链格孢菌侵染采后甜瓜细胞壁降解酶基因表达及活性变化分析

Gene Expression and Activity Change Analysis of Cell Wall Degrading Enzyme in Postharvest MelonInfected by Alternaria alternata

目的:探讨链格孢菌侵染采后甜瓜果实过程中细胞壁降解酶活性及基因表达的变化规律,揭示链格孢菌的致病机理。方法:以伽师瓜和86-1甜瓜果实为实验材料,损伤接种链格孢菌(Alternaria alternata),室温(25℃),相对湿度35%~40%贮藏,测定病斑直径,观察病斑组织显微结构,测定果胶裂解酶(Pectin lyase,PL)、纤维素酶(Cellulase,Cx)、β-葡萄糖苷酶(β-Glucosidase,CB)、多聚半乳糖醛酸酶(Polygalacturonase,PG),采用荧光定量聚合酶链式反应(Polymerase chain reaction,PCR)的绝对定量方法对AaPL、AaCx、AaPG、AaCB表达量的变化进行分析,并对四种细胞壁降解酶的活性与其对应基因的表达量进行相关性分析。结果:在相同的侵染天数下,在86-1甜瓜果肉、果皮组织的病斑直径大于伽师瓜;观察病斑组织显微结构,对比两种甜瓜,链格孢菌在86-1甜瓜细胞中繁殖更快,对86-1甜瓜细胞结构的破坏能力更强;比较四种细胞壁降解酶在两种甜瓜上活性和基因表达量的变化规律,相同天数下总体来说在86-1甜瓜接种组中的酶活性和基因表达量更高,在伽师瓜接种组中活性、表达量达到峰值的天数更晚。结论:在侵染过程中由于甜瓜自身抗病机制,使细胞壁降解酶活性及基因表达的变化趋势在6~10 d有所波动;链格孢菌在86-1甜瓜中的侵染能力更强,也体现了伽师瓜对链格孢菌侵染的抵抗能力更强。

Objective:To investigate the changes of cell wall degrading enzyme activity and gene expression during the infection of postharvest melon fruit by Alternaria alternata,and to reveal the pathogenic mechanism of Alternaria alternata.Methods:Jiashi melon and 86-1 melon fruits were used as experimental materials.Alternaria alternata was inoculated and stored at room temperature(25℃)and relative humidity of 35%~40%.The diameter of lesions was measured,and the microstructure of lesions was observed.Pectin lyase(PL),cellulase(Cx),β-glucosidase(CB)and polygalacturonase(PG)were determined.The expression changes of AaPL,AaCx,AaPG and AaCB were analyzed by absolute quantitative method of fluorescence quantitative polymerase chain reaction(PCR),and the correlation between the activity of four cell wall degrading enzymes and the expression of their corresponding genes was analyzed.Results:Under the same infection days,the lesion diameter of 86-1 melon pulp and peel tissue was larger than that of Jiashi melon.The microstructure of the lesion tissue was observed.Compared with the two melons,A.alternata propagated faster in 86-1 melon cells and had a stronger ability to destroy the cell structure of 86-1 melon.The changes of activity and gene expression of four cell wall degrading enzymes on two melons were compared.Under the same number of days,the enzyme activity and gene expression were higher in the 86-1 melon inoculation group,and the number of days when the activity and expression reached the peak in the Jiashi melon inoculation group was later.Conclusion:During the infection process,due to the melon's own disease resistance mechanism,the change trend of cell wall degrading enzyme activity and gene expression fluctuated from 6 to 10 days.The infection ability of Alternaria alternata in 86-1 melon was stronger,which also reflected that Jiashi melon had stronger resistance to Alternaria alternata infection.