利用原位杂交及原位RT-PCR技术检测梨树组织中苹果茎痘病毒的分布

A Study of the Distribution of Apple Stem Pitting Virus in Tissues of Pear Tree Using in situ Hybridization and in situ RT-PCR

【目的】搞清苹果茎痘病毒在梨树组织中的分布,为茎尖脱毒提供直接的理论依据和技术支撑。【方法】以库尔勒香梨茎尖、叶片为材料,利用非放射性标记物地高辛(DIG),通过RT-PCR反应体系,合成了苹果茎痘病毒的cDNA探针,利用核酸探针斑点杂交检测方法对该探针特异性及灵敏度进行验证。研究制作了适合原位PCR及原位杂交的石蜡切片,利用原位反转录酶聚合酶链式反应(IS-RT-PCR)技术检测石蜡切片组织中苹果茎痘病毒RNA的定位及分布,并对影响实验结果的重要因素进行优化。【结果】梨树中的苹果茎痘病毒RNA阳性信号主要位于叶片的叶肉细胞、茎尖的外围皮层组织及相应的初生维管束。蛋白酶K消化时间以20 min为宜,要获得良好的扩增效果,RT反应体系中RNasin的量需大于0.2 U.μl-1,dNTPs大于0.4 mmol.L-1,SuperScriptⅡ在0.1～1.3 U.μl-1,引物在0.9μmol.L-1以上。PCR反应体系中适宜的退火温度为60℃,循环35次,引物浓度应在0.8～1.2μmol.L-1,LA Taq酶浓度大于0.5 U.100.μl-1。【结论】梨树茎尖顶端分生组织0.25 mm的区域为无病毒区域。

【Objective】To understand the distribution of ASPV in tissues of pear tree and provide direct theoretical and technical support for viruses-elimination of shoot tips.【Method】With shoot pits and leaves of Korla pear trees,labeled by non-radioactivity Digoxigenin（DIG）,cDNA probe for ASPV was synthesized through RT-PCR reaction system,and the characteristics and sensitivity of probe was verified using blot hybridization.Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA was detected in paraffin slices using inverse transcriptase RNA polymerase reaction（IS-RT-PCR）,and the factors affecting experimental results were optimized.【Result】Positive signals of ASPV were mainly distributed in mesophyll cells of leaves,outside cortex and corresponding primary vascular bundles of shoot tips.Twenty minutes was the suitable digestive time for proteinase K.For the better amplification,the amount in RT reaction system should be above 0.2 U.μl^-1 for RNasin,above 0.4 mmol.L^-1 for dNTPs,0.1 to 1.3 U.μl^-1 for SuperScriptⅡ,and beyond 0.9 μmol.L^-1 for primer.The suitable annealing temperature in PCR reaction was 60 ℃,with 35 times cycles,primer concentration 0.8 to 1.2 μmol.L^-1,and LA Taq over 0.5 U.100 μl^-1.【Conclusion】Shoot tip within 0.25 mm of pear trees was virus-free area.