RNA interference of the nicotine demethylase gene CYP82E4v1 reduces nornicotine content and enhances Myzus persicae resistance in Nicotiana tabacum L.
The CYP82E4v1 gene was identified to encode nicotine demethylase, which catalyzed the conversion of nicotine to nornicotine. In this study, we constructed CYP82E4v1-RNAi vector and genetically transformed tobacco variety K326. The determination results of nicotine and nornicotine content via HPLC demonstrated that there was significant increase of nicotine content and reduction of nornicotine content in transgenic plants compared with those in wild-type plants. Exogenous application of IAA or GA3 could reduce the nicotine content in tobaccos, while ABA or 6-BA could increase the content of nicotine. And the more significant difference of nicotine content change in transgenic plants. Aphid-inoculation experiment demonstrated the number of aphid population in transgenic plants was significantly lower than wild-type plants at 12 d after aphid-inoculation. Meanwhile, the activity of AOEs and PAL in transgenic and wild-type tobacco plants after aphid-inoculation was measured. At 3 d after aphid-inoculation, both AOEs and PAL activity were significantly higher than controls, including wild-type plants with aphid-inoculation and transgenic plants with mock-inoculation. Also, the relative expression of these genes involved in salicylic acid/jasmonic acid (SA/JA) signaling pathways was analyzed at different stages after aphid-inoculation and the results demonstrated that there was significantly higher expression of JA-induced LOX gene in both transgenic and wild-type plants inoculated by aphid than the non-inoculated ones while no significant difference in the expression of SA-induced PR-1a gene among them was found, which indicated the JA-mediated resistance response was activated during aphid infestation. Moreover, although the expression level of BGL (another JA-induced gene) was less significant between the two inoculated tobaccos, it was significantly higher than the plant without inoculation, which was 1.4 and 2.2 folds higher than the non-inoculated controls respectively. To sum up, the improvement of aphid-resistance in transgenic tobaccos was based on nicotine accumulation which might cause nerve and antifeed toxicity and JA-mediated resistance response by enhancing the activities of AOEs and PAL.
The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, Guizhou Province, PR China; Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang, Guizhou Province, PR China.
Dan
D
Zhao
The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, Guizhou Province, PR China; Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang, Guizhou Province, PR China.
Li-Jun
LJ
Qin
The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, Guizhou Province, PR China; Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang, Guizhou Province, PR China. Electronic address: dgzhao@gzu.edu.cn.
De-Gang
DG
Zhao
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001042:Aphids / Q000502:physiology*; D003517:Cyclopentanes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005822:Genetic Vectors / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D054883:Oxylipins / Q000378:metabolism; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D020156:Salicylic Acid / Q000378:metabolism; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics* / Q000469:parasitology*
D000975:Antioxidants; D003517:Cyclopentanes; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D009538:Nicotine; C011006:jasmonic acid; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System; C516919:nicotine demethylase, tobacco; D010650:Phenylalanine Ammonia-Lyase; D020156:Salicylic Acid
CYP82E4v1 gene; Myzus persicae; Nicoine; RNA interference; Tobacco