关键词: 葡萄浆果内坏死病毒, 逆转录实时荧光定量PCR, 葡萄砧木, 时空分布

Abstract:

【Background】 Grapevine berry inner necrosis virus (GINV) is a positive single-stranded RNA virus reported in China in recent years, which is widespread and harmful. Highly sensitive detection technology is the key for field monitoring of the virus and the cultivation of virus-free seedlings.【Objective】 The objective of this study is to establish a high-sensitivity reverse transcription real-time quantitative PCR (RT-qPCR) detection system for GINV, clarify the sensitivity of different grape rootstocks to GINV, and to clarify the spatial and temporal distribution of GINV in host plants, so as to provide technical support for the monitoring and early warning of the virus.【Method】 Six sets of primers were designed according to the conserved sequences of replicase (RP), movement protein (MP) and coat protein (CP) genes registered in GenBank. The primers with strong specificity and good amplification effect were screened by conventional RT-PCR and RT-qPCR. Then, the annealing temperature and concentration of the primers were optimized to establish the SYBR Green I dye RT-qPCR detection system for GINV, and the sensitivity, specificity and field applicability of this system were further evaluated. GINV was inoculated into five grape rootstocks, including Beta, SO4, 101-14, 140R and 1103P, for symptom observation and virus detection to screen indicator plants with high GINV sensitivity. Based on the established RT-qPCR technology, samples of different grape rootstocks inoculated with GINV were detected at different stages and different parts, so as to clarify the spatial and temporal distribution of GINV in different grape rootstocks.【Result】 The SYBR Green I dye RT-qPCR detection technology system for GINV was established, and the optimal primer was GINVRPYGF2/R2 and the optimal primer concentration was 300 nmol·L^-1, the optimal annealing temperature was 58.4 ℃. This technique had strong specificity and high sensitivity for GINV detection, and its detection sensitivity was 1 000 times that of conventional RT-PCR. The observation results of GINV inoculation into different grape rootstocks showed that Beta was the most severe in GINV infection, which was manifested as systemic necrosis of leaves, while the leaves of other rootstocks only showed the symptoms of green mottling and ring spots. The results of RT-qPCR showed that the relative content of GINV was the highest in EL27 (setting stage), and there was no significant difference in the relative content of GINV among the five varieties during EL12 (inflorescence clear stage) and EL27, and there were significant differences in the relative content of GINV between Beta and SO4, 101-14, 140R and 1103P in EL31 (berries pea size stage), and the relative content of GINV varied greatly in different tissues, and the order from high to low was lower leaves, upper leaves, upper stems, lower stems and roots.【Conclusion】 A high-sensitivity and strong-specificity RT-qPCR method for the detection of GINV was established, and the spatial and temporal distribution of GINV in different grape rootstocks was clarified by this method.

Key words: grapevine berry inner necrosis virus (GINV), reverse transcription real-time quantitative PCR (RT-qPCR), grape rootstock, spatial and temporal distribution