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以苹果感病品种‘红星’和抗病品种‘红玉’为材料,研究了苹果与斑点落叶病菌(Alternaria alternata apple pathotype)互作过程中超微结构,细胞Ca~(2+)分布,以及在钙信号转导途径中具有重要作用的钙依赖蛋白激酶基因(CDPK)的表达,探讨钙信号在苹果防御斑点落叶病菌侵染中的作用。结果表明,在未接种状态下,叶肉细胞结构完好,叶绿体呈卵圆形沿细胞边缘排列,Ca~(2+)主要分布在细胞间隙和液泡中,‘红玉’细胞间隙Ca~(2+)密度比‘红星’大。接种斑点落叶病菌8h,‘红星’叶肉细胞中的Ca~(2+)沉淀主要分布在胞质中,而液泡等细胞器中减少;‘红玉’的Ca~(2+)沉淀主要集中在胞质和筛管分子中,液泡和细胞间隙中减少,且趋向于在细胞壁外围和液泡膜上沉积。接种18 h,‘红星’叶肉细胞间隙Ca~(2+)沉淀密度增加,而‘红玉’中的Ca~(2+)沉淀主要集中在叶肉细胞的胞质和液泡,以及筛管分子中。接种24 h,‘红星’叶肉细胞结构已发生形变,质膜发生裂解,筛管壁木质化加厚,Ca~(2+)沉淀主要分布在液泡中;‘红玉’叶片细胞结构完好,Ca~(2+)沉淀主要分布在液泡和胞质中。接种36 h,‘红星’叶肉细胞受到菌丝入侵,结构和形态遭到破坏,在未受损叶肉细胞中Ca~(2+)沉淀主要集中在液泡中,在受损的细胞中Ca~(2+)沉淀无序地散布在受损细胞周围及细胞间隙;此时‘红玉’叶肉细胞中Ca~(2+)沉淀主要集中在胞质和液泡中,并且能够保持Ca~(2+)动态平衡。在接种后不同阶段,‘红星’和‘红玉’叶片中MdCPKs基因呈现不同的表达特点:大多MdCPKs在‘红玉’中的表达量在24 h达到最高值;‘红星’中在36 h达到表达峰值,且表达量也比‘红玉’中低得多。上述结果表明,钙信号响应斑点落叶病菌侵染,在抗病苹果品种‘红玉’中,Ca~(2+)内流是细胞质Ca~(2+)上升的主要来源;在感病品种‘红星’中,细胞器Ca~(2+)释放是细胞质Ca~(2+)的主要来源。‘红玉’苹果MdCPKs基因响应病菌侵染比‘红星’苹果早而且强烈。
The ultrastructure and Ca ~ (2+) distribution of apple during the interaction between apple and Alternaria alternata apple pathotype were studied using ’Red Star’, an apple susceptible cultivar ’Red Star’ The expression of calcium-dependent protein kinase gene (CDPK), which plays an important role in the signal transduction pathway of calcium, explores the role of calcium signaling in the defense of apple against Spot Blight. The results showed that under uninoculated condition, the mesophyll cell structure was intact, and the chloroplast was oval and arranged along the cell edge. Ca 2+ mainly distributed in the intercellular space and vacuole. Density is greater than ’Red Star’. Inoculation of S. difficile 8h, the Ca ~ (2+) precipitation in ’Red Star’ mesophyll cells mainly distributed in the cytoplasm, while vacuoles and other organelles decreased; Ca ~ (2+) precipitation in ’Hongyu’ mainly concentrated in the cytoplasm In both the vacuole and the intercellular space, in the plasmid and scaffold molecules, they tend to deposit on the periphery of the cell wall and on the tonoplast. After 18 h of inoculation, the density of Ca2 + in interstitial cells of ’Red Star’ increased, while the Ca2 + in ’Red Jade’ mainly concentrated in the cytoplasm and vacuole of mesophyll cells, . At 24 h after inoculation, the cell structure of ’Red Star’ mesophyll was deformed, the plasma membrane was lysed, and the lignification of the screen wall was thickened. The Ca2 + precipitation was mainly distributed in the vacuole. The cell structure of ’Hongyu’ ~ (2+) precipitation is mainly distributed in the vacuole and cytoplasm. After 36 h of inoculation, the mesophyll cells of ’Red Star’ invaded by mycelium, the structure and morphology were destroyed. The Ca2 + precipitates in the uninjured mesophyll cells were mainly concentrated in the vacuole, 2 +) precipitates disorderly spread around the damaged cells and interstitial cells. At this time, Ca2 + precipitation in ’red’ leaf mesophyll cells mainly concentrated in the cytoplasm and vacuole, and Ca2 + )dynamic equilibrium. At different stages after inoculation, the expression of MdCPKs in the leaves of ’Red Star’ and ’Red Globe’ showed different expression characteristics: the expression level of MdCPKs in ’Red Globe’ reached its maximum at 24 h, reached at 36 h in ’Red Star’ Expression peak, and the expression of the amount is also much lower than ’red jade’. The above results showed that Ca2 + influx was the main source of Ca2 + in cytoplasm in the disease-resistant apple cultivar ’Hongyuanyu’. In the susceptible variety ’ Red Star ’, the release of organelle Ca ~ (2+) is the main source of cytoplasmic Ca ~ (2+). ’Red jade’ apple MdCPKs gene response to bacteria infection than ’Red Star’ apple early and strong.