Effects Of Calcium And Peroxidase Involved In Stone Cells Formation In Pear Fruit

China is one of the most important botanical origins of the Pyrus L,also pear variety and cultivation area are in the first place in the world.There are lots of combined factors impacts the eating quality of the pear and the stone cell is one of the crucial factors.The density and size of the stone cell play a primary role in the fruit quality of the pear while the lignin biosynthesis process directly affect the differentiation and development of the stone cell.Calcium is one of the indispensable trace elements in pear trees.Researchs found that calcium is widely involved in the physiological and biochemical processes of plants,affecting the activity of enzyme in tissues,which contain peroxidase.Peroxidases catalyze lignin monomer polymerization of oxidation on the cell wall,thus regulate the synthesis of lignin,affect the cell wall also affected the formation of stone cells.In this paper,we performed bioinformation analysis of the Class ? Peroxidase gene family involved in lignin metabolism in pear fruit,and screened out the key genes.Through the sequence analysis,protein sequence analysis,pear fruit transcript analysis at different times to map gene expression heat map,so as to screen out the key genes.In this paper,we used field and laboratory callus to study the effects of calcium on peroxidase production.In field trial,Dangshan pear with spraying of different concentrations(0.25%,0.5%,0.75%)of calcium chloride was used as the material to study the content of stone cells,lignin content,activity of peroxidase ? and peroxidase ? in lignin metabolism,key gene expression patterns in the whole growth and development of pear fruits and its impact on the fruit quality of ripe fruit.The callus culture system of young fruit was carried out.Callus growth process was used as materials to study the effects of different concentration of calcium on the activity of peroxidase and lignin content.The key genes screened out were cloned and subcellular localization was performed.The results were consistent with the prediction.The main test results are as follows:1.In this study,114 peroxidase genes were identified from pear genome by systematic analysis to form PRX gene family.These peroxidase genes can be divided into 12 groups based on phylogenetic relationships.These peroxidase genes are unevenly distributed on 17 chromosomes of pears and some are distributed on other scaffolds.WGD is the main way of the peroxidase gene family expanding in pear.Ka/Ks analysis showed that most of the replicated PbPRXs were undergoing purification.The gene expression analysis was carried out by transcriptome sequencing in the process of fruit growth.It was presumed that some high expression genes may be involved in the lignification of stone cells.The high expression genes were validated by real-time fluorescence quantitative PCR.It was found that the PRX gene family in pear was a multi-gene family,and PbPRX2,PbPRX3,PbPRX6,PbPRXl 7 and PbPRX25 genes could be the key genes in the synthesis of lignin in pear fruit.2.Dangshan pear was used as the experimental material to analyze the effects of different concentrations of calcium chloride sprayed on the stone cell content,lignin content,the quality of ripe fruit,the activity of different forms of peroxidase,H2O2 content and expression of the key genes in fruit.The results showed that the content of stone cell and lignin in the fruits increased first and then decreased in the whole growth and development process after spraying calcium chloride,and the relationship between stone cell and lignin content and calcium concentration;Spraying calcium chloride significantly increased the fruit weight,vertical and horizontal diameter of mature fruit;reduced the hardness of ripe fruit and fruit acid content,improved the soluble solids content in pear fruit.Combined with the requirements of fruit quality,tree growth and production costs and other factors,spraying 0.5%calcium chloride during the flowering and young fruit period is the best.3.Dangshan pear young fruit pulp was used as the experimental material to carried out the callus induction experiment.The effects of different levels of calcium chloride on the content of lignin,POD and hydrogen peroxide in callus were analyzed by adding different levels(0.05%,0.25%,0.5%,0.75%)of calcium chloride in subculture medium.The results show that low concentration of calcium treatment can reduce the activity of peroxidase,thereby inhibiting the oxidation polymerization of lignin monomer,thus reducing lignin.4.In order to study the function of key PRX gene in pear,the open reading frame(ORF)of PbPRX6 with restriction site was amplified by PCR and the fusion of PbPRX6 and green fluorescent protein(GFP)of the plant expression vector p35S-PRX6-GFP was constructed by using Dangshan pear as the experimental material.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 showed that the recombinant expression vector of PbPRX6 gene was located on the cell membrane.