Cloning And Functional Analysis Of Xyloglucan Endotransglycosylase/hydrolase In Kiwifruit Upon Botryosphaeria Dothidea Infection

Fruit ripe rot of kiwifruit caused by Botryosphaeria dothidea is an important fungal disease of kiwifruit from the near-mature stage to the storage stage.Previously,we obtained some varieties such as “Jinyan” and “Yunhai No.1” that were resistant to fruit rot through resistance selection.In order to clarify the disease resistance mechanism and identify disease-resistance genes of these varieties,the resistant variety “Jinyan” and susceptible variety “Hongyang” were inoculated with Botryosphaeria dothidea,and the RNA-seq technique was used to perform transcriptome sequencing of the treated samples.Through analysis and comparison,significant differentially expressed genes associated with fruit ripe rot resistance were screened out.On this basis,the genes were cloned,and the structure of gene products were analysed and expression level of genes were detected to speculate the function of the gene.The results were as following:1.Differential expression analysis of XTH genes.Using the data obtained from RNA-Seq technology,differential expression analysis of XTH gene was performed on the "Jinyan" kiwifruit and "Hongyang" kiwifruit during the harvest period.The results showed that there were 26 XTH genes differentially-expressed in "Hongyang",of which 13 genes were up-regulated and 13 genes were down-regulated.There were 24 differentially-expressed genes in "Jinyan",including 24 up-regulated genes and 0 down-regulated genes.The XTH genes were differentially expressed in varying degrees in this two kiwifruit varieties,and the differentially-expressed XTH gene species were only partially identical.Based on these differences,the three genes XTH5,XTH7 and XTH14 were finally screened for further study.The study of the differentially-expressed genes was helpful to elucidate the differences in disease resistance of "Jinyan" and "Hongyang" on fruit ripe rot.2.Sequence cloning of XTH genes.The full-length ORF sequences of these three genes XTH5,XTH7 and XTH14 were cloned from disease-resistant cultivar “Jinyan” by RT-PCR.The sequences were more than 90% homologous to the corresponding sequence from the Actinidia deliciosa in GenBank.The highest homology corresponding to that of Actinidia deliciosa is 99%.3.Bioinformatics analysis of XTH genes.A phylogenetic tree was established and the physicochemical properties of the amino acid sequence were analyzed.The prediction of hydrophobic regions/hydrophilic regions,the analysis of gene signal peptides and transmembrane structures and the prediction of secondary and tertiary structure of proteins for the three genes XTH5,XTH7 and XTH14 were performed.According to previous studies,it can be inferred that all three genes exert XTH activity,but it is worth noting that XTH14 may have different functions with the other two genes because of different length of Loop 2.This preliminary understanding of the basic function of these three genes andthe physicochemical properties of the protein can lay the foundation for future protein research.4.Expression level analysis of XTH genes.Real-time fluorescence quantitative PCR(qPCR)analysis of the XTH5,XTH7 and XTH14 genes showed that the expression of XTH5 and XTH7 genes in the resistant variety "Jinyan" was higher than that of susceptible variety “Hongyang ",while the expression of XTH14 gene in susceptible variety “Hongyang" was higher than the resistant variety "Jinyan",especially on the 6th day.After previous analysis,the functions of these XTH genes were related to fruit softening.XTH has the basic function of maintaining the structural integrity of the cell wall during fruit growth and maturation.The differential expression of these XTH genes produced during the process of fungal infection of the susceptible variety “Hongyang” will make its fruit softer than the resistant variety “Jinyan” and eventually the harm caused by fungus to “Hongyang” is more serious.In summary,the XTH gene has very close relationship with the severity of the fruit ripe rot of kiwifruit.The type of differential expression and the level of expression are important factors affecting the resistance of of kiwifruit to fruit ripe rot.