Cloning And Functional Analysis Of LpEIL1 In Perennial Ryegrass

Perennial ryegrass is a plant of the gramineae,which is widely cultivated and used in China.Perennial ryegrass likes warm and humid environments,and is not resistant to stress.It is susceptible to high temperature,cold,drought and other biological and abiotic stresses,leading to premature leaf senescence,which seriously reduces the quality of turf and pasture.Researching the molecular mechanism of ryegrass leaf senescence has important theoretical and practical significance for preventing and improving the quality and quality of ryegrass products.Ethylene is one of the main plant hormones that promote leaf senescence.Its signal transduction pathway and its molecular regulation mechanism are one of the research hotspots in the field of plant molecular biology.According to previous research in our laboratory,an EIN3-like gene EIN3-LIKE(LpEILl)was cloned from perennial ryegrass,which was involved in the ethylene signal transduction process,but the specific molecular regulatory mechanism was still unclear.In this study,by analyzing the leaf senescence phenotype of LpEIL1 transgenic Arabidopsis thaliana and ryegrass,the expression level of chlorophyll-degrading genes and their promoter activation activity,the molecular mechanism of LpEIL1 regulation of leaf signaling by the ethylene signal pathway transcription factor was further clarified.Main results of this study are shown as follows:(1)In the perennial ryegrass genomic cDNA library,the LpEIL1 open reading frame(ORF)is 1827bp in length and encodes 608 amino acids.Phylogenetic tree analysis and multiple sequence alignment results showed that LpEIL1 has a typical domain of EIL-like transcription factors and has high homology with EIN3 protein of corn and rice.(2)LpEIL1 is mainly expressed in leaves,but low in leaf sheaths,roots and stems.LpEIL1 is induced by ethylene,and AVG(an ethylene synthesis inhibitor)inhibits the expression of LpEIL1,indicating that LpEIL1 is a regulator of the ethylene signal pathway.(3)Through the ryegrass protoplast transformation system,the functional localization of LpEILl-GFP in the nucleus conformed to the nuclear localization characteristics of transcription factors.(4)LpEIL1 was overexpressed in WT and ein3 homozygous mutant background plants by Agrobacterium-mediated transformation.Compared with wild-type nuclear mutants,LpEIL1 overexpression transgenic lines under dark treatment conditions significantly accelerated leaf senescence and AtEIN3 mutant background transgenic lines can better compensate mutant aging phenotype.(5)LpEILl overexpression and RNA interference ryegrass transgenic lines were obtained respectively.Compared with the wild type,the chlorophyll content and Fv/Fm of leaves of LpEIL1 overexpressing transgenic lines were significantly reduced,and senescence was significantly accelerated.In contrast,RNA interference transgenic lines remained high,and the leaf senescence was significantly delayed.(6)Transient transformation of tobacco and yeast one-hybrid system proved that LpEIL1 can bind and activate the promoter of LpNAL directly,and indirectly provesd that LpEIL1 can activate the expression of chlorophyll-degrading genes such as LpSGR,LpNYC1,and LpPPH.The experimental results clarified the function of LpEILl and initially analyzed the molecular mechanism of leaf senescence.It has laid a theoretical foundation for the molecular breeding to delay the senescence of ryegrass.