Cloning And Functional Analysis Of DREPPs From Puccinellia Tenuiflora

Developmentally regulated plasma membrane polypeptide(DREPP)protein is a kind of plant-specific protein associated with plasma membrane,which it has many functions such as binding phosphatidylinositol phosphate(PtdInsPs),Ca2+/calmodulin(CaM)complex,microtubules and microfilaments.DREPP plays an important role in plant development and stress response.The molecular mechanism of DREPP in response to low temperature,drought and other stresses has been studied,but the molecular mechanism of its saline-alkali response has not been reported.In this study,Puccinellia tenuiflora was used as experimental material to analyze the PutDREPP family composition at genome-wide scale and clone the PutDREPP1 and PutDREPP2 genes.Furthermore,biochemical and molecular genetic strategies were used to analyze the expression characteristics of PutDREPP1 and PutDREPP2 genes,subcellular localization,and their ability to bind to Ca2+/CaM.By analyzing PutDREPP1 overexpressing Arabidopsis thaliana plants,the biological functions in the process of stress response were preliminarily analyzed.Using the newly obtained Puccinellia tenuiflora genome-wide database,we analyzed the PutDREPP gene family composition,including PutDREPP1 and PutDREPP2,and cloned the PutDREPP1 and PutDREPP2 genes.Bioinformatics analysis showed that the length of PutDREPP1 ORF was 609 bp,encoding 202 amino acids.The length of PutDREPP2 ORF was 651 bp,encoding 216 amino acids.Both PutDREPP1 and PutDREPP2 have DREPP family protein functional domains.PutDREPP1 and PutDREPP1 have the highest homology with OsDREPP1 and OsDREPP2 of Oryza sativa,respectively.The expression of PutDREPPl and PutDREPP2 in roots,stems,leaves,flowers,sheaths,spikes,stems and seeds of Puccinellia tenuiflora was detected by real-time PCR.PutDREPP1 had the highest expression in root and the lowest expression in spike.PutDREPP2 had the highest expression in seed and the lowest expression in leaf;In 75 mmol/L Na2CO3?100 mmol/L NaHCO3?200 mmol/L NaCl?10 mmol/L H2O2?160 ?mol/L CdCl2 and low temperature stresses,addition to the root of PutDREPPl suppressed expression in low temperature stress,the stress conditions are induced by PutDREPP1 expression of leaves and roots.The roots and leaves of PutDREPPl suppressed expression in 100 mmol/L NaHCO3?200 mmol/L NaCl?160 ?mol/L CdCl2 and low temperature stresses.This suggests that PutDREPPs may be involved in the response to salt,alkali,heavy metals,hydrogen peroxide and low temperature stresses.We by constructing pBl121-PutDREPP1-GFP and pBI121-PutDREPP2-GFP vectors,introduced them into Nicotiana tabacum leaf cells and Allium cepa epidermal cells for transient expression.Subcellular localization results indicated that PutDREPP1 and PutDREPP2 were mainly located in the plasma membrane under normal conditions.His-PutDREPP1 and His-PutDREPP2 fusion proteins were induced and purified by PutDREPPs gene prokaryotic expression system.Protein purity was verified by Western blotting,and the ability of PutDREPPs to bind Ca2+/CaM was preliminarily confirmed by Ca2+/CaM binding experiments.The overexpression PutDREPPl and PutDREPP2 genes plant vectors were constructed and the overexpression PutDREPPl and PutDREPP2 genes Arabidopsis thaliana were obtained by agrobacterium infection method respectively.The results showed that the root length and fresh weight of overexpression PutDREPPl gene Arabidopsis thaliana were superior to wild-type Arabidopsis thaliana under the stress of 100 mmol/L and 125 mmol/L NaCl.This suggests that PutDREPP1 has a positive regulatory effect on the salt response.Our findings provide insight into the molecular mechanisms by which DREPPs regulate stress responses,and further research on the molecular mechanism of saline-alkali response of halophytes such as Puccinellia tenuiflora provided new information.