Identification And Functional Analysis Of Suberin Formation Related Genes In Peel Of Sand Pear(Pyrus Pyrifolia)

Fruit pericarp is an important character of fruit crops,pericarp russet is an important commercial defect.Russet is mainly caused by the rupture of pericarp cells and the accumulation of suberin.It is of great significance to analyze and understand the related genes controlling the production and formation of suberin in pear for breeding and improving pear varieties with excellent fruit characteristics.In this study,we analyzed transcriptome data of ’Huanghua’ pear(russet pericarp)and its budding mutant ’Green huanghua’(green pericarp)obtained previously and obtained the differentially expressed genes between’Huanghua’ and ’Green huanghua’ in different periods of fruit development.Based on the gene information of differentially expressed genes and previous reports,we analysied five gene family genes closely related to the synthesis and regulation of suberin in pear and also conducted the related bioinformatic analysis.Furthermore,we analyzed the gene function of two Arabidopsis homologous genes of pear GDSL genes by CRISPR gene editing technology and T-DNA insertion mutation technology,which laid a good foundation for the application of these candidate differentially expressed genes in pear molecular breeding and related research.The main results are as follows:1.The number of reads in transcriptome samples of ’Huanghua’ pear(russet pericarp)and its bud mutant ’Green huanghua’(green pericarp)were 57444787,and the average ratio of reads to reference genome was 74.43%.The average comparison rate to exon region was 94.14%.Through comparison with seven protein databases,35381 pear genes were annotated and 26637 genes involving 54 main biological functional classes,14186 genes involving 25 classes and 35354 genes through pathway annotation were classified.The number of differentially expressed genes related to suberin formation,cuticle and wax formation,lipid formation and cell wall modification formation were 16,14,75 and 3,respectively,and 66 genes were found to be differently expressed in the key period of peel color transformation of ’Huanghua’ and ’Green huanghua’ pear.These results provide a reference for further study on the function of genes related to suberin formation in pear peel.2.According to the results of transcriptome analysis and literature reports,five gene families,GDSL,FAR,GPAT,KCS and BAHD,which are closely related to the synthesis and regulation of suberin,were further identified at the whole genome level and the corresponding bioinformatic analysis was also conducted.One hundred and thirteen GDSL genes,7 FAR genes,22 GPAT genes,28 KCS genes and 47 BAHD genes were identified in pear genome.The phylogenetic tree analysis,gene structure,conserved motifs,chromosome distribution and expression patterns in stem,leaf,petal,sepal,ovary,bud,and fruit of these gene families were analyzed.Thirty GDSL genes co-expressed with three known suberin biosynthesis genes ASFT,GPAT5,and CYP86B1 were screened.According to the coexpression analysis results and gene tissue specific expression results,12 GDSL genes,other 2 GPAT genes and 7 KCS genes were further screened.RT-qPCR was used to analyze the expression profile of these genes between russet pericarp of ’Huanghua’ pear and bud mutant’Green huanghua’ in different stages of fruit development.The results provide an important basis for further study on the relationship between these five gene family members and pear pericarp russet trait.3.Four candidate genes AtGDSL1,AtGDSL2,AtGDSL3 and AtGDSL4,which are closely related to the suberin formation,were obtained by phylogenetic tree analysis and coexpression analysis in Arabidopsis thaliana.The mutant gdsl1 was obtained by CRISPR gene editing technology,and the double mutant gdsl1 gdsl4 was obtained by hybridization with the existing T-DNA insertion mutant gdsl4.Through phenotypic study,it was found that compared with the wild type,the seed coat permeability of mutants gdsl1,gdsl4 and gdsl1gdsl4 increased,the salt tolerance of seeds of mutants gdsl1,gdsl4 and gdsl1gdsl4 decreased,the root permeability of mutants gdsl1,gdsl4 and gdsllgdsl4 increased,it was suggested that AtGDSLl and AtGDSL4 played an important role in the formation of suberin.