Preliminary Study On The Interaction Between CmNRAMP5 And CmSCAMP5 Protein In Melon

Previous studies have shown that overexpression and CRISPR-Cas9 gene editing of the CmNRAMP5 gene in melons resulted in significant early ripening and late ripening.Transcriptome and proteome of T₃ fruits of the two transgenic melons and control were sequenced at different developmental stages.Based on the multi-omics sequencing data,the interacting protein CmSCAMP5 of melon CmNRAMP5 was screened by bioinformatics analysis.In this study,the interaction between CmNRAMP5 and CmSCAMP5 in melon was verified by bimolecular fluorescence complementation experiment,yeast two-hybrid experiment,GST-Pull down experiment and Western blot experiment.In order to elucidate the specific functions of melon CmSCAMP5 and CmNRAMP5 proteins in plants at the level of late protein interaction.The experimental content is as follows:（1）Bioinformatics analysis:The evolutionary tree analysis of CmSCAMP5 protein revealed that the main body of the evolutionary tree was divided into two branches.In the first branch,jute and Cacao had the highest homology.In the second branch,the homology was the highest with white gourd（Benincasa hispida）.As can be seen from the heat map of gene expressions,the expression level of CmSCAMP5 was higher in the late respiratory saltation stage,but on the contrary in the growing stage.The analysis of protein interaction network revealed that CmSCAMP family interacting proteins were mainly involved in protein transport pathway.（2）BiFC-C-NRAMP5 and BiFC-N-SCAMP5 vectors were constructed to transform Agrobacterium GV3101,and tobacco leaves were injected and observed under confocal microscope.The results show that CmNRAMP5 and CmSCAMP5 were interacted.（3）Yeast two-hybrid experiment:pBT3-STE-NRAMP5 vector and pPR3-N-SCAMP5 vector were constructed to transform yeast NMY51,complete self-activation detection and interaction verification.It was found that there was no self-activation phenomenon at 0 mmol/L 3AT,so 0 mmol/L 3AT was selected for follow-up point plate verification.Positive controls pBT3-STE-PT2 and pPR3-N-PP95 grew normally on SD-TL,SD-TLH and SD-TLHA plates.Negative controls pBT3-STE-Os PT2 and pPR3-N-Os PP95CT grew normally on SD-TL plates,but could not grow on SD-TLH and SD-TLHA plates.The growth of pBT3-STE-NRAMP5 and pPR3-N-SCAMP5 were proved to be consistent between the experimental group and the positive control group.Therefore,it was further concluded that CmNRAMP5 interacts with CmSCAMP5 protein in melon.（4）GST-Pull down and Western blot experiment:pGEX-4T-NRAMP5 and p ET-28a-SCAMP5 vectors were constructed to transform E.coli BL21（DE3）and Rosetta（DE3）,and the optimal conditions for inducing expression were explored with different concentrations of IPTG and different induction times.In the Rosetta（DE3）host at 16℃,1.0 mmol/L IPTG for 11 h could induce a larger band than the target band（84.1KDa）.In Rosetta（DE3）,1.0 mmol/L IPTG at 16℃for 4 h induced larger bands than SCAMP5.The induced expression products were crushed by ultrasonic wave and GST column was used to perform Western-blot detection by Mouse Anti Gst-tag m Ab and HRP Goat Anti-Mouse Ig G（H+L）.The bands were about 25 KDa.It was speculated that the protein purified by GST purification column was 25 KDa GST Tag protein,and the capture protein band of GST tag protein was 37.9 KDa too large.It was preliminatively determined to be CmSCAMP5,which required Mouse anti His Tag m Ab.HRP Goat Anti-Mouse Ig G（H+L）was detected by Western-blot.The above experiments preliminarily verified that CmSCAMP5 and CmNRAMP5had protein interaction.