GrainGenes
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D. Gruszka, J. Zbieszczyk, M. Kwasniewski, I. Szarejko and M. Maluszynski
Department of Genetics, Faculty of Biology and Environment Preservation, University of Silesia, 40-032 Katowice, Poland
e-mail of main author: HYPERLINK "mailto:wintergrun@op.pl" wintergrun@op.pl
Brassinosteroids are growth-promoting natural products, found at low levels in pollen, seeds and young vegetative tissues throughout the plant kingdom. They play an essential role in diverse developmental processes including cell expansion, vascular differentiation, etiolation and reproductive development (Clouse and Sasse, 1998). It has been found that semidwarf barley accessions carrying the uzu gene are non-responding to brassinosteroids. The synteny suggested that the uzu gene may be homologous to rice D61, which is a homolog of Arabidopsis thaliana BR-insensitive1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1, HvBRI1 (acc. no. AB088206) was isolated (Chono et al., 2003). The sequence analysis showed that uzu phenotype was caused by single-nucleotide substitution A > G at the position 2612 of the HvBRI1 gene. This mutation resulted in an amino acid change at the highly conserved residue (His-857 to Arg-857) of the kinase domain of BRI1 receptor protein and caused the reduced sensitivity to BRs and reduced plant height (Chono et al., 2003). We have found a semidwarf mutant 093AR, produced by MNU (N-methyl-N-nitrosourea) treatment of variety Aramir, that proved to be allelic to the uzu form. The aim of this work was to determine and compare the sequences of HvBRI1 gene between spontaneous mutant uzu, induced mutant 093AR and its parent Aramir variety in order to identify the putative mutation(s).
DNA extraction was performed according to micro C-TAB protocol (Doyle and Doyle, 1987). Six primer pairs were designed on the basis of HvBRI1 sequence (GenBank acc. no. AB088206) utilizing the Primer3 program ( HYPERLINK "http://frodo.wi.mit.edu/cgi-bin/primer3" http://frodo.wi.mit.edu/cgi-bin/primer3). PCR reactions were performed using the touch-down strategy. Three independent PCR reactions were performed for each primer pair and amplification products were extracted from agarose gels using QIAEX II Gel Extraction Kit (QIAGEN) and cloned into pGEM-T Easy vector (Promega). Plasmid purification was carried out using QIAprep Spin Miniprep Kit (QIAGEN). Inserts were sequenced using SequiTherm EXCEL II DNA Sequencing Kit (EPICENTRE) utilizing LI-COR IR2 sequencer.
We determined the sequences of HvBRI1 gene in the genotype uzu, 093AR and Aramir variety. The single-nucleotide substitution has been confirmed at the position 2612 (A-2612 to G-2612) in the spontaneous mutant uzu, which proved to be specific for this genotype. This mutation is localized in highly conserved kinase domain of receptor protein. The comparison of sequences of HvBRI1 gene between mutant 093AR and Aramir variety, from which this mutant was obtained, led to the identification of the C to A substitutions at the positions 1760 and 1761 in the mutant (Fig. 1).
1 cgcttctcgcatggtctcaaggtagagcgagcgagctcccacatggattg
1701 gatgccgagcaagaagctgtgcaacttcacaaggatgtacatggggagca
1751 cagagtataccttcaacaagaatggctccatgatatttctggatttgtca
1801 tttaatcagcttgactcggagatacccaaggagcttggcaatatgttcta
2551 gtgagtggccagggtgaccgggagttcacagcggaaatggagaccattgg
2601 caagatcaaacaccgcaaccttgttccgctcctcggctactgcaagatcg
2651 gcgaggagcggctgctgatgtatgacttcatgaagtatggcagcttggag
3501 gcctctcagatgattgatgatgatgtgatgataccattctgaggttccca
3551 cgcaagct
Fig. 1. The sequence of HvBRI1 gene (acc. no. AB088206). cc- at the positions 1760 and 1761 indicate C > A substitutions in the 093AR sequence; a- at the position 2612 indicates A > G substitution in spontaneous mutant uzu.
On the basis of in silico translation using Jellyfish program it was postulated that this mutations lead to the replacement Thr-573 by Lys-573 (Fig. 2).
1 mdclrlavaaaalllaalaaaaddaqlldefrmalpsqaplegwtarega
501 ippelaeqsgkmtvgliigrpyvylrndelssqcrgkgsllefssirsed
551 lsrmpskklcnftrmymgsteytfnkngsmifldlsfnqldseipkelgn
601 mfylmimnlghnllsgaiptelagakklavldlshnrlegqipssfssls
1101 pgfgvmdmtlkeakeekd
Fig. 2 The sequence of HvBRI1 protein (acc. no. BAD01654). t indicates the Thr-573 to Lys-573 conversion, deduced by in silico translation, caused by C > A substitutions at the positions 1760 and 1761.
The threonine residue is localized between last LRR (Leucin-Rich Repeat) domain and pair of cysteines participating in protein dimerization, followed by transmembrane domain. It was shown utilizing ClustalW program that Thr-573 residue is highly conserved among BRI1 homologs from barley, rice, arabidopsis and tomato. As threonine and lysine have different chemical properties, their substitution within the conserved domain may lead to the change in protein activity.
References:
Chono, M., I. Honda, H. Zeniya, K. Yoneyama, D. Saisho, K. Takeda, S. Takatsuto, T. Hoshino and Y. Watanabe. 2003. A semidwarf phenotype of barley uzu results from a nucleotide substitution in the gene encoding a putative brassinosteroid receptor. Plant Physiology. 133:1209-1219
Clouse, S. D. and J. M. Sasse. 1998. Brassinosteroids: essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 427-451.
Barley Genetics Newsletter 36:1-2 (2006)
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