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本研究根据番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)分离物-SH2基因组序列,设计了一对引物,以番茄25S rRNA基因为内参,建立了番茄黄化曲叶病毒SYBR Green I实时荧光定量PCR检测方法。该方法可检测到浓度为4.64×10~(-7)ng/μL的植物DNA中含有TYLCV,其灵敏度是常规PCR的1000倍。利用该方法,研究了温室条件下以侵染性克隆接种TYLCV后的番茄植株中病毒DNA含量的变化情况。根、茎、叶中病毒DNA定量检测结果表明,TYLCV在3种植物器官中都呈现一个上升、稳定和下降的变化规律;病毒DNA在植株根部最早累积,累积的速度较慢,在叶部和茎部累积较快;叶部和茎部接种18 d后病毒DNA含量达到稳定期;在不同的器官中,病毒的含量不同,在茎部的含量最高,接种33 d后茎部病毒DNA的含量约为根部的100倍。本研究通过对TYLCV含量的动态监测,明确了病毒DNA在番茄植株中的累积和变化规律,为研究TYLCV侵染机制、病毒与寄主互作及病害防治提供了理论依据。
In this study, a pair of primers was designed based on the sequence of the SHH2 gene of Tomato yellow leaf curl virus (TYLCV). Tomato Tomato yellow leaf curl virus (SYBR Green I Real-time fluorescence quantitative PCR detection method. The method can detect TYLCV in plant DNA with the concentration of 4.64 × 10 ~ (-7) ng / μL, and its sensitivity is 1000 times higher than that of the conventional PCR. Using this method, we studied the changes of viral DNA content in TYLCV-infected tomato plants in greenhouse under inoculated clones. The results of quantitative detection of virus DNA in roots, stems and leaves showed that TYLCV showed a rising, steady and declining pattern in all three plant organs. The virus DNA accumulated and accumulated slowly at the root of plant, The accumulation of stems was faster; the content of virus DNA reached stable stage after 18 days inoculation of leaves and stems; the content of virus was different in different organs and the highest in stems; the content of DNA in stems after 33 days of inoculation About 100 times the root. In this study, through the dynamic monitoring of TYLCV content, the regularity of accumulation and variation of viral DNA in tomato plants was clarified, which provided a theoretical basis for studying TYLCV infection mechanism, virus-host interaction and disease prevention and control.