Cloning and functional analysis of leaf number gene phosphoproteinase 1 from Erigeron breviscapus
[Objective]The paper aimed to explore the function of the phosphoproteinase 1(EbPP1)gene related to the number of leaves in Erigeron breviscapus,which lays the foundation for revealing the molecular mechanisms of E.breviscapus leaf number and flowering time.[Method]The EbPP1 gene from the leaves of E.breviscapus was cloned and constructed into the plant overexpression vector RP101.The dip-ping flower method was used to infect newly bolted Arabidopsis thaliana plants for heterologous expression,several T1,T2 and T3 generations of A.thaliana positive plants were obtained through screening.Finally,the number of leaves and flowering time of T3 generation positive plants were counted.The expression level of EbPP1 was detected by RT-qPCR,and the endogenous hormone content of A.thaliana plants was measured.[Result]The full-length EbPP1 gene was successfully cloned and overexpression vector RP101-GFP EbPP1 was constructed for this gene,cloned stable genetic transformation had been achieved in A.thaliana,and 24 positive EbPP1 transgenic plants were obtained in the T3 generation.Real time fluorescence quantitative analysis was performed on the T3 generation positive plants,and RT-qPCR results showed that the EbPP1 gene was overexpressed in the transgenic A.thaliana,with its expression level increasing from 1.034 in the wild type to 136.33;Transgenic A.thaliana exhibited phenotypic characteristics of increased leaf number and delayed flowering time;Quantitative determination of endogenous hormone content in plants by LC-MS showed that the content of various hormones such as indole-3-carboxylic acid(ICA)and gibberellin-24(GA24)in T3 transgenic A.thaliana were significantly higher than that in the wild type,with the precursor substance of growth hormone L-tryptophan(TRP)being particularly significant,reaching three times that of the wild type.[Conclusion]Suc-cessfully,the heterologously EbPPl gene is expressed in A.thaliana plants.Compared with wild A.thaliana plants,transgenic A.thaliana plants have an increased number of leaves,increase its expression of EbPP1 gene,and increase endogenous hormone content.Based on the above results,it is suggested that EbPP1 may affect the number of leaves and flowering time of E.breviscapus by regulating plant hormone levels.The study lays the foundation for further analyzing the mechanisms related to the development of leaf number and flowering molecular mechanisms of E.breviscapus,and also provides a certain genetic basis for the breeding of high-quality E.breviscapus varieties.
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