| Under the constantly changing conditions of the global climate,crop growth needs to needs to deal with various abiotic resistance(low temperature,drought and soil alkalization)conditions.Abiotic stress can damage DNA directly or indirectly,so all species have a DNA repair system.The excision repair(BER)pathway is an important repair pathway in the DNA repair system.The first step of the pathway is initiated by a specific enzyme-DNA glycosylase.A lot of research on the BER pathway has been carried out in China,but instead of the research reports on the BER pathway in plants,we try to find a specific mechanism for responding to DNA damage in plants.The beet M14 line is a monomer addition line obtained by crossing and backcrossing cultivated beet and wild white-flowered beet.It absorbs abundant high-quality genetic resources and has overcoming tolerance to hydrochloride.It is an extremely rare material for studying adversity enhancement.In the early stage,the DNA glycosylase gene(Bv M14-Fpg X2)was cloned in the M14 strain of sugar beet using the homologous sequence cloning method.This study first tried to clone the Bv M14-Fpg X2 gene into the p LATE31 vector to obtain the recombinant plasmid p LATE31-Bv M14-Fpg X2,and transfer it into E.coli XL1 Blue,and found that it could not grow,therefore,the DNA glycosylase Bv M14-Fpg X2 is truncated and its C-terminal region contains a DNA sequence that may affect cell growth and cell expression,and two gene splicing sequences Bv M14-Fpg X2 RKS and Bv M14-Fpg X2 VQA are obtained,of which Bv M14-Fpg X2 RKS compared with Bv M14-Fpg X2 VQA leaves more C-terminal region.They were constructed into the p LATE31 prokaryotic expression vector,and the induction conditions were optimized in E.coli BL21(DE3)cells.The Bv M14-Fpg X2 RKS protein was not successfully induced.The Bv M14-Fpg X2 VQA recombinant protein in the form of inclusion bodies was subjected to denaturation and dissolution,nickel ion affinity chromatography,protein elution and renaturation dialysis.For the first time,the Bv M14-Fpg X2 VQA recombinant protein was isolated and purified in E.coli prokaryotic expression system.This study provides a guarantee for the follow-up study of the DNA damage repair function of Bv M14-Fpg X2 VQA,and lays a foundation for revealing the possible stress resistance of sugar beet M14 strain and the molecular mechanism of the increase in the activity of key enzymes in the BER repair system. |
