Study On Cloning And Expression Of Endo-1,4-β-Glucanase In Persimmon Fruit

Cellulose and hemicelluloses are the bones of cell wall,while endo-1,4-β-glucanase（EG）can catalyse the hydrolysis of polysaccharide with1,4-β glucosidic bond, cellulose andxyloglucan e.g,which plays an important role in cell enlongation,fruit ripening,vegetationabscission and sythesis and degradation of cellulose.A full length of endo-1,4-β-glucanase gene was isolated from maturepersimmon（Diospyros kaki L.‘Fuping Jianshi’）fruit by RT-PCR and RACE（rapidamplification of cDNA ends） method,and its mRNA expression level of differenttreatments（GA₃,ABA,1-MCP at room temperature）on persimmon fruits were furtherinvestigated by the Real-time Fluorescence qPCR.The results showed that:1.The obtained EG gene,named DKEG1（GenBank accession number: HQ222561）,was2011bp in length,and encoded545amino acid,with CBD residue in the deduced EG aminoacid sequences,whose5’ end is shorter than other blusted ones and is concluded to be signalpeptide,but the deduced EG protein has high homology with others on the conrespondingstructure domain, including typical4Cys residues and2promoter sites and the deduced EGis belong to the Glycosyl hydrolase family9.2.Real-time quantitative PCR（RT-qPCR） showed that：the expression of DKEG1incontrol fruits increased to maximum at3d after harvest，then decreased rapidly and rised to asecond expression peak at12d after harvest； while in GA3treatment, it was supressedobviously through the whole ripening and softening progress,though its expression levelincreased at6d after harvest, generally it’s still lower than that of control. The DKEG1wasabundantly expressed in ABA treatment from6to18days after harvest with a expressionpeak at9d after harvest，but it didn’t show an expression peak at3d after harvest as thecontrol. The expression peak of DKEG1in1-MCP treatment was delayed9days than thecontrol, and its expression were significantly inhibited at earlier stage but increased at laterstage. Therefore，the expression of DKEG1in persimmon fruits is concluded to be regulatedby ethylene and the ethylene signal transduction，and then participates in the ripening andsoftening progress.