Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

Abstract

Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.

Fig 1. Size distribution of small RNA sequences.

Sequence reads and unique sequence distribution showing the predominance of the 24 and 21nt length reads in Grand Naine (A) and Rasthali (B) small RNA libraries.

Fig 2. Generation of transgenic banana plants overexpressing MusamirRNA156.

(A) Schematic representation of the T-DNA region of the binary vector p1301-MusamiRNA156 depicting the insertion of the stem-loop sequence of MusamirRNA156 primary transcript in the multiple cloning site of modified pCAMBIA-1301 vector downstream of Zea mays polyubiquitin promoter. Putatively transformed embryos selected on the medium supplemented with hygromycin (B) followed by germination. The shoots were multiplied in medium containing BAP to form the clonal copies of the shoots (C) which were further rooted in NAA based medium (D).

Fig 3. Expression analysis of the T-DNA genes in transgenic banana lines.

(A) The expression of the GUS cassette in transgenic banana lines was determined by histochemical staining of the leaf discs and showed the presence of intense blue coloration in the transgenic lines (OV) whereas no colour was observed in the untransformed control plants (UC). (B) The quantum of the primary transcript of MusamirRNA156 in the transgenic banana line and untransformed control plant was determined by RT-PCR wherein a 175 bp product was seen in both untransformed control plant and selected transgenic line with later being brighter. (M- 1kb DNA ladder; N- RT-PCR control)

Fig 4. Phenotypes of the untransformed banana plant and pMusamiRAN156-1301 derived transgenic banana plant.

(A) Gross difference in the growth of the untransformed banana plant and p1301-MusamiRNA156 derived transgenic banana plant. (B) Poor development of the root system in the two plants 1 month post hardening. Difference in the leaf anatomy of the untransformed banana plant and p1301-MusamiRNA156 derived transgenic banana plant (C, D and E). Growth difference seen in the untransformed banana plant and p1301-MusamiRNA156 derived banana plant, three months post hardening (F). Microscopic examination of the leaf cellular pattern seen in the untransformed banana plant and p1301-MusamiRNA156 derived transgenic banana plant (G and H).