关键词: 西瓜, 小西葫芦黄花叶病毒, 原核表达, 外源喷施, 双链RNA, 防治效果

Abstract:

【Objective】Zucchini yellow mosaic virus (ZYMV) is the most prevalent virus that harms watermelon. The objective of this study is to realize the prevention and treatment of ZYMV by external application of viral gene dsRNA on watermelon. 【Method】Based on the existing ZYMV virus sequence in the laboratory, the NCBI database was used to find the similar sequences for different gene fragments. Then the software DNAMAN8 was used to perform multiple alignment analysis to find the conserved regions. Based on the analysis results, 5 gene fragments of ZYMV 3′UTR, 6K2, HC-Pro, P3 and NIb with a length range of 200-350 bp were selected, at the same time, a GUS gene fragment with a length of 190 bp was selected as a control. After the 6 fragments were amplified by PCR, they were inserted into the vector L4440 containing double T7 promoters by homologous recombination method. The RNAIII enzyme-deficient strain E. coli-HT115 was used to construct a prokaryotic expression system for dsRNA production, dsRNA was released by ultrasonic disruption and dissolved in TE buffer (10 mmol·L ^-1 Tris-HCl and 1 mmol·L ^-1 EDTA). The concentration of IPTG, induction time and ultrasonic breaking time in the production process were compared and analyzed. The dsRNA was applied on watermelon plants by spraying method, two experiments for spraying dsRNA first, then inoculating virus (prevention), and inoculating virus first, then spraying dsRNA (treatment) were designed, and the efficacy of dsRNA prevention and treatment against ZYMV was evaluated by statistical comparison and analysis of the incidence at 21 days after virus inoculation. 【Result】The production system for ZYMV 3′UTR, 6K2, HC-Pro, P3, NIb 5 gene fragments and GUS gene fragment was established, which could express and release dsRNA efficiently and stably. The cells could efficiently produce and release dsRNA after IPTG induced concentration of 8 mmol·L ^-1, induction time of 7 h, and disruption for 15 min under the condition of Ningbo Xinzhi brand ultrasonic cell disrupter 3ø and 60% output power. In the prevention experiment, it was found that the disease incidence of both GUS gene fragment dsRNA and TE buffer spraying treatments was 100%. The best control efficacy on ZYMV was HC-Pro gene fragment, after 21 days of virus inoculation, the efficacy reached 95%, the control efficacy of NIb gene fragment (80%) was relatively poor. On this basis, the efficacy of prevention and treatment of HC-Pro fragment on ZYMV was analyzed in depth. When the ZYMV was inoculated on the 3rd, 5th and 7th day after spraying the dsRNA of HC-Pro fragment, the incidence of disease was 16%, 63% and 63%, respectively. The dsRNA of the HC-Pro fragment was sprayed on the 1st, 3rd, 5th, and 7th day after inoculation of ZYMV, the results show that there is no obvious therapeutic efficacy, and only the onset of disease was delayed.【Conclusion】A prokaryotic expression system was established for dsRNA targeting different gene fragments of ZYMV, and it was found that dsRNA based on HC-Pro gene fragment has the best control efficacy on ZYMV, which can reduce the incidence of disease, delay the onset time of disease, and have the potential for application.

Key words: watermelon, Zucchini yellow mosaic virus (ZYMV), prokaryotic expression, external application, double- stranded RNA (dsRNA), control efficacy