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Identification of microRNAs potentially involved in male sterility of Brassica campestris ssp. chinensis using microRNA array and quantitative RT-PCR assays

Abstract

microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensis cv. ‘Aijiaohuang’ genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Of these, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of ‘Bcajh97-01A’ sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5’ modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development.

Abbreviations

BLAST:

Basic Local Alignment Search Tool

qRT-PCR:

quantitative reverse-transcription polymerase chain reaction

RACE:

rapid amplification of cDNA ends

References

  1. Sun, G. MicroRNAs and their diverse functions in plants. Plant Mol. Biol. 80 (2012) 17–36.

    Article  CAS  PubMed  Google Scholar 

  2. He, S., Yang, Z., Skogerbo, G., Ren, F., Cui, H., Zhao, H., Chen, R. and Zhao, Y. The properties and functions of virus encoded microRNA, siRNA and other small noncoding RNAs. Crit. Rev. Microbiol. 34 (2008) 175–188.

    Article  CAS  PubMed  Google Scholar 

  3. Pfeffer, S., Zavolan, M., Grasser, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C. and Tuschl, T. Identification of virusencoded microRNAs. Science 304 (2004) 734–736.

    Article  CAS  PubMed  Google Scholar 

  4. Siomi, H. and Siomi, M.C. Posttranscriptional regulation of microRNA biogenesis in animals. Mol. Cell. 38 (2010) 323–332.

    Article  CAS  PubMed  Google Scholar 

  5. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism and function. Cell 116 (2004) 281–297.

    Article  CAS  PubMed  Google Scholar 

  6. Chen, X. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303 (2004) 2022–2025.

    Article  CAS  PubMed  Google Scholar 

  7. Voinnet, O. Origin, biogenesis and activity of plant microRNAs. Cell 136 (2009) 669–687.

    Article  CAS  PubMed  Google Scholar 

  8. Lee, R.C., Feinbaum, R.L. and Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75 (1993) 843–854.

    Article  CAS  PubMed  Google Scholar 

  9. Floyd, S.K. and Bowman, J.L. Gene regulation: ancient microRNA target sequences in plants. Nature 428 (2004) 485–486.

    Article  CAS  PubMed  Google Scholar 

  10. Guo, H.S., Xie, Q., Fei, J.F. and Chua, N.H. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development. Plant Cell 17 (2005) 1376–1386.

    Article  CAS  PubMed  Google Scholar 

  11. Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B. and Bartel, D.P. Prediction of plant microRNA targets. Cell 110 (2002) 513–520.

    Article  CAS  PubMed  Google Scholar 

  12. Yamasaki, H., Abdel-Ghany, S.E., Cohu, C.M., Kobayashi, Y., Shikanai, T. and Pilon, M. Regulation of copper homeostasis by micro-RNA in Arabidopsis. J. Biol. Chem. 282 (2007) 16369–16378.

    Article  CAS  PubMed  Google Scholar 

  13. Sunkar, R., Chinnusamy, V., Zhu, J. and Zhu, J.K. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 12 (2007) 301–309.

    Article  CAS  PubMed  Google Scholar 

  14. Ruiz-Ferrer, V. and Voinnet, O. Roles of plant small RNAs in biotic stress responses. Annu. Rev. Plant Biol. 60 (2009) 485–510.

    Article  CAS  PubMed  Google Scholar 

  15. Katiyar-Agarwal, S. and Jin, H. Role of small RNAs in host-microbe interactions. Annu. Rev. Phytopathol. 48 (2010) 225–246.

    Article  CAS  PubMed  Google Scholar 

  16. Grant-Downton, R., Le Trionnaire, G., Schmid, R., Rodriguez-Enriquez, J., Hafidh, S., Mehdi, S., Twell, D. and Dickinson, H. MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana. BMC Genomics 10 (2009) 643–658. DOI:10.1186/1471-2164-10-643.

    Article  PubMed  Google Scholar 

  17. Chambers, C. and Shuai, B. Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR. BMC Plant Biol. 9 (2009) 87–96. DOI:10.1186/1471-2229-9-87.

    Article  PubMed  Google Scholar 

  18. Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J.D., Feijo, J.A. and Martienssen, R.A. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136 (2009) 461–472.

    Article  CAS  PubMed  Google Scholar 

  19. Borges, F., Pereira, P.A., Slotkin, R.K., Martienssen, R.A. and Becker, J.D. MicroRNA activity in the Arabidopsis male germline. J. Exp. Bot. 62 (2011) 1611–1620.

    Article  CAS  PubMed  Google Scholar 

  20. Sunkar, R. and Jagadeeswaran, G. In silico identification of conserved microRNAs in large number of diverse plant species. BMC Plant Biol. 8 (2008) 37–49. DOI:10.1186/1471-2229-8-37.

    Article  PubMed  Google Scholar 

  21. Glowacki, S., Macioszek, V.K. and Kononowicz, A. R proteins as fundamentals of plant innate immunity. Cell. Mol. Biol. Lett. 16 (2011) 373–396.

    Article  Google Scholar 

  22. He, X.F., Fang, Y.Y., Feng, L. and Guo, H.S. Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIR NBS-LRR class R gene-derived novel miRNA in Brassica. FEBS Lett. 582 (2008) 2445–2452.

    Article  CAS  PubMed  Google Scholar 

  23. Wang, L., Wang, M.B., Tu, J.X., Helliwell, C.A., Waterhouse, P.M., Dennis, E.S., Fu, T.D. and Fan, Y.L. Cloning and characterization of microRNAs from Brassica napus. FEBS Lett. 581 (2007) 3848–3856.

