【作者】 李文龙；

【导师】 王冬梅；

【作者基本信息】 河北农业大学 ， 植物学， 2012， 博士

【摘要】 本实验室前期工作已经证明,在大豆-大豆花叶病毒(Soybean mosaic virus,SMV)组成的不亲和组合中胼胝质在胞间连丝上的沉积是阻碍病毒进行胞间转运的关键。如在不亲和组合中预注射2-脱氧葡萄糖(胼胝质合成抑制剂)后再接种病毒,在病毒侵染部位仍能观察到坏死斑但胼胝质消失,且上位叶片也能观察到坏死斑出现并能够检测到病毒外壳蛋白基因。为了进一步探究在该互作体系中胼胝质积累的分子调节机制,我们以冀豆7号分别与SMV株系N3和SC-8组成不亲和和亲和组合,通过免疫组化检测、实时定量PCR、Western Blotting检测、酶活性测定和免疫金标记技术分别在转录水平、翻译水平、酶活性和原位水平对调控胼胝质积累的两种酶即β-1,3-葡聚糖酶(β-1,3-glucanase, BG)和胼胝质合酶(callose synthase, CalS)进行了系统研究。主要结果如下:1、利用免疫组化技术检测了大豆与SMV互作过程中BG和CalS定位。试验结果表明:坏死斑出现前,接种叶片上能够标记上SMV的细胞也能标记上BG和CalS;坏死斑出现后,BG和CalS主要标记在坏死斑内部及周围。2、通过实时定量PCR技术,研究了大豆与SMV互作过程中BG和CalS在转录水平的变化。试验结果表明:BG基因在亲和组合和不亲和组合中在转录水平都有上调表达,并且在不亲和组合中上调表达的时间早于亲和组合;CalS基因在不亲和组合中接种病毒后48 h就开始上升表达而在亲和组合中的表达量一直比较低。3、利用Western Blotting技术检测了大豆与SMV互作过程中BG和CalS在翻译水平的变化。试验结果表明:BG抗体能够检测出3种BG亚型,其中28 kDa和37 kDa的两种亚型在不亲和组合、亲和组合和模拟接种对照中随接种后时间的延长均没有明显变化,33 kDa的亚型在不亲和组合和亲和组合中的表达量随接种时间的延长都逐渐升高,且在不亲和组合中的上调比亲和组合提前了24 h,但在模拟接种对照中没有表达;CalS在不亲和组合中接种病毒后48 h就开始表达并且表达量逐渐升高,而在亲和组合中接种病毒后96 h才开始表达并且表达量一直比较低。4、对大豆与SMV互作过程中BG活性和CalS活性的测定结果表明:不亲和组合和亲和组合中BG活性都比较高并且在不亲和组合中酶活升高的更快;CalS在不亲和组合中酶活性比较高,而在亲和组合中酶活性一直比较低。5、利用免疫金标记技术对BG和CalS在大豆与SMV互作过程中的时空表达特征进行了系统研究。试验结果表明:BG在不亲和组合中主要定位在液泡中,而在亲和组合中主要定位在细胞壁和胞间连丝上;CalS在不亲和组合和亲和组合中都定位在细胞壁和胞间连丝上。综合以上结果,我们认为在大豆与SMV互作过程中胼胝质积累的基本调控模式为:不亲和组合中,BG被高度诱导表达但被限制在液泡中不能运输到细胞壁及胞间连丝周围从而不能够水解胼胝质;CalS在细胞壁及胞间连丝周围高度诱导表达促进胞间连丝上胼胝质的合成。因此,不亲和组合中胞间连丝上积累大量胼胝质导致病毒不能够进行细胞间运输。亲和组合中,BG被高度诱导表达并且能够运输到细胞壁及胞间连丝周围促进了胞间连丝处胼胝质的水解;而CalS在细胞壁及胞间连丝周围的表达量较低导致胼胝质合成少。因此,亲和组合中胞间连丝上没有胼胝质积累从而使病毒能够进行细胞间运输。更多还原

【Abstract】 Our previous work has proven that callose deposition at plasmodesmata is a critical factor in restricting the cell-to-cell movement of Soybean mosaic virus. We also found that in the incompatible combination the fluorescence spots due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-D-glucose (DDG, an inhibitor of callose synthesis) and necrosis was observed and the expression of virus coat protein gene was detected in the upper leaf. These results showed that, during the defensive response of soybean to viral infection, callose deposition at Plasmodesmata (PD) was mainly responsible for restricting the movement of the virus between cells. In the present work, the regulation of individualβ-1,3-glucanase (BG) and callose synthase (CalS) were analyzed by immunohistochemistry, real-time RT-PCR, Western Blotting, activity determination and transmission electron microscopic immunolocalization in the incompatible combination between the Soybean mosaic virus (SMV) strain N3 and the soybean cv. Jidou 7 and in the compatible combination between the SMV strain SC-8 and the soybean cv. Jidou 7. The main results are as follows:1. CalS and BG were examined using immunohistochemistry in combination between the SMV and soybean. Results revealed that CalS and BG were located in the virus infected cells before necrotic spots appeared; CalS and BG were located in and around the necrotic spots after necrotic spots appeared.2. Changes in steady state levels of transcripts from CalS and BG were examined using real-time RT-PCR. Real-time RT-PCR analysis revealed that BG was upregulated in both incompatible combination and compatible combination; CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination.3. Changes in steady state levels of posttranslation from CalS and BG were examined using Western Blotting. Western Blotting revealed that BG was upregulated in both incompatible combination and compatible combination; CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination.4. Changes in steady state levels of activity from CalS and BG were examined. Our results showed that the activity of BG was upregulated in both incompatible combination and compatible combination; the activity of CalS was upregulated in the incompatible combination and was not upregulated in the compatible combination. 5. To directly test whether these enzymes target to PD, immunolocalization experiments were conducted with antibody raised against BG and CalS. Our results show that BG targeted to PD in the compatible combination and targeted to vacuole in the incompatible combination. CalS targeted to PD in both incompatible combination and compatible combination.Taken together, we get a basic model for mechanisms of callose turnover at plasmodesmata in SMV-soybean interaction. In the incompatible combination, BG was upregulated and it targeted to vacuole; CalS was upregulated and it targeted to PD. So there was lots of callose, which was responsible for restricting the movement of the virus between cells, deposited at PD in the incompatible combination. In the compatible combination, BG was upregulated and it targeted to PD; CalS was not upregulated and it targeted to PD. So there was no callose deposited at PD and viruses could transport between cells in the compatible combination.更多还原