Regulating Mechanism Of Calcium And Peroxidases On Lignin Formation In Pear Fruit

Pear is one of the most widely distributed fruit trees in China,and its output ranks third.An important feature of pear fruit that distinguishes it from other fruits is the stone cell content.The content of stone cells a great influence on the taste of the fruit,so reducing the content of stone cells is an important research topic in the pear industry.According to reports,spraying calcium-containing agents in the field can reduce the content of stone cells,but the effect is mixed.Therefore,studying the mechanism of calcium regulating the formation of stone cells and lignin has become a hot spot in pear quality research.This study uses a combination of field trials and indoor trials.Using’Dangshansuli’ as material,to study the relationship between calcium and stone cell content,lignin content,POD Ⅰ/Ⅱ activity and PRX-related gene expression patterns during fruit development of ’Dangshansuli’,and the effects on the quality of ripe fruits.Collect the’Dangshansuli’ fruit 21 days after full bloom to induce pear pulp callus.Using callus as material,to study the relationship between different concentrations of calcium ions and the lignin content,POD Ⅰ/Ⅱ/Ⅲ activity in ’Dangshansuli’ callus and the expression pattern of PRX-related genes.And the relationship between the distribution of calcium ions in cells and different types of POD,the distribution of lignin.Using ’Dangshansuli’ cDNA as a template,PbPRX8 was successfully cloned,and PbPRX8-positive Arabidopsis seedlings were obtained.The relationship between PbPRX8 and lignin was studied in Arabidopsis and pear fruits,respectively.The main test results are as follows:1.The experiment used ’Dangshansuli’ as the material,and sprayed water and 0.5%calcium nitrate on the leaves,on 7d,14d,21d,and 28d after flowering,to determine the pear fruit stone cells,lignin and other related indicators.The results show that 0.5%calcium nitrate can increase the total calcium content of the pulp,and 7 days after the treatment is stopped,the total calcium content in the pulp returns to normal;it will increase the fruit shape index,hardness,soluble sugar content,pulp stone cells content of the ripe pear to a certain extent;can increase the lignin content of juvenile pulp;can increase the activity of POD Ⅰ and POD Ⅱ of pear pulp,and mainly act on POD Ⅱ;up-regulate the expression of PbPRX8 gene,and down-regulate the expression of PbPRX75,PbPRX2,PbPRX3 gene.These experiments indicated that 0.5%calcium nitrate would reduce fruit quality and promote the formation of pear pulp lignin;POD Ⅱ may be a key enzyme controlling lignin formation,and PbPRX8 may be a key gene regulating POD Ⅱ activity.2.The experiment uses ’Dangshansuli’ callus as the material,and uses different concentrations of calcium nitrate(0%,0.05%,0.1%,0.2%)medium for cultivation to determine the lignin,POD activity and other indicators of callus,To observe the distribution area of calcium ions and lignin.The results show that three concentrations of calcium nitrate can inhibit the formation of lignin in callus,of which 0.1%calcium nitrate treatment has the lowest lignin content;it effectively inhibits the activity of POD Ⅰ/Ⅱ/Ⅲ,in which G-POD Ⅱ is consistent with the change of lignin;Up-regulation of PbPRX2,PbPRX3,PbPRX61 gene expression,down-regulation of PbPRX8,PbPRX13,PbPRX20,PbPRX21,PbPRX91 gene expression,of which PbPRX8,PbPRX21,PbPRX48 and lignin changes consistent.In addition,calcium ions absorbed by calcium-treated callus are mainly distributed on the cell wall;lignin mainly accumulates on the cell wall.These results indicate that 0.1%calcium nitrate has the best and stable inhibition effect and maintains a good growth state;G-POD Ⅱ is a key enzyme controlling lignin formation,and PbPRX8,PbPRX21,PbPRX48 are the key genes regulating POD activity;the cell wall may be the site of lignin synthesis.3.The experiment used ’Dangshansuli’ fruit cDNA as a template to successfully clone PbPRX8 with a CDS sequence of 1053 bp,and obtained PbPRX8 positive Arabidopsis seedlings.PbPRX8 subcellular localization results indicate that it is a membrane protein.In PbPRX8 overexpressing Arabidopsis thaliana plants,the Arabidopsis plants were dwarfed and the stem lignin content increased significantly.PbPRX8 transient over-surface experiments in ’Cuiguan’ and ’Dangshansuli’ fruits showed that the lignin content in the region injected with PbPRX8 overexpression vector increased significantly.These results prove that PbPRX8 promotes the formation of pear fruit lignin.