RNA干扰(RNA interference,RNAi)是由双链RNA(double-stranded RNA,dsRNA)诱发的、同源mRNA高效特异性降解的现象,引发细胞内同源靶基因特异性抑制或沉默。RNAi技术已应用在害虫防治、抑菌、抗病毒等多个领域,但在控制农产品品质劣变中应用较少。该研究以大肠杆菌RNase Ⅲ缺陷型菌株HT115(DE3)为发酵平台,以L4440载体为骨架构建重组载体,进行马铃薯多酚氧化酶StPPO编码基因的dsRNA表达与发酵条件优化,并进行dsRNA-StPPO生物学功能验证。结果表明,异丙基硫代半乳糖苷(isopropyl thiogalactoside,IPTG)诱导剂浓度为0.6 mmol/L,诱导时间为6 h,外加酵母浸粉0.48%,温度为39 ℃,装瓶量为45 mL/250 mL,转速为189 r/min时,目标dsRNA的产量最高,达3.95 μg/mL。浸泡dsRNA-StPPO溶液后,处理组鲜切马铃薯较对照组有较高的L*、较低的a*和褐变指数,且组织中的多酚氧化酶酶活力较低,说明施用dsRNA-StPPO可以抑制马铃薯polyphenol oxidase StPPO基因表达有效控制鲜切褐变现象。
RNA interference (RNAi) is a phenomenon of highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which triggers specific cellular inhibition or silencing of homologous target genes.RNAi technology has been applied in many fields such as pest control, bacterial inhibition, antiviral, etc., with less application in controlling the quality deterioration of agricultural products.To optimize the expression and fermentation conditions of dsRNA for the gene encoding potato polyphenol oxidase StPPO, Escherichia coli HT115 (DE3) RNase III deficient strain was employed as the fermentation platform and recombinant vector was constructed using the L4440 vector as the backbone.Furthermore, the biological function of dsRNA-StPPO was validated.The results showed that the yield of the target dsRNA was the highest at 3.95 μg/mL at the concentration of isopropylthiogalactoside (IPTG) inducer at 0.6 mmol/L, induction time of 6 h, plus yeast extract of 0.48%, temperature of 39 ℃, bottling volume of 45 mL/250 mL, and rotation speed of 189 r/min.After immersion in dsRNA-StPPO solution, fresh-cut potatoes in the treatment group had higher L*, lower a* and browning index, and lower polyphenol oxidase enzyme activity in the tissues compared with the control group, suggesting that the application of dsRNA-StPPO can inhibit the expression of the StPPO gene and effectively control the browning phenomenon of fresh-cut potato.
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