    Article  CAS  PubMed  Google Scholar 

  24. Huang, L., Cao, J., Ye, W., Liu, T., Jiang, L. and Ye, Y. Transcriptional differences between the male-sterile mutant bcms and wild-type Brassica campestris ssp.chinensis reveal genes related to pollen development. Plant Biol. 10 (2008) 342–355.

    Article  CAS  PubMed  Google Scholar 

  25. Huang, L., Ye, W., Liu, T. and Cao, J. Characterization of the male-sterile line ‘Bcajh97-01A/B’ and identification of candidate genes for genic male sterility in Chinese cabbage-pak-choi. J. Am. Soc. Hortic. Sci. 134 (2009) 632–640.

    Google Scholar 

  26. Bolstad, B.M., Irizarry, R.A., Astrandand, M. and Speed, T.P. A comparison of normalization methods for high-density oligonucleotide array data based on variance and bias. Bioinfo 19 (2003) 185–193.

    Article  CAS  Google Scholar 

  27. Gao, X., Gulari, E. and Zhou, X. In situ synthesis of oligonucleotide microarrays. Biopolymers 73 (2004) 579–596.

    Article  CAS  PubMed  Google Scholar 

  28. Zhu, Q., Hong, A., Sheng, N., Zhang, X., Jun, K.Y., Srivannavit, O., Gulari, E., Gao, X. and Zhou, X. Microfluidic biochip for nucleic acid and protein analysis. Methods Mol. Biol. 382 (2007) 287–312.

    Article  CAS  PubMed  Google Scholar 

  29. Mathews, D.H., Sabina, J., Zuker, M. and Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288 (1999) 911–940.

    Article  CAS  PubMed  Google Scholar 

  30. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (2003) 3406–3415.

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, B.H., Pan, X.P., Cox, S.B., Cobb, G.P. and Anderson, T.A. Evidence that miRNAs are different from other RNAs. Cell Mol. Life Sci. 63 (2006) 246–254.

    Article  CAS  PubMed  Google Scholar 

  32. Meyers, B.C., Axtell, M.J., Bartel, B., Bartel, D.P., Baulcombe, D., Bowman, J.L., Cao, X., Carrington, J.C., Chen, X., Green, P.J., Griffiths-Jones, S., Jacobsen, S.E., Mallory, A.C., Martienssen, R.A., Poethig, R.S., Qi, Y., Vaucheret, H., Voinnet, O., Watanabe, Y., Weigel, D. and Zhu, J.K. Criteria for annotation of plant MicroRNAs. Plant Cell 20 (2008) 3186–3190.

    Article  CAS  PubMed  Google Scholar 

  33. Dai, X. and Zhao, P.X. psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res. 39 (2011) W155–159. DOI:10.1093/nar/gkr319.

    Article  CAS  PubMed  Google Scholar 

  34. Livak, K.J. and Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta CT) method. Methods 25 (2001) 402–408.

    Article  CAS  PubMed  Google Scholar 

  35. Wu, M.F., Tian, Q. and Reed, J.W. Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression and regulates both female and male reproduction. Development 133 (2006) 4211–4218.

    Article  CAS  PubMed  Google Scholar 

  36. Wei, L.Q., Yan, L.F. and Wang, T. Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa. Genome Biol. 12 (2011) R53. DOI: 10.1186/gb-2011-12-6-r53.

    Article  CAS  PubMed  Google Scholar 

  37. Moxon, S., Schwach, F., Dalmay, T., Maclean, D., Studholme, D.J. and Moulton, V. A toolkit for analysing large-scale plant small RNA datasets. Bioinformatics 24 (2008) 2252–2253.

    Article  CAS  PubMed  Google Scholar 

  38. Xie, F. and Zhang, B. Target-align: a tool for plant microRNA target identification. Bioinformatics 26 (2010) 3002–3003.

    Article  CAS  PubMed  Google Scholar 

  39. Bonnet, E., He, Y., Billiau, K. and Van de Peer, Y. TAPIR, a web server for the prediction of plant microRNA targets, including target mimics. Bioinformatics 26 (2010) 1566–1568.

    Article  CAS  PubMed  Google Scholar 

  40. Ohta, T. The nearly neutral theory of molecular evolution. Annu. Rev. Ecol. Sys. 23 (1992) 263–286.

    Article  Google Scholar 

  41. San, M.D. and Agorreta, A. Molecular systematics: A synthesis of the common methods and the state of knowledge. Cell. Mol. Biol. Lett. 15 (2010) 311–341.

    Article  Google Scholar 

  42. Aukerman, M.J. and Sakai, H. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15 (2003) 2730–2741.

    Article  CAS  PubMed  Google Scholar 

  43. Jones-Rhoades, M.W. and Bartel, D.P. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14 (2004) 787–799.

    Article  CAS  PubMed  Google Scholar 

  44. Song, C., Jia, Q., Fang, J., Li, F., Wang, C. and Zhang, Z. Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags. Plant Biol. (Stuttgart) 12 (2010) 927–934.

    Article  CAS  Google Scholar 

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Correspondence to Jiashu Cao.

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Jiang, J., Jiang, J., Yang, Y. et al. Identification of microRNAs potentially involved in male sterility of Brassica campestris ssp. chinensis using microRNA array and quantitative RT-PCR assays. Cell Mol Biol Lett 18, 416–432 (2013). https://doi.org/10.2478/s11658-013-0097-9

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  • DOI: https://doi.org/10.2478/s11658-013-0097-9

Key words

  • Brassica campestris
  • Chinese cabbage
  • Brassica rapa
  • Microarray
  • microRNA
  • Pollen development
  • Quantitative RT-PCR
  • 5’ modified RACE
  • Male sterile line
  • Male fertile line