Screening And Analysis Of Soybean Salt-Resistant PM18 Mutant Protein

The inherent disorder protein is a class of proteins that are not stable in the natural state of secondary or tertiary structures,local or integral,but are still capable of exercising normal biological functions.This protein is involved in a variety of physiological processes,through the protein,nucleic acid,lipids,metal ions and small molecules and other molecules in the molecular control network to play a variety of functions,but also in the plant response to stress,So IDPs have potential application value in improving crop stress resistance.LEA protein is a kind of typical intrinsic disorder protein,which can be induced by abiotic stress such as low temperature,drought and high salt.Under stress conditions,LEA protein has the functions of maintaining protein activity,inhibiting protein aggregation,stabilizing protein conformation and protective membrane structure.A large number of transgenic experiments have confirmed that overexpression of Group 3 LEA protein in plants can improve the resistance of host plants.Soybean PM18 protein belongs to group 3 LEA protein.Our laboratory preliminary study has found that this gene can improve the salt tolerance of Escherichia coli,yeast and Arabidopsis thaliana,but the mechanism of action has not been reported.Due to the disordered nature of the inherent disorder protein,it is often used in molecular networks to interact with different target proteins and participate in different physiological processes,so we speculate that it can interact with other proteins in plant resistance to stress When the protection function.Therefore,we screened the soybean salt-tolerant protein PM18 as the entry point to further reveal the salt-resistant mechanism of LEA protein.In order to further explore the salt tolerance mechanism of PM18 protein,the PM18 protein was predicted by bioinformatics.The results showed that soybean PM30 and PM32 protein and soybean PM18 protein were most likely to be predicted by the online site STRING.There is an interaction relationship.The PM18 protein was subcellularly mapped by PSORT II and Plant-PLoc.The predicted results showed that the PM18 protein was predicted by the three different software metaPrDOS,DISOPRED and IUPred.The results showed that the N-terminal(0-30 position)and C-terminal(300-309)showed a strong disorder,and its region containing 8 conserved domains(113-205)should be ordered.Then,the soybean cDNA library was constructed by using the method of SMART method.The fragment size of the inserted fragment was between 0.5 and 5.OKbp,and the titer was titer.For the 4x104cfu/ml,the library quality is better,in line with the late yeast two-hybrid experimental requirements.Then,the PM18 gene was cloned from the soybean cDNA by PCR and identified as vector pGBKT7 after sequencing.The pGBKT7-PM18 and pGADT7 plasmids were co-transformed into yeast strain AH109 to detect the absence of self-activation of bait protein,which could be screened by yeast two-hybrid system and no need to add 3AT.Finally,the yeast two-hybrid system was used to screen the plasmid pGBKT7-PM18 and the plasmid of soybean cDNA library to yeast strain AH109.Twenty-three positive clones were screened and 17 valid positive clones were obtained by rotary validation.The full-length sequences were LOC100170728,MYB173,LOC100499708,LOC100306286,LOC100500540,LOC100038325,and the full-length sequences were searched by NCBI.The full-length sequences of the five gene fragments were successfully obtained from the soybean cDNA by PCR method,and successfully constructed on the pGADT7 vector.The method of rotary verification was used again.The results were as follows:,Which was co-transferred to yeast with pGBKT7-PM18,respectively.The results showed that the proteins encoded by soybean LOC100170728 and MYB173 could interact with soybean salt-resistant protein protein.