柿果实内切-1,4-β-葡聚糖酶基因克隆与定量表达分析

Studies on Cloning and Real-time Expression of Endo-1,4-β-glucanase Gene in Persimmon Fruit

以成熟期‘富平尖柿’(Diospyros kakiL.‘Fuping Jianshi’)为材料,采用RT-PCR和RACE相结合的方法,克隆柿果实中内切-1,4-β-葡聚糖酶基因(EG)cDNA,并在此基础上采用实时荧光PCR定量技术检测采后常温下柿果及GA3、ABA和1-MCP处理柿果软化过程中该EG基因转录水平的表达特点。试验结果:获得了一个内切-1,4-β-葡聚糖酶基因cDNA,命名为DKEG1(GenBank accession number:HQ222561),长2011bp,编码545个氨基酸,末端含有CBD区域。实时荧光PCR定量技术检测发现常温下对照组柿果采后3d时出现DKEG1基因表达高峰,下降后又逐渐上升,并在12d时出现第2个高峰;赤霉素明显抑制了柿果实在整个后熟过程中DKEG1基因的表达,尽管在6d时也有升高,但与对照相比全程表达量均极低;ABA处理大大提高了DKEG1基因的表达量,没有出现采后3d的表达高峰,但在6～18d期间其表达量均高于对照的最高峰值,其表达高峰出现采后9d;1-MCP处理的柿果表达高峰比对照推迟9d,且前期表达量显著降低,但后期又上升。据此推断柿果实DKEG1的表达受乙烯生成和乙烯信号转导的调控,进而参与果实的后熟软化过程。

A full length of endo-l,4-β-glucanase gene was isolated from mature persimmon （Diospyros kaM L. ＇Fuping Jianshi＇ ） fruit by RT-PCR and RACE （rapid amplification ofcDNA ends） method, and its mRNA expression level of different treatments （ GA3, ABA, 1-MCP at room temperature） on persimmon fruits were further investigated by the Real-time fluorescence qPCR. The results showed that the obtained EG gene, named DKEG1 （GenBank accession number： HQ222561 ）, was 2 011 bp in length, and encoded 545 amino acid, with CBD residue in the deduced EG protein. Real-time fluorescence qPCR（RT-qPCR）showed that the expression of DKEG1 in control fruits increased to maximum at 3 d after harvest, then decreased rapidly and rised to a second expression peak at 12 d after harvest; While in GA3 treatment, it was suppressed obviously through the whole ripening and softening progress. Though its expression level increased at 6 d after harvest, generally it＇s still lower than that of control. The DKEG1 was abundantly expressed in ABA treatment from 6 to 18 days after harvest with an expression peak at 9 d after harvest, but it didn＇t show an expression peak at 3 d after harvest as the control. The expression peak of DKEGI in 1-MCP treatment was delayed 9 days than the control, and its expression were significantly inhibited at earlier stage but increased at later stage. Therefore, the expression of DKEG1 in persimmon fruits is concluded to be regulated by ethylene and the ethylene signal transduction, and then participates in the rinenin~ and softening progress.