Screening Of Wheat Dehydrin WZY1-2 And WZY2 Interacting Proteins Under Drought Stress

Wheat is an important food crop and is often subjected to environmental factors such as drought,high temperatures,salting,and heavy metals during its growth,resulting in severe reductions in production.As an induced protein during plants suffering from stress,dehydrins can reduce the damage of plant cells and protect plants.Dehydrin,a member of the second subfamily of LEA protein,is highly hydrophilic.It has been proved that dehydrin plays an important role in the resistance of plants to drought stress.In this paper,the WZY1-2 and WZY2(wheat dehydrin protein)genes were cloned from Zhengyin No.1 wheat.Through the construction of prokaryotic expression vector and bait carrier,the proteins interacting with dehydrin WZY1-2 and WZY2 were screened by Pull-down technology and yeast two hybrid technology,and the function of the protein was further studied.The results lay a foundation for further analysis of molecular biological mechanisms of wheat drought resistance and molecular breeding of wheat.The following results were obtained through the research:1.The dehydrin genes WZY1-2 and WZY2 were cloned from wheat Zhengyin No.1 and the gene coding regions were 789bp and 459bp,encoding 262 and 152 amino acids respectively.The molecular weights of the proteins were 28KD and 15.522KD,belonging to the SK3 and YSK2 types dehydrin.The prokaryotic expression vector was successfully constructed and transformed into E.coli BL21(DE3).The optimal expression of WZY1-2 was found to induce 6h at 0.1 mM at 37 C,and WZY2 at IPTG induced 1Oh for 0.2mM at 37.2.After the purification of WZY1-2 and WZY2 by GST chromatography column,there are 132 potential interaction proteins from total protein of wheat through Pull-down,of which 96 are unknown structural proteins,36 are known structural proteins,and 75 potential interaction proteins with WZY2,of which 68 are unknown structure egg white and 7 are known structural proteins.7 proteins related to WZY1-2 and drought resistance were selected.There were 6 proteins related to drought resistance,which were interacted with WZY2.By constructing bait carrier and prey carrier,their interaction was further verified in yeast strain Y2HGold.3.through the construction of PGBKT7-WZY1-2 and PGBKT7-WZY2 vector,the yeast strain Y2HGold was transformed to verify its self activation and toxicity,and it was found that when the concentration of AbA was 200ng/ml,it could inhibit the yeast’s background expression,and WZY1-2 and WZY2 were not toxic to yeast when they were expressed in yeast.4.the cDNA library was transformed into the Y2HGold yeast strain containing PGBKT7-WZY2.By detecting its conversion efficiency,8.7*106 McAbs were found,indicating that almost all the library plasmids had been converted into.Through subsequent drying experiments,a total of 21 interacting proteins were obtained,9 of which were proteins associated with stress resistance,and their interactions need to be further verified in yeast.