Study On Iron Nutrition Funcition Of Specific Calmodulin-like Protein(CML) Of Medicago

Medicago falcata L.and Medicago sativa L.are important leguminous forages,they are high-quality forage plants with rich nutrients and high crude protein content.Medicago falcata L.has strong stress resistance and good qualities of drought,cold and salt-alkali resistance.Medicago sativa L.has high yield and strong adaptability,so they are natural gene resource pool.Calmodulin-like protein,as an important Ca²⁺binding protein,plays an important role in cell signal transduction.However,there are few reports on Calmodulin-like protein in Medicago falcata L.and Medicago sativa L..In this study,Calmodulin-like protein genes in Medicago falcata L.and Medicago sativa L.were identified by transcriptome sequencing and 12specific predictive coding Calmodulin-like protein genes were discovered by analyzing transcriptome sequencing data.12 genes were transfered into the Col-0genome of wild-type Arabidopsis thaliana by Agrobacterium-mediated inflorescence infection.Finally,stable genetically modified Arabidopsis thaliana was obtained.In this study,the physiological functions of two genes MfCML29 and MfCML1 in Medicago falcata L.during plant growth were discussed,which could provide reference for the research of Calmodulin-like protein in Medicago.The main results of this paper are as follows:⑴12 genes were linked to binary expression vectors pGWB605 and pGWB617 by Gateway cloning technique.Agrobacterium-mediated inflorescence infection was used to transfer genes into wild-type Arabidopsis thaliana.12transgenic lines were obtained by screening on the resistant medium containing glufosinate ammonium.⑵Bioinformatics analysis of 12 proteins was carried out.The bioinformatics predictions of MfCML29 and MfCML1 indicate that both contain an EF-hand conservative domain.Subcellular localization results showed that both MfCML29 and MfCML1 proteins were localized in the endoplasmic reticulum of plant cells.⑶Phenotypic analysis of MfCML29 and MfCML1 genetically modified Arabidopsis thaliana were carried out on different mineral cation media.The results showed that there was no significant difference in seedling growth phenotype between transgenic plants and wild-type Arabidopsis thaliana Col-0 on medium containing different concentrations of Ca²⁺,Zn²⁺,Mg²⁺,Mn²⁺.But there are differences in different Fe²⁺concentration media.Transgenic plants are resistant to low iron.Compared with Col-0,the leaves of transgenic plants are larger and greener,the root length and fresh weight of transgenic plants are also significantly higher than those of Col-0.It is speculated that MfCML29 and MfCML1,candidates of Calmodulin-like proteins in Medicago falcata L.,are involved in regulating plant Fe nutritional balance.⑷In order to further explore the mechanism of low iron sensitive phenotype of MfCML29 genetically modified Arabidopsis,the Fe content of transgenic Arabidopsis and Col-0 seedlings was measured.The results showed that the Fe content of genetically modified Arabidopsis thaliana was significantly higher than Col-0.It indicates that MfCML29 participates in the accumulation of Fe elements.