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大豆抵抗SMV长距离运输机制的研究

标题: 大豆抵抗SMV长距离运输机制的研究
英文标题: Study on the Mechanism of Soybean Resistant to SMV Long-distance Transport
作者: 姚贵滨
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 胼胝质,大豆花叶病毒,嫁接,长距离运输
英文关键词: callose,SMV,grafting technique,long distance transport
论文级别: 硕士
学位: 学位论文
导师: 王冬梅
专业: 植物学
提交时间: 2011
摘要: 大豆花叶病毒(SMV)侵染大豆植株后,通过胞间连丝进行胞间运输,通过维管束系统完成长距离运输.目前,关于病毒在胞间运输方面的研究报道较多,而在病毒的长距离运输方面,由于对维管束直接接种病毒存在一定的难度,人们对病毒长距离运输的研究还较少.本实验室前期工作已经证明,SMV接种大豆叶片后,在不亲和组合中胼胝质通过对胞间连丝颈部的修饰作用限制了病毒在胞间的转运,使病毒不能进入维管系统进行长距离运输,从而将病毒限制在叶片接种部位的很小区域内.为此我们提出,胼胝质在胞间连丝上的沉积是限制大豆花叶病毒在胞间转运的主要因素.本试验正是基于上述研究基础开展工作的,利用嫁接技术将大豆花叶病毒接种到维管束中,通过对嫁接部位的上位叶发病情况的观察、大豆花叶病毒外壳蛋白基因的检测、胼胝质专一性染料苯胺蓝染色并辅以荧光显微镜观察以及药物学试验,探讨了胼胝质在大豆抵抗大豆花叶病毒长距离运输中的作用及其机制.本试验选用大豆品种冀豆7号、冀黄13号和nf-58为材料,大豆SMV株系为SC-8和N3,其中冀豆7号和冀黄13号分别与SMV株系SC-8组成亲和组合,冀豆7号和nf-58分别与SMV株系N3组成不亲和组合和亲和组合.获得的主要试验结果如下:1.通过对砧木和接穗苗龄的选择以及嫁接后温度和湿度的控制,成功将冀黄13号和nf-58嫁接到冀豆7号.2.在接穗冀黄13号叶片上进行摩擦接种SMV株系SC-8,在接种后6天在砧木冀豆7号的茎和上位叶中能检测到SMV的CP基因,并随后在上位叶出现发病症状.表明利用嫁接技术已成功将病毒接种到砧木的维管束中,由于砧木冀豆7号与SMV株系SC-8为亲和组合,所以病毒经维管束的韧皮部运输到上位叶片中.3.在接穗nf-58叶片上进行摩擦接种SMV株系N3,在接种后6天在砧木冀豆7号的上位叶中未能检测到SMV的CP基因,上位叶也一直不表现发病症状,但是在冀豆7号的茎的下部检测到了SMV的CP基因,而茎的上部没能检测到SMV的CP基因.表明虽然利用嫁接技术成功将病毒接种到砧木的维管束中,但由于砧木冀豆7号与SMV株系N3为不亲和组合,所以病毒在通过维管束的韧皮部向上运输过程中受到了限制.4.对结果2和3中的冀豆7号茎部制作石蜡切片,通过胼胝质专一性染料苯胺蓝染色观察发现,结果3中(冀豆7号与SMV株系N3为不亲和组合)茎的下部维管束筛板处能够观察到较强的胼胝质荧光,而在其茎的上部及结果2中(冀豆7号与SMV株系SC-8为亲和组合)茎部的维管束筛板处只观察到微弱的胼胝质荧光(或者没有观察到胼胝质荧光).5.为了进一步探讨胼胝质在筛板上的沉积对限制SMV在维管束中完成长距离运输的作用,将nf-58嫁接到冀豆7号后,在nf-58叶片上预注射500μmol/L 2-DDG(即胼胝质合成抑制因子)再经摩擦接种SMV株系N3, 6天后在冀豆7号的上位叶中即能检测到SMV的CP基因.对冀豆7号的茎部制作石蜡切片利用胼胝质专一性染料苯胺蓝染色观察发现,注药处理后在维管束的筛板处观察到的胼胝质荧光较不注药对照减弱了许多(或没有观察到胼胝质荧光).综合上述试验结果,我们认为胼胝质对筛板的修饰是大豆限制SMV在维管束中进行长距离运输的主要因素.
英文摘要: After infected the plants of soybean, soybean mosaic virus ( SMV) will be transported between the cell through the plasmodesmata and complete the long distance transport via the sieve tubes of the phloem. There have been many investigations concerning the transportation through the plasmodesmata, but little known about the long distance transport because it is diffcult to let the SMV inoculate to the vascular.Preliminary work in our laboratory has showed that in the resistant varieties the modifications in the neck of the plasmodesmata restrict cell-to-cell transport through the plasmodesmata,blocking the SMV into the vascular for the long distance transport, which limited the virus to a small area within the inoculation region. So we propose that callose deposition in plasmodesmata plays an important role in SMV transport.In the present study, we has study the mode of the action of the callose in the long distance transport of the SMV,by inoculating the SMV to the vascular using grafting techniques, then observe the disease of the grafting leaf,detect the gene of the SMV coat protein(SMV-CP), fluorescence and pharmacological experiment using a specific dyeing on callose. The materials used in this study were soybean JD-7、JH-13 and nf-58. the soy bean mosaic virus were SC-8 and N3. Soybean JD-7 and JH-13 were susceptible to SMV SC-8 respectively, JD-7 was resistant to SMV N3 and nf-58. was susceptible to SMV N3. The results are as follows:1. The soybean JH-13 and nf-58 has been grafted to JD-7 sucessfully at the appropriate instar of the rootstock and the ingraftment and by controlling the temperature and humidity.2. The SMV SC-8 was inoculated to the JH-13 leaves, six days later the SMV-CP gene was detected in the stem and second leaves of JD-7 respectively, then symptom of disease appeared in the second leaves which showed that the SMV was inoculated to the vascular successfully. The SMV transport from the sieve tubes of the phloem.to the upper leaves because JD-7 are susceptible to SMV SC-8 .3. The SMV N3 was inoculate to the leaves of nf-58, six days later, the SMV-CP gene was not detected in the lobus superior of JD-7, also no symptom of disease was found,however,the SMV-CP gene was detected in the bottom of stem of JD-7, there is no CP gene in the superior part of stem. This has showed that the SMV was inoculated to the vascular successfully,but the SMV transport was blocked because the JD-7 is resistant to SMV N3.4. By staining with aniline blue,we also observed strong florescence spots due to callose formation on sieve plate in the bottom stem of JD-7 but no upper stem after SMV N3 was inoculated to the leaves of nf-58.Weak fluorescence or no fluorescence on sieve plate in the upper or bottom stem of JD-7 after SMV SC-8 was inoculated to the leaves of JH-13.5. To further examine whether the callose deposition observed in result 4 was involved in plant resistance against virus in phloem, inoculation experiments were performed by,prior to inoculation, injecting plant tissues with DDG which is a callose synthesis inhibitor. Injecting with 500μM DDG prior to inoculation found that the virus was unable to induce callose deposition on sieve plate, and the virus was able to transport via the sieve tubes of the phloem as judged by the appearance of foliar symptoms as well as by the expression of SMV-CP gene from the upper leaves.These results have showed that callose is the main factor in blocking the SMV into the vascular for the long distance transport.