Differential expression analysis of microRNA in banana different organs

Abstract:【Objective】Micro RNAs(mi RNAs) are endogenous non- coding small RNAs(s RNAs) with a wide range of regulatory functions in plant development and stress responses. The banana(Musa acuminata, AAA group) is one of the most important fruit crops in tropical and subtropical countries with a high nutritional value for human health. It comprises an important part in the diet of millions of people around the world. Thousands of mi RNAs have been identified in many plant species, whereas only a limited number of mi RNAs have been predicted in Musa acuminata(A genome) and Musa balbisiana(B genome). To obtain more insight into the role of mi RNAs in banana growth and development, we implemented conserved mi RNAs express patterns in four tissues.【Methods】Roots, leaves, flowers, and fruits were collected from banana plants(Musa acuminata, AAA group,‘Brazilian') grown at the fruit science experimental station ofthe Chinese Academy of Tropical Agricultural Sciences(Haikou, Hainan province, China). After collection,all the samples were immediately frozen in liquid nitrogen and stored at-80 ℃ until use. Total RNA was isolated from different tissues following their reference(Chai et al., 2014) and briefly exposed to RNAasefree DNAase I. c DNAs were synthesized by M-MLV according to the manufacturer's instruction, using the specific stem-loop primers for mi RNAs. Stem-loop RT-PCR and q RT-PCR were applied to detect the tissue-specific expression levels of conserved mi RNA in banana roots, leaves, flowers, and fruits. After reverse transcription, the products of each reaction were diluted 10 times to avoid potential primer interference in the q RT-PCR reactions. Each reaction consisted of 2 μL of product from the diluted reverse transcription reaction, 2 μL of primers(forward and reverse, 10 μmol·L-1), 10 μL of Fast Start Universal SYBR Green Master(ROX), 0.5 μL Reference Dye Ⅱ, and 5.5 μL of nuclease-free water. The reaction was initiated with pre-denaturation at 95 ℃ for 2 min, followed by 40 cycles of denaturation at 95 ℃ for 5 s, annealing at 50 ℃ for 15 s, and extension at 72 ℃ for 20 s. The q RT-PCR reactions were performed using FastStart Universal SYBR Green Master(ROX) on a Mx3005P? Real-Time PCR System. Reactions were done in triplicate and the controls with no template and no reverse transcription were included for each gene.The banana 5S r RNA was used as an internal control. The relative expression quantification was calculated using the equation 2- ΔΔCTmethod.【Results】In order to examine ten conserved mi RNAs(mi R156 d,mi R162 a, mi R164 e, mi R166 c, mi R167 c, mi R169 h, mi R319 m, mi R399 a, mi R4995 and mi R5538), a stem-loop RT-PCR was performed using a total RNA isolated from roots, leaves, flowers and fruits. Except for mi R166 c, mi R319 m and mi R5538, 7 mi RNAs showed the expected size at 70 nt among four tissues.Mi R166 c was detected in roots, leaves and flowers. Mi R319 m and mi R5538 were detected in single tissue,respectively in fruits and flowers. All the PCR products were cloned and verified by sequencing. Meanwhile, the expression level of 4 randomly selected mi RNAs was analyzed by quantitative real-time PCR.Mi R156 d and mi R162 a presented similar orders of expression in leaves, roots, flowers and fruits, with both presenting the highest expression levels in flower tissue. The expression of mi R156 d in flower was 2.25,obviously higher than the quantity in roots(0.17),fruits(0.34) and leaves(1.00). The expression of mi R162 a in flowers was 4.36,obviously higher than the quantity in roots(1.72),fruits(0.99) and leaves(1.00). As for mi R164 e, the expression level in roots was highest(2.37), followed by that in flowers(1.93), fruits(0.98),and leaves(1.00). The expression of mi R169 h was not significantly different in these four tissues. Thus, the conserved mi RNAs were expressed in a tissue-specific manner.【Conclusion】Our approach is a suitable and effective method for identification of conserved mi RNAs from the banana's four tissues with stemloop q RT-PCR and total nucleic acid as the template. The expression patterns of the 4 randomly selected mi RNAs were tissue-specific, suggesting that mi RNAs may play an important role in banana development.Future work to characterize the mi RNAs' targets and their mutual relationship is necessary to fully elucidate the functions of mi RNAs in the development and physiology of bananas(Musa spp.).