The Role Of StCDPK29 Production Of Reactive Oxygen Species Of Wound Healing Potato Tubers

Wound-healing is a typical reaction of potato tubers after harvest.The formation of wound healing is involved in various metabolisms.Active oxygen species plays an active role in the polymerization of wound healing substance.However the regulation mechanism is unknown.In this study,the"Atlantic"potato tubes were used as materials.StCDPK29 was screened by transcpritome of wound healing tubers.Overexpression and interference expression vectors were constructed.The target gene was transfered into potato by Agrobacterium,and StCDPK29transgenic plants and tubers were obtained.The role of StCDPK29 was evaluated by determining the production of reactive oxygen species,observing the accumulation of suberin and lignin at wound sites,and analyzing the expression of downstream StNOXs and StSODs family genes in transgenic tubers.The results showed as below:1.The RNA-seq combined with biosignal analysis showed that the StCDPKs gene include subfamilies I,II,III and IV.Among them,StCDPK29 had the highest gene expression,which was located in the III subfamily,and had the highest homology with Le CDPK29.StCDPK29 encoded a stable,hydrophobic protein that consisted of 705 amino acids with a relative molecular weight of172545.9 k Da.2.The StCDPK29 overexpression vector PBI121-CDPK29 and interference expression vector p Hellsgate-CDPK29 was constructed by double enzyme digestion and Gateway technology.The transgenic plants and tubers were obtained by Agrobacterium infection method.By constructing the e GFP-CDPK29 fusion expression vector and observing with laser scanning confocal microscope,the protein was found to locate in the plasma membrane and cell nucleus.3.The content of O₂^-and H₂O₂ at wound sites of overexpressed tubers was significantly higher than wild type,also was the accumulation of suberin and lignin.The accumulation speed of suberin and lignin was rapider than wild type.However,the interference expression tubers had less the content of O₂^-and H₂O₂,and less accumulation of suberin and lignin than wild type.The accumulation speed was slowly.4.Gene expressed of StNOXs in overexpressed tubers were significantly up-regulated,however interference expression tubers were significantly down-regulated.St NOXA had the muximum expression after 24h of healing.And St NOXB and St NOXC were significantly expression during the whole healing.The results indicated that StCDPK29 regulated St NOXA expression in early stages of healing,and regulated St NOXB and St NOXC expression during healing.5.Gene expressed of StSODs in overexpressed tubers were significantly up-regulated,however,interference expression tubers were significantly down-regulated.The first expression peak of StSODs showed within 24h of healing,and the second peak appeared at 14d of healing.The maximum expression of St[Cu-Zn]-SOD,St Fe-SOD and St Mn-SOD was observed at 8h,8h and 12h after healing,respectively.It is suggested that StCDPK29 regulates expression of StNOXs and StSODs in time sequence,directly affects the accumulation of reactive oxygen species at wound sites of tubers,and plays an important role in the formation of wound healing tissue of potato tubers.