Cloning And Functional Analysis Of LLG1 From Puccinellia Tenuiflora

LORELEI-like GPI-anchored protein?LLG?is a type of protein located on the outer surface of the cell membrane.It acts as a molecular chaperone of the CrRLKIL family of receptor kinases and participates in regulating various biological processes such as growth and stress response.However,the molecular mechanism involved in the salt-alkali response has not been reported.We used the genetics method to clone the PutLLG1 gene using the gramineous halophyte Puccinellia tenuiflora as material.Bioinformatics analysis showed that its open reading frame is 501 bp and encodes 166 amino acids.PutLLG1 has the structural characteristics of the GPI anchor protein family.The amino acid sequence of PutLLGl includes the N-terminal signal peptide,the central region,and the C-terminal GPI anchor.The C-terminal has a conformational variable region.Real-time fluorescence quantitative analysis showed that PutLLG1 gene was expressed in the roots,stems,leaves,flowers,ears,sheaths,and seeds of Puccinellia tenuiflora,with the highest expression in roots and the lowest expression in ears.When subjected to stress?50 mmol/L Na2CO3,100 mmol/L NaHCO3,200 mmol/L NaCl,5 mmol/L H2O2,160 ?mol/L CdCl2 and 4??,PutLLG1 gene is induced in roots and leaves.This implies that PutLLG1 gene may be involved in the response process of salt alkali,low temperature,oxidation,and heavy metal stress.Through the subcellular localization analysis of the tobacco?Nicotiana tabacum?leaf cells and the onion?Allium cepa?epidermal cells of PutLLG1,we found that under normal conditions,PutLLG1 is localized on the cell membrane and a small amount is located in the nucleus.Using the yeast in vitro expression system,the yeast with empty pYES2 as a negative control,under salt?NaCl?,alkali?NaHCO3,Na2CO3?,oxidation?H2O2?and osmotic?Sorbitol?stress,PutLLG1 gene-transgenic S.cerevisiae tolerance of the strain was investigated.The results show that under normal conditions,the growth status of the PutLLG1 gene-transformed yeast and the control group are basically the same.Under stress conditions,the transgenic yeast grows better than the control yeast strain.Therefore,PutLLG1 gene may increase the resistance of yeast strains to stress.The PutLLG1 gene was constructed on the pBI121 plant expression vector by homologous recombination technology,and the recombinant plasmid was transferred into wild-type Arabidopsis using Agrobacterium infection method to obtain Arabidopsis plants overexpressing the PutLLG1 gene.Under stress of 100 mmol/L NaCl and 125 mmol/L NaCl,the root length and fresh weight of Arabidopsis plants overexpressing PutLLG1 gene were better than those of wild-type Arabidopsis plants.The Arabidopsis atllgl mutant plants?SALK₀8036.C?have lower root length and fresh weight than wild-type Arabidopsis thaliana under normal conditions or under salt stress.In summary,our results indicate that PutLLG1 gene may be involved in the process of salt response regulation,which plays an important role in plant growth and development and in the process of resisting adversity.These results provide information for the in-depth study of the molecular mechanism of Puccinellia tenuiflora LLG involved in the salt-alkali response.