Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (8): 1524-1531.doi: 10.3864/j.issn.0578-1752.2020.08.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Splicing Property Analyses of the NRSE1 Element from Tobacco PR3b mRNA After Fusion Expression with GUS Gene

ZHAO Xue1,WANG Feng2,WANG WenJing1,LIU XiaoFeng1,BIAN ShiQuan1,LIU YanHua1,LIU XinMin1,DU YongMei1,ZHANG ZhongFeng1,ZHANG HongBo1()   

  1. 1 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong
    2 College of Agronomy and Biotechnology, Southwest University, Chongqing 400716
  • Received:2019-09-05 Accepted:2019-12-18 Online:2020-04-16 Published:2020-04-29
  • Contact: HongBo ZHANG E-mail:zhanghongbo@caas.cn

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Abstract:

【Objective】Previously, a post-transcriptional splicing of tobacco PR3b gene was observed in the low-nicotine mutants (nic1, nic2) of Nicotiana tabacum L. cv. Burley 21, yet the mechanism underlying this phenomenon is still unclear. In this study, we developed transgenic plants expressing the fusion of the alternative splicing element NRSE1 (nicotine-synthesis related splicing element 1) from PR3b and the GUS gene to investigate the splicing properties of the NRSE1 element after excising from PR3b mRNA, in order to reveal its regulatory mechanism. 【Method】The NRSE1 element was amplified from PR3b cDNA by PCR amplification, and the vector for expressing the fusion of NRSE1 element and the GUS gene was constructed by molecular methods. And, the vector was used to develop transgenic plants expressing the fusion of NRSE1 element and GUS gene with wild type tobacco and the low-nicotine mutants nic1 and nic2 via agrobacterium (LBA4404) mediated transformation method. The transgenic plants were identified by RT-PCR and GUS staining, and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants of wild type tobacco and low-nicotine mutants were then analyzed by RT-PCR. Seedlings of the transgenic plants were treated with ethylene (ET) and jasmonic acid (JA), respectively. And, the effects of ET and JA treatment on the GUS activity and the splicing of the fusion of NRSE1 element and GUS gene in the transgenic plants were analyzed by GUS staining and RT-PCR, respectively. The effects of ET and JA treatment on the expression level of the fusion of NRSE1 element and GUS gene were analyzed as well. 【Result】A set of transgenic wild type tobacco and low-nicotine mutants expressing the fusion of NRSE1 element and GUS gene were identified by RT-PCR and GUS staining. Further RT-PCR and sequencing analyses showed that the NRSE1 element could be alternatively spliced at higher levels in the low-nicotine mutants after fusion with the GUS gene, in a pattern consistent with its alternative splicing in the PR3b mRNA as previously report. ET and JA treatments could alter the GUS activity in the transgenic plants, but did not affect the inducible splicing of the NRSE1 element or the expression level of the fusion of NRSE1 element and GUS gene in the transgenic plants. 【Conclusion】 A highly splicing of the NRSE1 element was observed in the low-nicotine mutants after fusion expression with GUS gene. The alternatively splicing of NRSE1 element is independent of the rest regions of PR3b mRNA. ET and JA treatments had an effect on the GUS activities of the transgenic plants expressing the fusion of NRSE1 element and GUS gene, which may result from a translational regulation.

Key words: Nicotiana tabacum L., PR3b gene, low-nicotine mutant, alternative splicing, jasmonic acid, ethylene

Fig. 1

Schematic diagram of vector construction for the fusion of NRSE1 element and GUS gene A: The diagram of PR3b gene and the alternative splicing region, and the first spliced base are separated by 4 bases from the left 64 bases; B: Structural diagram of the vector expressing the fusion of NRSE1 element and GUS gene"

Fig. 2

PCR test of E. coli clones with recombination plasmids Marker indicates DNA molecular standards"

Fig. 3

Identification of transgenic plants A: Identification of transgenic plants by RT-PCR; B: Identification of transgenic plants by GUS staining. Ctrl indicates untransgenic wild type tobacco. WT+ indicates transgenic wild type tobacco; nic1 and nic2 indicate the transgenic plants of low-nicotine mutants. PR3b-GUS indicates the fusion of NRSE1 element and GUS gene"

Fig. 4

Molecular splicing analysis of the fusion of NRSE1 element and GUS in nic1 and nic2 mutants WT+ indicates transgenic wild type tobacco; nic1 and nic2 indicate the transgenic plants of low-nicotine mutants. Ctrl indicates untransgenic wild type tobacco. PR3b-GUS indicates the fusion of NRSE1 element and GUS gene. Asterisk indicates primer dimers"

Fig. 5

Effects of ET and JA treatment on the GUS activities of transgenic tobacco plants A: GUS activity of transgenic tobacco; B: Splicing analysis of the fusion of NRSE1 element and GUS. Asterisk indicates primer dimers; C: Relative expression of GUS gene after transgenic plants treated with ET and JA. WT+ indicates transgenic plant of wild type; nic1 and nic2 indicate transgenic plant of low-nicotine mutants. Mock indicates control treatment; ET and JA indicate ethylene and jasmonate treatment, respectively"

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