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A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion

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Abstract

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using HmR Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA3) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F2 from OsAPC6 × Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.

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Abbreviations

GA:

Gibberellic acid

APC:

Anaphase-promoting complex

sd1 :

semidwarf 1

TPR:

Tetratricopeptide repeat

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Acknowledgments

We thank Vajinder Kumar, Research Scholar, NRC on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi and Tripta Jhang, Scientist at Central Institute of Medicinal and Aromatic Plants, Lucknow for assistance in the isolation of flanking sequences and in BLAST analysis. This research was funded by CSIR, New Delhi, India.

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Authors and Affiliations

  1. Indian Institute of Technology Roorkee, Roorkee, 247 667, Uttarakhand, India

    Mankesh Kumar, P. Osman Basha, Anju Puri, Deepak Rajpurohit, Gursharn Singh Randhawa & Harcharan Singh Dhaliwal

  2. National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110012, India

    Tilak Raj Sharma

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  1. Mankesh Kumar
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  2. P. Osman Basha
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  7. Harcharan Singh Dhaliwal
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Correspondence to Harcharan Singh Dhaliwal.

Additional information

The nucleotide sequence reported in this paper has been submitted to Bankit (database no. 1164269; http://www.ncbi.nlm.nih.gov/BankIt) under accession numbers: FJ548927.

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Table S1

Result of FST search for the candidate query gene NC_008396.1 in OrygeneDB database (DOC 47 kb)

Fig. S1

Comparative anatomy of Basmati 370 and OsAPC6 mutant. (a) LS of second internode in Basmati 370 and (b) OsAPC6, at 10× magnification. (c) TS of second internode in Basmati 370 and (d) OsAPC6, at 10× magnification. (e) TS of leaf–blade of fourth leaf in Basmati 370 and (f) OsAPC6, at 10× magnification (DOC 100 kb)

Fig. S2

Germination of OsAPC6 mutant and Basmati 370 on 80 ppm hygromycin solution (DOC 746 kb)

Fig. S3

TAIL-PCR products amplified from OsAPC6. Lane, M = 100-bp DNA ladder; I, II, and III: products of primary, secondary, and tertiary reactions with RB1, RB2, and RB3, respectively, using AD1, AD2, and AD3 primers (DOC 79 kb)

Fig. S4

Amplification with gene-specific primer and T-DNA-specific primers using different primer combinations in Basmati 370 and OsAPC6. M = 100-bp ladder: lanes 1–10, T-DNA-specific primer and gene-specific primer combinations; 1 DTF1 + RB1, 2 DTF1 + RB2, 3 DTF1 + RB3, 4 DTF2 + RB1, 5 DTF2 + RB2, 6 DTF2 + RB3, 7 DTF1 + DTR1, 8 DTF1 + DTR2, 9 DTF2 + DTR1, 10 DTF2 + DTR2. Lanes: M, 100-bp ladder. 1 OsAPC6, 2 = Basmati 370 (DOC 134 kb)

Fig. S5

Schematic representation of the T-DNA constructs HmR Ds that was used for Basmati 370 transformation. LB left border, RB right border, SPT streptomycin phosphotransferase, NPT neomycin phosphotransferase, HPT hygromycin phosphotransferase. NotI, HO nuclease, and Dpn/ClaI methylase: rare cutter sites within the T-DNA and within the Ds element. SacI is the unique restriction site in the construct (DOC 46 kb)

Fig. S6

Genomic sequence of the candidate gene (Os03g0236900) from O. sativa cv. Nipponbare between position 7,178,921–7,187,583 bp of chromosome 3 (black-colored base pairs in small letter are intronic and red-colored capital letter base pairs are exonic regions in the sequence; DOC 54 kb)

Fig. S7

Characteristics of protein sequence of APC6 in rice obtained from NCBI protein ID BAF11405. The protein is made up of 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain as indicated by stretch of black-colored letters. The 34-amino acid TPR domain consists of 12 copies of the TPR motif as indicated by blue-colored letters and 16 TPR binding sites as indicated by red-colored letters. The whole sequence consists of two TPR domain superfamily that are overlapping with each other as indicated by underlined and italics letter (DOC 38 kb)

Fig. S8

Phylogenetic rooted tree of 12 protein amino acid sequences showing homology to APC6 of O. sativa (NP_001049491, red box; DOC 180 kb)

Fig. S9

Multiple sequence alignment of APC6 protein in rice run under ClustalW against the human, Drosophila melanogaster, Ratus norvegicus, Saccharomyces cerevisae, and Mus musculus APC/Cdc16. The shaded boxed amino acids are the conserved region within the TPR domain among all the species (DOC 907 kb)

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Kumar, M., Basha, P.O., Puri, A. et al. A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion. Funct Integr Genomics 10, 349–358 (2010). https://doi.org/10.1007/s10142-009-0155-6

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