Construction Of Potato Transient Expression System And Study Of The Mechanism Of StPCR1 Response To Cd Stress In Soi

Potato（Solanum tuberosum L.）is an important food crop,and its yield and edible safety are seriously affected by heavy metals.Plant Cd Resistance（PCR）is a class of transmembrane proteins with PLAC8 domain.This gene family has been reported in Arabidopsis thaliana and rice,but there are few reports on the function of PCR gene in potato.In order to facilitate the analysis of StPCR gene function,the young buds of"Yunshu 505"were first used as explants in this study to study the effects of different concentrations of hormone treatment combinations on callus induction of potato young buds.On this basis,the transient expression system of potato was optimized.Secondly,21 PCR genes were identified and screened from potato genome files,and through molecular biology and toxicology methods,the key gene StPCR1 that may actively participate in Cd stress was discovered.The transient expression system was used to observe the functional position of StPCR1 and detect its expression level and physiological indicators under Cd stress.The feasibility of this system is verified.Finally,two interacting target proteins of StPCR1 were screened by yeast two-hybrid,which laid a theoretical foundation for further elucidate the molecular mechanism of StPCR1 gene involved in potato stress response.The main conclusions are as follows:（1）Establishment of tissue culture system of potato sprout explants.The comparison of medium with different disinfection time and different hormone ratio showed that the Browning rate and contamination rate of explants were lower and the survival rate was higher when 75%Na Cl O was sterilized for 10 min.The results showed that MS+0.2 mg/L NAA+0.7 mg/L 6-BA medium was effective,and the explants had early germination,bright green leaves and large leaf area.（2）Establishment of instantaneous expression system mediated by Agrobacteriu m Tumefaciens GV3101.Vacuum time,pressure and concentration of Agrobacterium solution were compared.The results showed that when the vacuum pressure was 1.0Kpa,the vacuum time was 20 min and the concentration of bacterial solution OD₆₀₀=1.2,the expression of GFP protein in potato was the most obvious.（3）Identification of potato PCR family members and discovery of key genes.Twenty-one members of the PCR gene family were identified from potato genome,distributed on 10 chromosomes,all with PLAC8 domain and conserved domain sequences.The evolutionary tree was divided into three branches.The first branch had the largest number of members（10）,and StPCR1 was closely related to At PCR1gene in Arabidopsis Thaliana,while the second branch had the smallest number of members（4）.Analysis of cis-acting elements showed that StPCR1 had the most cis-acting elements in abiotic stress,indicating that StPCR1 may play a unique role in coping with abiotic stress.（4）Verification and application of StPCR1 transient expression system.The overexpression vector of potato StPCR1 gene was constructed,and the potato tissue culture plant/soil culture plant was transformed by Agrobacterium-mediated transient expression system.Under the stress of heavy metal Cd,the StPCR1 gene expression and the contents of physiological indexes in the transient transformed potato tissue culture seedlings were significantly increased,and the activity of antioxidant enzyme was significantly higher than that in the control group.The activities of SOD,POD and CAT were about 2.2 times,1.5 times and 6 times of that in the control group,respectively.After transient conversion of StPCR1 into potato soil-cultured seedlings,the content of Cd in potato roots increased.SOD,POD and CAT also maintained the antioxidant system of potato soil-cultured seedlings and eliminated the damage of excessive ROS,indicating that plants with transient expression of StPCR1 could better cope with the ROS surge caused by Cd stress and reduce plant damage.（5）Two proteins interacting with StPCR1 were screened.Two proteins（StMAT E60/StMHC1）interacting with StPCR1 were screened by yeast two-hybrid,and the in teraction between them was further confirmed by bimolecular fluorescence compleme ntation technique.