| Based on the Rosaceae genome database,the classification and phylogenetic analysis of the protein sequences and conserved domains of pear and other three Rosaceae species were performed to identify the Rosaceae CAD gene family,and the evolutionary course of this gene was elucidated using bioinformatics analysis.The content of lignin,the content of stone cells and the changes of CAD activity were used to screen the appropriate calcium concentration in the formation of the stone cells in 'Dangshansu pear' and its callus.The expression of PbrCAD gene under calcium induction was analyzed by qRT-PCR.Cloning based on bioinformatics analysis and related experimental data,we screened the key gene PbrCAD1 affecting the formation of stone cells and carried out subcellular localization.The main results of the study are as follows:1.In this study,the bioinformatics analysis of the CAD gene family was carried out with pear(Pyrus bretschneideri),apple(Malus domestica),Chinese plum(Prunus mume)and strawberry(Fragaria vesca),designated as PbrCAD,MDPCAD,PmCAD,and mrnaCAD,respectively.The results showed that:the four Rosaceae species included 149 CAD gene members with ADH-N and ADH-zinc-N conserved domains,and phylogenetically clustered into six subfamilies.These genes distributed unevenly on the chromosome,colinearity analysis of the same species showed that there are nine pairs of collinear genes among the four Rosaceae species.The dispersed duplication arid WGD/segmental duplication account for the largest number of evolutionary events,which were the main method of CAD amplification in the Rosaceae species.2.57 CAD family genes were identified by bioinformatics in the pear genome,and their phylogenetic tree,gene structure,amino acid conservative sequence and Ka/Ks were analyzed and discussed.The PbrCAD gene that may be involved in lignin synthesis was screened by transcriptome data and stone cell distribution and stone cell content changes.The expression level of the candidate gene was detected by qRT-PCR to further determine the key genes that involved in lignin synthesis.The results showed that:these PbrCAD genes were divided into 7 subfamilies and the same family member contains similar gene structures and conserved sequences.All Ka/Ks ratios of the paralogous genes were less than one,implying that they had experienced strong purifying selective pressure.The transcriptional data revealed that 51 pear CAD genes were expressed during fruit development,and four PbrCAD genes may be involved in lignin formation were screened by quantitative fluorescence data.3.In order to analyze the fruit weight,the content of stone cells,the content of lignin,and the cinnamyl alcohol dehydrogenase activity,the‘Dangshansu pear'was used as a test material to spray different concentrations of calcium(CK,0.25%,0.5%,0.75%).The expression of PbrCAD gene in calcium treatment was measured.The results showed that:with the growth and development of fruit,the single fruit weight of spraying 0.5%concentration of calcium was always lower than the weight of other treatments.The content of stone cells and lignin content in the pear fruit increased first and then decreased,the content of lignin in stone cells tended to rise first and then tended to slow,and their content was the lowest in the 0.5%concentration calcium treatment.The activity of CAD decreased in the whole growth process of fruit,spraying 0.5%calcium could significantly inhibit its activity.PbrCAD1 gene expression decreased significantly in calcium treatment.In actual production and application,0.5%calcium fertilizer plays an important role in reducing the content of stone cells and improving fruit quality.PbrCADI may play a major role in the lignin synthesis of pear fruit.4.The explants were produced by inducing the young fruit of 'Dangshansu pear,' after several subcultures,the yellow and white callus with loose texture,strong growth,and uniform growth rate were selected as test materials.The results showed that:the medium suitable for inducing the callus formation from the young fruit of 'Dangshansu pear' was MS+2.0mg/L 2,4-D+0.5mg/L 6-BA+0.5mg/L NAA+75mg/L Vc,the induction rate was 95%.The suitable medium for subculture of callus was MS+2.0mg/L 2,4-D+1mg/L 6-BA+lmg/L KT,the proliferation rate of callus was 81.5%.5.With the callus and cell suspension as test materials of 'Dangshansu pear' as experimental materials,the effects of calcium concentration(CK,0.05%,0.1%,0.25%)on lignin content and CAD activity of callus were analyzed.The results showed that:low concentration of calcium ions can reduce the activity of cinnamyl alcohol dehydrogenase,inhibiting the oxidative polymerization of lignin monomer,thereby reducing the lignin content.High concentration of calcium inhibit the growth of callus,resulting in shortened callus subculture cycle and easy browning.6.In order to further study the function of key CAD gene in pear,the open reading frame(ORF)of PbrCADl with restriction site was amplified by PCR and the fusion of PbrCAD1 and green fluorescent protein(GFP)of the plant expression vector p35S-PbrCAD1-GFP was constructed.The plant expression vector was observed by fluorescence microscopy in tobacco leaf epidermal cells to determine the localization of the gene in the cells.The results show that:The recombinant expression vector of PbrCAD1 gene was located on the cell membrane. |
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