Cloning And Expression Analysis Of Xyloglucan Endotransglycosylase/hydrolase In Persimmon Fruit

Persimmon (Diospyros kaki L.) fruits naturally soften easily after harvesting. Xyloglucanis the predominant hemicellulose in the cell wall, limiting the expand of cell wall. In order toexplore the molecular regulation mechanism of xyloglucan endotransglycosylase/hydrolase(XTH) in persimmon fruit, persimmon (Diospyros kaki L.cv. Fuping Jianshi) fruit was used asmaterial in this research. The total RNA from different stages of persimmon fruit wereextracted, four full-length XTH cDNA were obtained by RT-PCR and RACE. Delay in fruitsoftening in relation to the expression of XTH genes was investigated after treatments withpropylene, GA3and1-MCP. The results are as follows.1. Using the total RNA from mature stage and colour-changed stage of persimmon fruit asthe template, four full-length XTH cDNA were cloned by RT-PCR and RACE. DkXTH1(1082bp, GenBank accession number JN605344) and DkXTH2(1243bp, GenBank accessionnumber JN605345) from mature stage, DkXTH3(1244bp, GenBank accession number JN605346) and DkXTH4(1059bp, GenBank accession number JN605347) from colour-changedstage. The result of Blastn Alignment in the NCBI showed that the sequences of DkXTHs hadhigh identities with XTH genes of other species on the nucleotide level. All four deducedpolypeptides contained the conserved DEIDFEFLG motif of XTH, a potential N-linkedglycosylation signal (N-X-S/T) site after the motif and two pairs of cysteines were present inthe carboxyl-terminal region.2. The real-time quantitative PCR analysis to the XTH genes indicated that accompanyingwith fruit ripening four XTH genes showed different expression patterns. The expressionlevels of DkXTH1and DkXTH2were high in control fruit and propylene-treated fruit. Theexpression levels of DkXTH3and DkXTH2were low during fruit softening. The expression ofXTH genes was related to persimmon fruit softening. The peaks of DkXTH1and DkXTH2were appeared in the early stage and middle stage, respectively; DkXTH1might involve in theearly stage while DkXTH2was associated with the later stage of fruit softening.3. In this study, treatment with propylene promoted the softening of persimmon fruit, increased the ethylene production, advanced the onset of climatic of ethylene production andpromoted the expression of XTH genes. While treatments with GA3and1-MCP delayed thesoftening of persimmon fruit, inhibited ethylene production and suppressed the expression offour XTH genes. Therefore, the expression of XTH genes in persimmon fruit was concluded tobe regulated by ethylene.