Abstract
Developing gene constructs with the barnase gene and propagating them in Escherichia coli or Agrobacterium tumefaciens is difficult as unintended leaky expression leads to the death of the bacterial cells. In the present work, we circumvented this problem by developing intron containing barnase genes. We tested the use of two different introns viz., (i) the first intron (190 bp) of the catalase gene of castor bean and (ii) the twelfth intron (92 bp) of Pyruvate ortho-phosphate dikinase 2 (PPDK2) gene from cotton. Due to the absence of splicing in bacterial cells the unintended expression of the barnase protein was blocked allowing easy development and propagation of constructs. Further, we demonstrated that the intron introduced in the barnase gene was efficiently spliced out in the tapetum tissue of the anther in transgenic tobacco lines leading to male-sterility.
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Abbreviations
- Bsnp :
-
Barstar gene under its native promoter
- int:
-
Intron
- SOE-ing:
-
Gene Splicing by Overlap Extension
- 35S:
-
Cauliflower Mosaic Virus 35S promoter
- pA:
-
Polyadenylation signal of 35S
References
Bhullar S, Chakravarthy S, Advani S, Datta S, Pental D, Burma PK (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132:988–998
Eamens AL, Blanchard CL, Dennis ES, Upadhyaya NM (2004) A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.). Plant Biotechol J 2:367–380
Hartley RW (1988) Barnase and Barstar, expression of its cloned inhibitor permits expression of a cloned ribonuclease. J Mol Biol 202:913–915
Horton RM, Cai Z, Ho SN, Pease LR (2013) Gene splicing by overlap extension: tailor made genes using the polymerase chain reaction. BioTechn 54:129–133
Jagannath A, Bandyopadhyay P, Arumugam N, Gupta V, Burma PK, Pental D (2001) The use of a spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breed 8:11–23
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene. EMBO J 6:3901–3907
Jopcik M, Bauer M, Moravcikove J, Boszoradova E, Matusikova I, Libantova J (2013) Plant tissue-specific promoters can drive gene expression in Escherichia coli. Plant Cell Tiss Organ Cult 113:387–396
Koltunow MA, Truettner J, Cox HK, Wallroth M, Goldberg BR (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224
Mariani C, Beuckeleer MD, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737–741
Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31(6):805–813
Paul W, Hodge R, Smartt S, Draper J, Scott R (1992) The isolation and characterization of tapetum-specific Arabidopsis thaliana A9 gene. Plant Mol Biol 19:611–622
Prodromou C, Pearl HL (1992) Recursive PCR: a novel technique for total gene synthesis. Protein Eng 5:827–829
Ray K, Bisht NC, Pental D, Burma PK (2007) Development of barnase/barstar transgenics for hybrid seed production in Indian oilseed mustard (Brassica junceal.czern&coss) using a mutant acetolactate synthase gene conferring resistance to imidazolinone-based herbicide‘Persuit’. Curr Sci 93:1390–1396
SvabZ HP, Maliga P (1995) Generation of transgenic tobacco plants by co-cultivation of leaf disks with Agrobacterium pPZP binary vectors. In: Maliga P, Klessig DF, Cashmore AR, Gruissem W, Varner JE (eds) Methods in plant molecular biology, A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 55–77
Acknowledgments
The AEG1 (Accession No. KF742407) and PPDK2 (Accession No. KF742403) genes were isolated in the laboratory by Dr. Kumar Paritosh. This work was supported by grant-in-aids under New Millennium Indian Technology Leadership Initiative (NMITLI), Council of Scientific and Industrial Research CSIR), Govt. of India and from University of Delhi. AKM also acknowledges fellowship from CSIR.
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Mehrotra, A.K., Bhullar, S. & Burma, P.K. Development of intron-containing barnase gene (barnase-int) encoding a toxic protein to facilitate its cloning in bacterial cells. J. Plant Biochem. Biotechnol. 23, 435–439 (2014). https://doi.org/10.1007/s13562-014-0266-6
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DOI: https://doi.org/10.1007/s13562-014-0266-6