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园艺学报 ›› 2015, Vol. 42 ›› Issue (3): 489-495.doi: 10.16420/j.issn.0513-353x.2014-0791

• 蔬菜 • 上一篇    下一篇

间接原位RT-PCR法检测辣椒叶组织中辣椒轻斑驳病毒的分布

杨洪一,郭世辉,李丽丽   

  1. 1东北林业大学生命科学学院,哈尔滨 150040;2黑龙江省林业科学研究所,哈尔滨 150081
  • 出版日期:2015-03-25 发布日期:2015-03-25
  • 基金资助:

    中央高校基本科研业务费项目(DL13EA06-3)

Analysis on the Distribution of Pepper mild mottle virus in Leaf Tissues of Peppers with Indirect in situ RT-PCR

YANG Hong-yi1,GUO Shi-hui1,and LI Li-li2,*   

  1. 1College of Life Sciences,Northeast Forestry University,Harbin 150040,China;2Institute of Forestry Science of Heilongjiang Province,Harbin 150081,China
  • Online:2015-03-25 Published:2015-03-25

摘要: 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7 μm、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2.5 h、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。

关键词: 辣椒, 辣椒轻斑驳病毒, 原位RT-PCR, 地高辛, 组织定位, cDNA

Abstract: The aim of this study was to analyze the characterization of distribution of Pepper mild mottle virus(PMMoV)in tissue of peppers,and an in situ RT-PCR system was developed to locate PMMoV in tissue of peppers. Total RNA was extracted from pepper leaves with a modified CTAB method. The specific fragment was amplified by RT-PCR using total RNA as a temple,and it was testified that the fragment was PMMoV-specific one on the basis of cloning and sequencing. A specific digoxin-labeled cDNA probe of PMMoV was generated according to PCR labeling technique. A system for localization of PMMoV in tissue level was developed with indirect in situ RT-PCR,via optimization of in situ RT-PCR including section preparation(7 μm sections in the Superfrost plus positively charged slides),pre-treating(1 mg ? L-1 protease K digested for 5 min),RT-PCR(performing 2.5 h in 37 ℃ for reverse transcriptionand annealing in 58 ℃ for PCR),and detection of hybridization. The technique,which can analyze the distribution of PMMoV in tissue of peppers,was developed. The result of in situ RT-PCR showed that PMMoV-positive signals were the best in palisade tissues of pepper leaves,and it had lots of PMMoV-positive signals in palisade tissues. Epidermis had also few positive signals. In addition,few positive signals located mostly in epidermis could be found in petioles.

Key words: Capsicum, Pepper mild mottle virus, in situ RT-PCR, digoxin, location in tissue cDNA

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