The Molecular Basis Of Glycoside Hydrolase GH9 Gene Family In The Development Stages In Peel Of Ziziphus Jujube ‘Goutouzao’ Fruit

Chinese Jujube(Ziziphus jujube Mill.)is a perennial shrub or small trees belonging to Rhamnaceae.The development of jujube fruit involves the division and expansion of peel cells,which is the result of the dynamic interaction between cell wall biosynthesis and remodeled.Cellulase is a major member of the glycoside hydrolase 9(GH9)gene family and is closely related to cell wall decomposition.In order to explore the role of GH9 gene family in the development of jujube peel,we studied the cellulose content,histological structure,bioinformatics and gene expression of GH9 gene family in peel of Goutouzao at different developmental stages,including 6 developmental stages: young fruit StageⅠ,green fruit StageⅡ,enlarged StageⅢ,white ripening StageⅣ,semi-red StageⅤ,and full-red StageⅥ.The T-DNA insertion mutant with high sequence similarity between the GH9 gene of Arabidopsis thaliana and jujube was used to speculate the function of the GH9 gene of jujube.A cellulase gene Zj GH9C3 with the highest expression level was screened from the members of the GH9 gene family,and the function of this gene was studied.The main results obtained were as follows:(1)The transcriptome sequencing analysis of jujube ‘Goutouzao’ at six stages revealed that a total of 18 878 genes were detected,of which 7015 DEGs genes were up-regulated and 11 863 DEGs genes were down-regulated.During the early stage of jujube development(StageⅠ～ StageⅣ),KEGG enrichment showed that DEGs were significantly enriched in metabolic pathways such as phenylalanine metabolism and flavonoid biosynthesis;GO enrichment showed that DEGs were significantly enriched in molecular functions closely related to hydrolase activity.Thirty members of the GH9 gene family were identified from the jujube genome,which were divided into three subfamilies GH9 A,GH9B and GH9 C according to the phylogenetic relationship.The protein structure domain includes Glyco＿hydro＿9 superfamily,CBM＿2 superfamilies,among which 3 genes belong to CBM＿2 domains,which have the function of binding cellulose.The analysis of gene expression patterns showed that 17 of the 30 identified GH9 genes were expressed in StageⅠ～StageⅢ of peel development with high expression level,while in StageⅣ～StageⅥ expression level was decreasing;Real-time quantitative PCR assay showed that the expression levels of 8 differentially expressed genes were decreased gradually and rapidly with the development of jujube fruit,and the change trend was consistent with the expression level of transcriptome sequencing results.(2)During the development of jujube fruit,the cellulose content of peel showed a downward trend.It reached the highest at StageⅢ(217.33 mg/g),and then decreased rapidly and significantly to the lowest at StageⅥ(98.51 mg/g).The cellulase activity of jujube peel showed a significant upward trend,and reached the highest at StageⅥ(200.24 U/g).Correlation analysis of cellulose content and cellulase activity and GH9 gene family member expression in peel of jujube at different developmental stages showed that cellulose content in peel of jujube was significantly negatively correlated with cellulase activity,and GH9 gene family member expression was significantly positively correlated with cellulose content,but significantly negatively correlated with cellulase activity(p<0.05).Paraffin sections were used to observe the cell wall structure in peel of jujube at different developmental stages.After safranin fast green staining,the color gradually changed from blue-green to light blue and then to pink,and the cell structure was gradually loosely arranged.The results showed that the cellulose content of the cell wall in peel of jujube ‘Goutouzao’ gradually decreased during the development process,and the change trend was basically consistent with the q RT-PCR results of the 8 candidate genes and the expression level of the differential genes determined by transcriptome sequencing.(3)Select Arabidopsis GH9 T-DNA mutants with high sequence similarity to jujube GH9 genes.Phenotypic analysis of the At GH9 mutants showed that its number of rosette leaves,root length,plant height,pod length,and seed setting were significantly higher than those of the wild-type.The cellulose content in the pods of the At GH9 mutants was significantly reduced compared to the wild-type,while the cellulase activity in the pods was significantly increased compared to the wild-type(p<0.05),and the expression level of the At GH9 gene in the wild-type pods was low or even non-expressed.This indicates that the At GH9 genes mutation can affect the synthesis of cellulose in the cell wall of Arabidopsis pod.(4)Construct an overexpression vector of Zj GH9C3 and transform it into Arabidopsis.The overexpressed plant of p Zj GH9C3 gene was detected at relative m RNA expression and protein expression levels,that p Zj GH9C3 expressed highest in the pod.Expression of p Zj GH9C3 gene affects the phenotype of pod development in transgenic Arabidopsis.Analysis of pod cellulose content and cellulase activity showed that the pod cellulose content was significantly lower than the wild-type,while pod cellulase activity was significantly higher than the wild-type.This indicates that p Zj GH9C3 catalyzes the degradation of cellulose in the cell wall of pods and affects their growth and development.In conclusion,the research shows that GH9 gene family reduces the content of peel cellulose during jujube fruit development and participates in cell differentiation during peel development.