Isolation, cloning and expression of CCA1 gene in transgenic progeny plants of Japonica rice exhibiting altered morphological traits

PLoS One. 2019 Aug 5;14(8):e0220140. doi: 10.1371/journal.pone.0220140. eCollection 2019.

Abstract

Circadian clock genes holds tremendous potential for breeding crops better adapted to environmental fluctuations inherent to climate change. Endogenous TOC1 promoter and CCA1 gene from rice were isolated, cloned and mobilized into pCAMBIA1300 vectors and RNAi constructs A, B and C. Embryogenic calli of varying ages derived from mature seeds of Taipei 309 were employed for Agrobacterium-mediated genetic transformation generating T0, T1 and T2 independent transgenic lines were analyzed for over-expression and repression of CCA1 gene along with various morphological traits. Six hundred and thirty two T0 transgenic plants were generated from rice calli using constructs A, B and C. T0 progeny plants derived from constructs A, B and C did not show any considerable difference in morphological traits. T1and T2 progeny plants derived from construct A exhibited over-expression of CCA1 gene, on the contrary, progeny plants derived from RNAi constructs B and C exhibited repression of CCA1 gene in qRT-PCR analysis at different time points and showed rhythmicity peaking at dawn (6:00 AM) and lowest expression at 12:00 Noon. T1 and T2 progeny plants derived from construct A, namely, A-17 and A-45 exhibited reduced number of tillers/panicles (6-8), reduced thousand seed weight (10.1-16.6g), decreased seed length (4.98 to 6.58mm), decreased seed width (1.1-1.8mm) as compared to wild type plants. T1 and T2 progeny plants of construct B and C showed increased number of tillers/panicles (8-19), better seed yield (4.98-28.9g), increased thousand seed weight (15.6-29.03g), slightly increased seed length (5.7-7.43mm) and slightly increased seed width (1.7-2.98mm) as compared to wild type plants. Chlorophyll content in T1 and T2 progeny plants did not show any significant difference among the three constructs, however, rhythmicity was observed over the period of time in conjunction to CCA1 gene expression. Evidence has been presented which demonstrates that endogenous repression of CCA1 gene resulted in improved morphological traits: increased number of tillers/panicle, thousand seed weight, seed size; whereas, over-expression leads to diminution in morphological traits: decreased number of tillers/panicle, thousand seed weight, seed size as compared to the wild type in T1 and T2 progeny plants. This is first report of successful regulation of endogenous CCA1 gene under control of TOC1 promoter and its effect on improved growth vigor in Japonica rice.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Agrobacterium / genetics
  • Cloning, Molecular
  • Gene Expression Regulation, Plant*
  • Oryza / anatomy & histology
  • Oryza / genetics*
  • Plant Proteins / genetics*
  • Plants, Genetically Modified
  • Promoter Regions, Genetic / genetics
  • Transcription Factors / genetics*
  • Transformation, Genetic

Substances

  • Plant Proteins
  • Transcription Factors

Grants and funding

This work was supported by: 1. The Award of Fulbright Nehru Senior Research Fellowship to Prof A. Chaudhury by the USIEF (The United States-India Educational Foundation) New Delhi; Bureau of Educational and Cultural Affairs, U S Department of State, USA; International Educational Exchange Program, USA vide letter 1510/FN-SR/2011 Dated May 17, 2011; 2. FIST program, sanctioned to Prof. A. Chaudhury by the Department of Science & Technology, Ministry of Science & Technology, Government of India, New Delhi, India Vide Letter # No SR/FST/LSI-153/2003 Dated July 01, 2004; 3. SAP DRS-I and SAP DRS-II, sanctioned to Prof. A. Chaudhury by the University Grants Commission, New Delhi Vide Letter no. 3-18/2009 dated March 31, 2009 and Letter No F.3-4/2016/DRS-II (SAP-II) dated March 18, 2016; 4. JRF/SRF under Basic Scientific Research (BSR) to Ms. Anita Devi Dalal by the University Grants Commission, New Delhi Vide Letter no. DBNT146299 dated August 01, 2014.