Preliminary Study On The Interaction Between CmNRAMP5 Protein And CmCSOD1-1 Protein In Melon

Cucumis melo L.is a crucial cash crop in China and a valuable model plant in the Cucurbitaceae family for investigating the molecular mechanism of fleshy fruit development and ripening due to its unique botanical properties.Understanding the stress resistance of melon and the mechanisms governing fruit development and ripening is of paramount importance for agricultural planting and production.Melon copper-zinc superoxide dismutase CmCSOD1-1,a member of the melon superoxide dismutase（SOD）protein family,plays a pivotal role in various biological processes such as catalyzing the catabolic reaction of superoxide radicals,preventing cellular oxidative damage,responding to environmental stress,scavenging reactive oxygen species（ROS）poisoning,cell differentiation,and aging.Previous studies of yeast two-hybrid screening of the homogenized c DNA library during fruit ripening revealed that the protein interacting with the melon natural resistance-associated macrophage protein CmNRAMP5 contains CmCSOD1-1.CmNRAMP5 functions as a transporter of Cu²⁺,Fe²⁺,Mn²⁺and promotes fruit ripening in melon,while CmCSOD1-1 is a Cu²⁺and Zn²⁺containing metalloenzyme,and the absence of Cu²⁺and Zn²⁺results in reduced or lost enzyme activity.We further confirmed the interaction between CmCSOD1-1 and CmNRAMP5 protein by the yeast two-hybrid and bimolecular fluorescence complementation（Bi FC）technique.Our study yielded the following results:（1）Bioinformatics analysis identified seven CmSOD family members by whole-genome identification of melon.CmSOD family proteins mainly participate in catalase activity,ROS metabolic processes,SOD activity,oxidative stress response,tryptophan metabolic process,glyoxylate and tricarboxylate metabolic process,etc.Among them,CmCSOD1-1 protein is involved in all the above biological processes.CmCSOD1-1 is localized on melon chromosome 2 and encodes 459 amino acids.The total molecular weight of the encoded protein is approximately 37 k Da,the theoretical isoelectric point is 5.28,and the protein is acidic,with a hydrophilic coefficient of-0.163.It has no leading peptide,no signal peptide,no transmembrane structural domain,and is subcellularly localized in the plant cytoplasm.The evolutionary tree results indicated that the SOD gene family members of melon and watermelon were evolutionarily close,suggesting similar evolutionary origins.The promoter region of the CmCSOD1-1 gene contains cis-acting elements associated with abiotic stress response,hormone response,and light response.（2）We constructed the yeast two-hybrid vectors p PR3-N-CmCSOD1-1and p BT3-STE-CmNRAMP5 and found that there was an interaction between CmNRAMP5 and CmCSOD1-1 proteins.（3）Bimolecular fluorescence complementation（Bi FC）vectors,p SPYNE-CmNRAMP5（containing the N-terminal of CmNRAMP5 protein）and p SPYCE-CmCSOD1-1（containing the C-terminal of CmCSOD1-1protein）,were constructed and transiently transformed into the leaves of Benthamia using Agrobacterium tumefaciens.Laser confocal fluorescence microscopy was used to detect the fluorescence signal,which revealed an interaction between CmNRAMP5 and CmCSOD1-1 proteins.The reciprocal fluorescence was observed on the lower epidermal cell membrane and stomatal cell membrane of tobacco.（4）Prokaryotic expression vectors,p GEX-4T-CmNRAMP5 and pET-28a-CmCSOD1-1,were constructed.Optimize out the conditions for CmCSOD1-1 protein induction.（5）The expression of CmCSOD1-1 gene was analyzed using real-time quantitative polymerase chain reaction（RT-q PCR）at different ripening periods（18d,35d,40d,and 45d）of melon fruits.The results showed that the expression of CmCSOD1-1 was highest at 40d and lowest at 35d and 45d.