Functional Verification Of PsERF And PsCIPK Gene Related To Waterlogging Tolerance In Cherry

Cherry(Cerasus pseudocerasus)is a deciduous fruit tree which belongs to the genus Rosaceae.It has high nutritional value and good economic benefits.However,its root system is shallow,and it is easy to cause crop damage during the cultivation process,which affects its economic benefits.Therefore,the genetic improvement and germplasm innovation of cherry tolerance has important theoretical and practical significance.In the previous study,the waterlogging-tolerance cherry germplasm Prunus serrulata ' Yimeng' was used as a test material,PsERF and PsCIPK genes,which were found to be involved in regulating the waterlogging tolerance of cherry,were cloned.Based on this work,we further constructed the overexpression vectors and RNAi vectors of these two genes and used the Agrobacterium-mediated genetic transformation method to transfer the two genes into the Arabidopsis thaliana to obtain homozygous transgenic positive plants.Through waterlogging stress treatment,the phenotypic changes of Arabidopsis thaliana were observed and compared,and the changes of carbohydrate metabolism-related enzyme activities in transgenic and wild-type plants were determined to provide a theoretical basis for further revealing the molecular mechanism of cherry tolerance.This project will lay a foundation for the cultivation of new waterlogging-tolerance germplasm in cherry.The main results were shown as follows:(1)Cloning of waterlogging tolerance related genes in cherry.The total RNA was extracted from the leaves of Prunus serrulata 'Yimeng' and the first cDNA chain was synthesized by reverse transcription.The primers were designed according to the sequence of the PsERF and PsCIPK gene in Genebank.The full-length sequences of the two target genes were cloned and included complete coding regions.And it is estimated that the amino acid sequence similarity between the cloned amino acid sequence and the PsERF and PsCIPK genes registered in the NCBI database are 100%.(2)Constructing the over-expression vector and RNAi expression vector of PsERF and PsCIPK gene.After amplification of the coding region with appropriate restriction sites,the target gene PsERF and the binary expression vector pCAMBIA1304 were digested with BglII single enzyme,the target gene PsCIPK and the binary expression vector pCAMBIA1304 were digested withNcoI single enzyme,using AscI and SwaI positively excised the target gene PsERF and RNAi expression vector pFGC5941 positively,reversed and double-digested the target gene PsERF with SpeI and BamHI,and the RNAi expression vector with positive target gene obtained in the first step pFGC5941-PsERF-positive,gel recovery,connection of the target fragment and vector,successful construction of plant overexpression vectors pB1304-PsERF,pB1304-PsCIPK and RNAi expression vector pFGC5941-PsERF-RNAi,empty pB1304 and pG5941.After correct sequencing and verification,the recombinant plasmid and empty vector were transformed into Agrobacterium tumefaciens EHA105 by freeze-thawing method to obtain Agrobacterium transformants which can be used for genetic transformation.(3)Gaining Arabidopsis homozygote(PsERF and PsCIPK).Transformation of Arabidopsis was conducted by floral-dip with recombinant agrobacterium transformants.Finally,9 lines of overexpressionv PsERF transgenic homozygosis,16 lines of overexpression PsCIPK transgenic homozygosis,5 lines of silencing expression PsERF transgenic homozygosis,and 3 lines of vector-control transgenic homozygosis were gained through a series of tests,including resistance screening of T1 generation,GUS staining,isolation screening of T2,T3 generation.The results of GUS staining,PCR confirmed the integration of the target genes into the genome of Arabidopsis,and qRT-PCR showed high level expression of target genes in transgenic lines compared to the control.(4)Comparison and analysis of phenotype in Arabidopsis homozygote.Phenotypic analysis of the transgenic Arabidopsis line showed that the root system of overexpressing the PsERF and PsCIPK Arabidopsis was significantly longer than the wild type.After 2 weeks of waterlogging treatment,it was found that overexpression of PsERF and PsCIPK genes could enhance the tolerance of Arabidopsis thaliana.Silencing expression of PsERF in Arabidopsis thaliana caused the plants tolerance were inhibited and showed premature death.(5)The effects of waterlogging stress on the activity of carbohydrate-metabolizing enzymes in wild-type and transgenic Arabidopsis thaliana were determined.Five-week-old transgenic Arabidopsis plants were treated for 0 d,1 d,2 d,3 d,4 d,and 5 d with a wild-type control and their carbohydrate metabolism-related sucrose synthase(SUS)and ?-amylase(RAMY),alcohol dehydrogenase(ADH),pyruvate decarboxylase(PDC),and soluble protein content.The results showed that the activities of SUS,RAMY,ADH and PDC in Arabidopsis thaliana with overexpressing PsERF gene were significantly higher than those of the control and wild type during waterlogging stress,however,the changes in the activities of sugar metabolism-related enzymes in Arabidopsis thaliana with overexpressing PsCIPK genes were basically contrary to the trend of overexpression of PsERF genes.This suggested that overexpressing PsERF gene could increase the waterlogging resistance of plants by adjusting the sugar metabolism,and reduce the damage caused by waterlogging.Therefore,we think that the PsERF gene may be a key gene which regulates the tolerability of cherry.