摘要
苹果茎沟病毒(Apple stem grooving virus，ASGV)和苹果褪绿叶斑病毒(Apple
chlorotic leaf spot virus，ACLSV)是梨树上两种重要病毒，广泛分布于世界各梨、苹果种植区。在梨树上这两种病毒通常不产生明显的症状，但影响树势和降低产量。栽培无病毒种苗是减轻这些病毒危害的重要途径，热处理和化学处理是获得无病毒果树种质的重要途径。为探索适合这两种病毒的脱病毒处理条件，建立更加有效的脱病毒技术，本研究以砂梨离体植株为研究体系，对热处理和化学处理进程中ASGV和ACLSV在离体植株及其茎尖的分布动态变化进行了研究，取得如下进展。
1．采用酶联免疫吸附法(ELISA)和RT-PCR技术相结合，对砂梨‘黄花’的20个芽系的ASGV和ACLSV，筛选出带有这两种病毒的芽系共13个，通过在MS培养基上扩繁，建立了用于本研究的离体培养体系。
2．采用组织免疫印迹技术对ASGV和ACLSV在‘黄花’离体植株茎干各部位分布特点进行了分析，通过改进病毒抗体吸附方法和延长洗涤时间有效降低了非特异反应，根据最终产生紫色反应可以判断病毒存在部位。采用该方法对植株纵切面印迹分析的结果表明，ASGV和ACLSV在‘黄花’离体植株茎干的顶端和基部含量均很高，茎干中部也有病毒分布，但反应颜色相对较浅。
3．合成了这两种病毒的特异性生物素标记探针，建立了病毒斑点杂交和组织印迹原位杂交技术。采用组织印迹原位杂交技术对‘黄化’离体植株茎干纵切面和各部位的横切面印迹进行了病毒检测，其结果与组织免疫印迹相似，但特异性更强，杂交后免疫检测产生的紫色反应更明显。
4．采用恒温(37℃)和变温(32℃／39℃，8h／16h交替)热处理技术对‘黄化’离体植株进行了脱病毒处理，采用以上建立的免疫印迹和组织印迹原位杂交技术对处理不同时间进程中植株茎干病毒浓度变化进行了分析。结果显示，在热处理过程中植株茎干病毒含量随处理时间的延长而逐渐降低，且茎干顶端病毒浓度的变化较基部更大，恒温处理至35d或变温处理至50d后，茎尖基本无病毒产生的特异性紫色反应，而这种紫色反应在基部仍清晰可见。在处理不同时期，取1～5mm茎尖(37℃)或0.5～5mm(32℃／39℃)茎尖进行了再培养，采用ELISA对再生植株病毒检测的结果显示，所取茎尖越小或处理时间愈长，ELISA检测吸光值愈低，表明随着处理时间的延长，病毒含量自茎尖向基部逐渐降低，这种变化趋势与组织印迹观察到的现象一致。ELISA和斑点杂交的结果表明，恒温处理35d后取1mm茎尖或变温处理45d后取0.5mm茎尖可以脱除ASGV和ACLSV。
5．比较热处理再生植株的ELISA和斑点杂交检测结果显示，对未处理植株采用ELISA法可以有效检测这两种病毒，而经热处理后，植株体内病毒含量降低，此时需采用更灵敏的斑点杂交技术以确保获得无病毒种质。
Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are two important viruses in pear, which are worldwide distributed. In commercially cultivated pear, these two viruses usually do not cause visible symptoms, but they decrease the growth and productivity of infected trees. One of most effective measures for the controlling of these virus diseases is utilizing certified healthy propagation materials. Thermotherapy and chemotherapy are the most widely used measures for the production of virus-free plant materials. In these studies, for obtaining higher virus elimination efficiency, the virus distribution features in in vitro pear plants during thermotherapy and chemotherapy were analyzed in this study. Seeking results are listed as following.
1. 20 in vitro plant clones of Pyrus pyrifolia cv.'Huanghua'were detected by ELISA and RT-PCR for ASGV and ACLSV. Results showed that 13 clones were infected by both ASGV and ACLSV. These clones were propagated on MS medium for the following studies.
2. Tissue printing immunoassy was used for the detection of ASGV and ACLSV in different parts of stems of in vitro pear plants. Non-specific reaction was decreased by improving the absorbance protocols of polyclonal antibodies against these two viruses, prolonging the periods of washing steps. The distribution of viruses in vertical sections of plants could be easily observed based on the present of purples products. Results showed that both ASGV and ACLSV had a high concentration in the base and tip of these plants, and the intermediate section also had virus distribution with a lower amount.
3. Biotinylated cDNA probes specific to ASGV and ACLSV were prepared from cloned fragments. The dot-blot hybridization and tissue-printing hybridization methods were studies for the detection of these viruses in plant crude extracts and tissues, respectively. These viruses in both vertical and horizontal sections of the stem of in vitro plants were detected by in situ tissue-printing hybridization. Similar results were achieved as tissue printing immunoassy, but with a more intensive reaction-signal.
4. In vitro plant clones of Pyrus pyrifolia cv. 'Huanghua' were treated under 37℃ and 32℃/39℃ (8h /16h alteration) respectively. Changes of virus titers in stems of treated plants during thermotherapy were analyzed by tissue printing immunoassy and in situ tissue-printing hybridization established formerly. Results indicated that virus titers showed a decreasing tendency while the treatment periods were prolonged, and the tendency was more obvious in shoot tips than bases. When plants were treated for 35 days under 37℃ or 50 days under 32℃/39℃, no purple signal was observed in shoot tips,
引文
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