RNA干扰对烟草NtPPO8基因沉默的效应分析

Analysis of the Effects of RNA Interference on Tobacco NtPPO8 Gene Silencing

多酚氧化酶(PPO)是发生酶促褐变的关键酶,PPO活性过高会使烟叶变褐。为明确烟草NtPPO8基因对PPO活性的影响,建立降低NtPPO8基因表达量的病毒诱导的基因沉默(virus induced gene silencing,VIGS)体系,探究使用RNA干扰技术对烟叶NtPPO8基因表达量及PPO活性的影响。以中烟100和K326为材料,分析在移栽后50d进行VIGS载体侵染的植株,并在侵染后10、20、30、40d分别测定NtPPO8基因表达量及PPO活性。结果表明,接种VIGS载体的转化植株,其中在侵染后20d时NtPPO8基因的沉默效率最高,为78.46%;随着侵染时间推移,NtPPO8基因的沉默效率逐渐降低,在接种后40d内有效表达;且转化植株中的病毒载体会随着植株的生长而向上传导,随着接种部位的上移,沉默效率逐渐降低。因此,研究建立VIGS体系能够有效沉默NtPPO8基因的表达,并且有效降低PPO活性,为提高烟叶耐烤性及降低烟叶褐变比例提供了新的思路和方法。

Polyphenol oxidase(PPO)is a key enzyme for enzymatic browning and excessive PPO activity can brown tobacco leaves.To identify the effect of the tobacco NtPPO8 gene on PPO activity,a virus-induced gene silencing(VIGS)system was established to reduce the expression of the NtPPO8 gene.It was planned to explore the effect of RNA interference technology on the expression of the NtPPO8 gene and PPO enzyme activity in tobacco leaves.Using Zhongyan 100 as the material,the plants infected with the VIGS vector at 50d after transplantation were analyzed and the NtPPO8 gene expression level and PPO enzyme activity were measured at 10,20,30,and 40d after infection.The results showed that:in the transformed plants inoculated with VIGS vector,the highest silencing efficiency of the NtPPO8 gene was 78.46%at 20d after transplantation;the silencing efficiency of the NtPPO8 gene gradually decreased with time and it was effectively expressed within 40d after inoculation;the viral vector was transmitted upward with the plant growing and the silencing efficiency was gradually decreased as the inoculation site moves upward.Therefore,the establishment of the VIGS system could effectively silence the expression of the NtPPO8 gene and effectively reduce the activity of PPO.It provides new ideas and methods for improving tobacco leaf curing tolerance and reducing the proportion of tobacco leaf browning.