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Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

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Expression of selected novel miRNAs in response to B. japonicum inoculation in soybean roots at different time points. Corresponding ethidium bromide stained gels show equal loading of total RNA in all lanes. (Note: The precursors of miR171 seem to be evolutionarily related, but the mature miRNAs are different from each other and we classified this as a novel miRNA).
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Figure 4: Expression of selected novel miRNAs in response to B. japonicum inoculation in soybean roots at different time points. Corresponding ethidium bromide stained gels show equal loading of total RNA in all lanes. (Note: The precursors of miR171 seem to be evolutionarily related, but the mature miRNAs are different from each other and we classified this as a novel miRNA).

Mentions: We performed Northern analysis of selected miRNA candidates with two goals. 1. To experimentally validate these miRNAs and 2. To study the abundance of these miRNA during the early phases of nodulation. All of the 5 novel miRNAs tested were detectable by Northern expression analysis (Figure 4). Of the 10 conserved miRNAs tested, 9 were detectable by Northern expression analysis except miR167 (Figure 5). Thus, we were able to validate a majority of the identified miRNAs.


Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

Expression of selected novel miRNAs in response to B. japonicum inoculation in soybean roots at different time points. Corresponding ethidium bromide stained gels show equal loading of total RNA in all lanes. (Note: The precursors of miR171 seem to be evolutionarily related, but the mature miRNAs are different from each other and we classified this as a novel miRNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335117&req=5

Figure 4: Expression of selected novel miRNAs in response to B. japonicum inoculation in soybean roots at different time points. Corresponding ethidium bromide stained gels show equal loading of total RNA in all lanes. (Note: The precursors of miR171 seem to be evolutionarily related, but the mature miRNAs are different from each other and we classified this as a novel miRNA).
Mentions: We performed Northern analysis of selected miRNA candidates with two goals. 1. To experimentally validate these miRNAs and 2. To study the abundance of these miRNA during the early phases of nodulation. All of the 5 novel miRNAs tested were detectable by Northern expression analysis (Figure 4). Of the 10 conserved miRNAs tested, 9 were detectable by Northern expression analysis except miR167 (Figure 5). Thus, we were able to validate a majority of the identified miRNAs.

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

Show MeSH