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为了了解辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)在辣椒组织中的分布特点,探索利用间接原位RT-PCR技术对病毒在辣椒组织中进行定位。利用改进的CTAB法从辣椒叶片中提取总RNA,利用RT-PCR技术扩增出PMMoV的特异片段,并经克隆测序,证明其为PMMoV的特异片段。利用PCR技术,制备出了地高辛标记的特异cDNA探针。对原位RT-PCR反应体系进行优化,经过切片制备(切片厚度7μm、应用Superfrost plus正电荷防脱载玻片)、预处理(1 mg·L~(-1)蛋白酶K消化5 min)、RT-PCR(37℃反转录2.5h、PCR退火温度为58℃)、杂交检测,建立了应用间接原位RT-PCR技术检测PMMoV在辣椒组织中分布的方法。原位RT-PCR结果显示,叶片组织中PMMoV阳性信号主要分布于栅栏组织,其次是海绵组织,表皮组织中也有少量阳性信号。此外,叶柄中可见少量阳性信号,主要位于表皮组织。
In order to understand the distribution characteristics of pepper mottle virus (PMMoV) in pepper tissues, we explored the use of indirect in situ RT-PCR to locate the virus in pepper tissues. Total RNA was extracted from pepper leaves by improved CTAB method. The specific fragment of PMMoV was amplified by RT-PCR and cloned and sequenced to identify PMMoV-specific fragments. Digoxigenin labeled specific cDNA probes were prepared by PCR. The in situ RT-PCR reaction system was optimized and prepared by sectioning (slice thickness 7μm, applying Superfrost plus positive charge anti-shed glass slides) and pretreatment (1 mg · L -1 proteinase K digestion for 5 min) RT-PCR (reverse transcription at 37 ℃ for 2.5h, annealing temperature at 58 ℃) was used to detect the distribution of PMMoV in pepper tissue by indirect in situ RT-PCR. In situ RT-PCR results showed that PMMoV positive signals in leaf tissue were mainly distributed in palisade tissue, followed by sponge tissue and a small amount of positive signal in epidermal tissue. In addition, a small amount of positive signals were visible in the petiole, mainly in the epidermis.