Crops ›› 2022, Vol. 38 ›› Issue (3): 80-86.doi: 10.16035/j.issn.1001-7283.2022.03.011

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

Yao Yifan1(), Dai Zhuoyi1, Jiang Zhimin2, Xu Min3, Li Fangfang3, Zhang Xi4, Dang Wei1, Wu Zhaoyun1, Ding Yongle1, Yang Tiezhao1()   

  1. 1College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2Zhejiang China Tobacco Industry Co., Ltd., Hangzhou 310008, Zhejiang, China
    3China Tobacco Corporation Henan Company, Zhengzhou 450002, Henan, China
    4Technology Center, China Tobacco Shaanxi Industrial Co., Ltd., Baoji 721013, Shaanxi, China
  • Received:2021-07-07 Revised:2021-08-22 Online:2022-06-15 Published:2022-06-20
  • Contact: Yang Tiezhao E-mail:641429066@qq.com;yangtiezhao@126.com

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Abstract:

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.

Key words: RNA interference, Polyphenol oxidase, Virus induced gene silencing, Tobacco, VIGS

Table 1

Primer sequence"

引物Primer 引物序列Primer sequence
18S F GGCCGTTCTTAGTTGGTGGA
18S R GTCCCTCTAAGAAGCTGGCC
NtPPO8 F GCCACAATTAAACCCGGTGT
NtPPO8 R ACTCTTCATCCACGGCATGA

Fig.1

Vector construction colony PCR electrophoresis diagram M: DL2000 marker, 1-8: E. coli colonies transformed by the product of TRV-NtPPO8 ligation vector"

Fig.2

Results of TRV2-NtPPO8 vector sequencing vector sequencing comparison"

Fig.3

The effects of different treatments on PPO activity and NtPPO8 gene expression Lowercase letters indicate significant differences at the P < 0.05 level between different plants,“*”indicates significant differences at the P < 0.05 level between different treatments of the same plant,“**”indicates a significant difference at the level of P < 0.01 between different treatments of the same plant, the same below"

Fig.4

PPO activity and NtPPO8 gene expression in different parts of tobacco treated by expression vector S1: 8th leaf position, S5: 12th leaf position, S9: 17th leaf position, S13: 22nd leaf position"

Fig.5

PPO activity and NtPPO8 gene expression in tobacco treated with expression vector at different times"

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