Identification of Endogenous GUS Gene and Its Enzyme Activity Analysis in Larix kaempferi(Lamb.)Carr.
[Objective]The endogenous enzyme activity of β-glucuronidase(GUS)in Larix kaempferi(Lamb.)Carr.was detected,and the feasibility of exogenous GUS as a reporter gene in genetic engineer-ing was analyzed.[Method]GUS protein sequences from pCAMBIA1301 vector was used to blast the ref-erence genome of L.kaempferi,and then GUS gene in L.kaempferi(LaGUS)was identified and cloned.Expression pattern of LaGUS was analyzed using the published transcriptome data and quantitative re-verse transcription PCR.Enzyme activity of endogenous GUS during L.kaempferi embryogenesis was ex-amined with histochemical method with X-gluc as the substrate of enzyme reaction.[Result]The endo-genous GUS gene,LaGUS was identified in L.kaempferi with a coding sequence of 3 435 bp,encoding 1144 amino acids.It was found that sequences similar to LaGUS existed in at least 25 plant species.The expression level of LaGUS was higher in the active stage than in the dormant stage of L.kaempferi,and higher in somatic seedlings than in embryogenic callus and somatic embryo.Histochemical analysis showed that blue color was observed in embryogenic callus,somatic embryo and somatic seedlings after staining at room temperature for six days,and the degree of staining in somatic seedlings was higher than that in embryogenic callus and somatic embryo.[Conclusion]L.kaempferi has endogenous GUS activity,and using GUS as a reporter gene in genetic engineering may have certain interference.
万方数据
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