Study On The Binding Properties Of MfCML29 Zn²⁺/Fe²⁺

Medicago falcata L.has excellent nutritional value,high crude protein,and has excellent characteristics such as drought resistance,salt and alkali resistance.It is an important germplasm resource for resistance breeding.Calmodulin-like?CML?,as an important member of the Ca²⁺-binding protein family,plays an important role in transmitting Ca²⁺signals and regulating plant growth and development and adaptation to environmental stimuli.This paper is related to the study of the calfin-likeproteinencodinggeneMfCML29?Medicagofalcata CALMODULIN-LIKE?.The study found that the predicted calmodulin-like protein MfCML29 containing Ca²⁺binding domain can specifically bind to Zn²⁺/Fe²⁺and its physiological function was studied.The main findings are as follows:?1?At first,we focused on the analysis of the Ca²⁺-binding protein gene MfCML29 by gene synthesis technology and performed a series of bioinformatics functional analysis on the encoded MfCML29 protein.The results showed that MfCML29 is a hydrophilic protein containing two EF-Hand domains.?2?We constructed 35S-MfCML29-GFP vector that was cloned into pGWB605 vector and MfCML29 was ligated with green fluorescent protein?GFP?.The 35S-MfCML29-GFP vector was transformed into Agrobacterium,transiently transformed into tobacco,and MfCML29 was expressed in the epidermis of tobacco leaves.Subcellular localization showed that MfCML29 was localized in the endoplasmic reticulum.?3?MfCML29 was cloned into wild-type Arabidopsis thaliana by gene cloning,and MfCML29 was heterologously expressed in Arabidopsis thaliana.By phenotypic analysis of transgenic Arabidopsis thaliana,transgenic Arabidopsis thaliana was found to have stronger resistance to low iron than wild-type Arabidopsis thaliana,indicating that MfCML29 can enhance the resistance of transgenic Arabidopsis to low Fe²⁺stress.?4?The MfCML29 protein was expressed and purified,and the interaction between protein and ion was analyzed by Microcal ITC200 isothermal titration calorimetry.The results showed that MfCML29 could specifically bind to Zn²⁺/Fe²⁺but not to Ca²⁺.Thus,we found that MfCML29 can specifically bind to Zn²⁺/Fe²⁺.At present,there is no relevant report about MfCML29 specifically binding to Zn²⁺/Fe²⁺,so it has important research value.