of outgrowth.3.FaTB1 encodes a member of the TCP transcription factor family and is the homologous gene of teosinte branched1(tb1)in corn.This gene is the key integration factor that controls the development of lateral branches in axillary buds.Phylogenetic analysis showed that FaTB1 was most closely related to LpTB1,followed by BdTB1,OsTBl and ZmTB1.Amino acid sequence alignment found that FaTB1 contained SP domain specific to monocotyledonous plants,as well as typical TCP domain and R domain specific to TB1.Subcellular localization analysis showed that FaTBl was located in the nucleus.Expression pattern analysis showed that the gene had the highest expression in axillary bud and crown.FaTB1 was overexpressed in tall fescue,and it was found that the tillers of transgenic lines was significantly reduced compared with the wild type.Through the analysis of the dynamic changes of axillary buds and tillers of transgenic lines,it was found that FaTBl inhibited the development of tiller by affecting the outgrowth of axillary buds rather than the initiation of axillary buds.It was found that the content of ABA and the ABA synthesis and signal transduction genes were up-regulation in the crown of the transgenic lines.At the same time,the bud dormancy gene was up-regulated and the bud-activating gene was down-regulated in the transgenic lines,indicated that the transgenic lines inhibited the outgrowth of axillary buds and may be related to the axillary bud dormancy caused by the increase of ABA content.FaHOX12,a direct downstream gene of FaTB1,was identified in tall fescue,and the gene was up-regulated in transgenic lines.The FaB1 interacting proteins FaGID1L2 and FaUPL1 were screened and validated by yeast two-hybrid and two-molecule fluorescence complementation experiments,so as to provide a basis for further study of the potential mechanism of FaTB1 inhibition of tiller development. |
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