Study On The Function Of GID1 In Regulating Lignin Formation Of Pear Fruits By Gibberellin

Pyrus is the third largest fruit in China.It is widely planted worldwide.It is widely praised in the fruit market for its sweet taste and unique flavor.However,different varieties show wide differences in fruit morphology and physiological characteristics.In recent years,Western pear varieties have entered the Chinese market,while Oriental pears such as‘Dangshan Su’pear is gradually at a disadvantage due to poor taste.Stone cells are a peculiar characteristic of pear fruits and are formed by the lignification of cell walls.The high content seriously affects the taste and processing quality of the fruit.Therefore,inhibiting the formation of stone cells is of great significance to improving the quality of the fruit.Previous studies have shown that the formation of stone cells is comprehensively affected by itself and environmental factors,among which the regulation of the endogenous hormone gibberellin plays an important role.During the development of young pear fruit,the content of various endogenous hormones in the pulp is constantly changing.This study used‘Dangshan Su’and‘Cuiguan’pear as the test materials to determine the endogenous hormones during the growth and development of the two varieties.The content of hormones,stone cells and lignin,and the correlation between endogenous hormones and the two are discussed.In this project,the callus of‘Dangshan Su’pear was treated with exogenous gibberellin,and the effect of exogenous gibberellin on lignin metabolism was studied through the propagation of different concentrations of medium.The role of cell development and lignin formation is further explained.Gibberellin receptor（GID1）is a key mediator in the process of gibberellin signal transduction and plays an important role in regulating plant growth and development.Bioinformatics methods have been used to analyze the GID1 gene in the genome of Chinese white pear（Pyrus bretschneideri）and make whole-genome identification and expression analysis.The key gene PbGID1.25,which affects the development of stone cells,was screened through RNA-Seq data,and transgenic technology was used to make it stably expressed in Arabidopsis and pear callus,and paraffin sectioning,subcellular localization,q RT-PCR and other methods were used.The wild-type and transgenic plants were analyzed in terms of plant phenotype,tissue staining and molecular expression level.The main results are as follows:1.The changes in the content of auxin（IAA）and gibberellin（GA₃）in the pulp of the two varieties are the same,but the content of the two endogenous hormones in‘Dangshan Su’pear is higher than that in‘Cuiguan’,and the content of gibberellin is the same.In pear callus,the application of different concentrations of GA₃from external sources has varying degrees of influence on the content of lignin.The low concentration（1mg/m L,2 mg/m L）treatment of GA₃synergistically regulates the production of lignin,while the high concentration of GA₃（4 mg/m L）GA₃treatment played an antagonistic effect,and the expression levels of specific enzymes CCR and CAD in the lignin synthesis pathway showed the same trend.2.There are 50 GID1 genes Pyrus bretschneideri in genome,including three Arabidopsis orthologous genes.Its chromosomes are widely distributed,except for chromosomes 1 and 8.Prediction of subcellular location indicates that the protein is mostly located in the plasma membrane,most genes have only one exon,no introns,and the conserved motifs motif1,2,4,and 8 are shared by most genes;PbGID1 starts the sub-region contains multiple light and hormone response elements.The results of q RT-PCR showed that PbGID1 was highly expressed in roots,leaves and fruits,and almost not expressed in stems and pollen.The expression of PbGID1 in callus increased significantly after treatment with exogenous gibberellin.There are five PbGID1 genes that are significantly different in the transcriptome database of the fruit in the stone cell-enriched area and the stone-cell-deficient area.Among them,PbGID1.25 is significantly higher in the stone-cell-deficient area than in the enriched area,and its expression level is similar to that of‘Dangshan Su’pear.The expression trends of fruit stone cells and lignin are consistent.3.The gibberellin receptor gene PbGID1.25 was cloned from the fruit tissue of‘Dangshan Su’pear.The CDS sequence is 1089 bp in length and encodes 370 amino acids.The protein structure and domain analysis show that PbGID1.25 belongs to theα/βsheet Hydrolase superfamily;Hydrophilicity and hydrophobicity analysis show that PbGID1.25is a soluble protein;subcellular location shows that PbGID1.25 is a cell membrane protein;these fully prove that PbGID1.25 is a gibberellin receptor gene.The Agrobacterium-mediated method was used to transiently overexpress PbGID1.25 in pear fruits,and the lignin content at the injection site was significantly reduced.After the p35S-PbGID1.25-GFP fusion vector was stably transformed in Arabidopsis thaliana,the inflorescence stems of the transgenic positive lines were sectioned with safranin-fast green staining,showing that the intervascular fibers,wood fibers and vessel cell walls in the Arabidopsis stems were significantly changed Thinner,lower lignin content,lower lignin biosynthesis-related gene expression.At the same time,the gibberellin signaling pathway in the transgenic plants was promoted,the expression levels of RGA and GAI genes that inhibit stem development were reduced,and the endogenous active gibberellins GA₄and GA₇increased significantly.The p35S-PbGID1.25-GFP fusion vector was stably expressed in the callus,and the transgenic callus cell line also showed a decrease in lignin.DELLA protein is the downstream receptor of GID1.Overexpression of DELLA can make stalks dwarf and increase the degree of lignification.The overexpression of PbGID1.25 may inhibit the expression of DELLA in gibberellin signal transduction,thereby releasing the promotion of lignin metabolism.