Crops ›› 2021, Vol. 37 ›› Issue (5): 6-13.doi: 10.16035/j.issn.1001-7283.2021.05.002

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Cloning and Functional Analysis of Tobacco Nt14-3-3-like C Gene

Song Kunfeng1(), Hao Fengsheng2, Yao Xin1, Wang Jing1, Liu Weiqun1()   

  1. 1College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2College of Plant Protection, China Agricultural University, Beijing 100089, China
  • Received:2021-04-02 Revised:2021-05-06 Online:2021-10-15 Published:2021-10-14
  • Contact: Liu Weiqun E-mail:laurnin@foxmail.com;wqliu@henau.edu.cn

Abstract:

Tobacco mosaic virus (TMV) has a wide range of hosts and is highly infectious. TMV damages the yield and quality of crops. A protein 14-3-3-like C was screened as a candidate gene from different TMV resistant tobacco varieties. In this study, Nt14-3-3-like C was cloned and analyzed, transgenic plants were obtained by Agrobacterium-mediated transformation, qPCR was used to analyze the gene expression of transgenic plants. The results showed that Nt14-3-3-like C was 780bp in length encoding 260 amino acids, which had the typical groove structure of 14-3-3 protein, but had no transmembrane structure and signal peptide; 14-3-3-like C protein was localized in the nucleus and cell membrane. qPCR analysis of transgenic materials showed that the expression of genes related to pathogen defense and photosynthetic pathway decreased significantly in plants inhibited by Nt14-3-3-like C, these results suggested that Nt14-3-3-like C may participated in TMV infection through pathogen defense and photosynthetic pathways.

Key words: TMV, 14-3-3 protein, Pathogen defense, Gene cloning

Fig.1

Electrophoretogram of Nt14-3-3-like C PCR products M: DL2000 DNA marker, the same below; 1-3: PCR products"

Fig.2

Comparison of sequencing and alignment results Nt14-3-3-like C: sequence cloned; 14-3-3-like C: sequence in tobacco genome database"

Fig.3

Bioinformatics analysis of 14-3-3-like C protein a: prediction of transmembrane structure; b: prediction of signal peptide; c: prediction of phosphorylation sites; d: prediction of protein tertiary dimensional structure"

Fig.4

Phylogenetic tree analysis of 14-3-3-like C"

Fig.5

Electrophoretogram of recombinant expression vector 35S::14-3-3-like C-GFP PCR amplification 1-3: PCR products;“-”represents negative control"

Fig.6

Subcellular localization of 14-3-3-like C The green fluorescence is 14-3-3-like C-GFP; the red fluorescence is ARR-mcherry (ARR is a nuclear marker protein)"

Fig.7

Identification of Nt14-3-3-like C expression vector 1-3: bacterium solution PCR products (a); 1-2: agarose gel electrophoresis results of restriction enzyme digestion products (b)"

Fig.8

Agrobacterium-mediated genetic transformation"

Fig.9

Detection results of transgenic plants 1-6: agarose gel electrophoresis of PCR products"

Fig.10

Detection of gene expression inhibition at transcription level “**”indicates significant different at 0.01 level, the same below. CK: control line; L1-L3: RNAi lines"

Fig.11

Gene expression analysis of pathogen defense and photosynthetic pathway"

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