香蕉不同器官中microRNA差异表达分析

Differential expression analysis of microRNA in banana different organs

【目的】研究mi RNA在香蕉不同器官中的表达,初步探索mi RNA在香蕉生长发育过程中的可能作用。【方法】利用茎环引物的半定量和实时荧光定量PCR策略,检测香蕉中的保守mi RNA,并进一步研究mi RNA在香蕉根系、叶片、雄花和果实组织中的表达差异。【结果】mi RNA156d和mi RNA162a具有相似的表达模式,其最高表达量均出现在雄花组织中。mi R156d在雄花中的表达量(2.25)明显高于根系(0.17)、果实(0.34)和叶片(1.00);mi R162a在雄花中的表达量最高,为4.36,根系组织次之,为1.72,而在叶(1.00)和果实(0.99)中的表达几乎相同。mi R164e在根系中的表达量最高,为2.37,雄花次之,为1.93,而在叶(1.00)和果实(0.98)中表达几乎相同。mi R169h在4种组织中的表达量差异不明显。【结论】以香蕉各组织总RNA为模板,通过以SYBR Green为染料的Stem-loop q RT-PCR方法,成功地在香蕉根系、叶片、雄花和果实4种器官中均检测到了保守mi RNA的表达,表明本研究所建立的体系是定性或定量检测香蕉植株中保守mi RNA的准确而有效的方法。同时,在香蕉的不同组织中发现了具有显著表达差异的mi RNA,符合mi RNA在不同组织的表达活性具有多样性的规律。

[Objective] MicroRNAs （miRNAs） are endogenous non-coding small RNAs （sRNAs） 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 miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in Musa acuminata （A genome） and Musa balbisiana （B genome）. To obtain more insight into the role of miRNAs in banana growth and development, we implemented conserved miR- NAs 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 of the 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 （Chaiet al., 2014） and briefly exposed to RNAasefree DNAase I. cDNAs were synthesized by M-MLV according to the manufacturer＇s instruction, using the specific stem-loop primers for miRNAs. Stem-loop RT-PCR and qRT-PCR were applied to detect the tissuespecific expression levels of conserved miRNA 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 qRT-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 FastStart 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 qRT-PCR reactions were performed using Fast- Start Universal SYBR Green Master （ROX） on a Mx3005PTM 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 rRNA was used as an internal control. The relative expression quantification was calculated using the equation 2^- △△CT method. [Results] In order to examine ten conserved miRNAs （miR156d, miR162a, miR164e, miR166c, miR167c, miR169h, miR319m, miR399a, miR4995 and miR5538）, a stem-loop RT-PCR was performed using a total RNA isolated from roots, leaves, flowers and fruits. Except for miR166c, miR319m and miR5538, 7 miRNAs showed the expected size at 70nt among four tissues. MiR166c was detected in roots, leaves and flowers. MiR319m and miR5538 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 miRNAs was analyzed by quantitative real-time PCR. MiR156d and miR162a presented similar orders of expression in leaves, roots, flowers and fruits, with both presenting the highest expression levels in flower tissue. The expression of miR 156d in flower was 2.25, ob- viously higher than the quantity in roots （0.17）, fruits （0.34） and leaves （1.00）. The expression of miR162a in flowers was 4.36, obviously higher than the quantity in roots （1.72）, fruits （0.99） and leaves （1.00）. As for miR164e, 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 miR169h was not significantly different in these four tissues. Thus, the conserved miRNAs were expressed in a tissue-specific manner. [Conclusion] Our approach is a suitable and effective method for identification of conserved miRNAs from the banana＇ s four tissues with stemloop qRT-PCR and total nucleic acid as the template. The expression patterns of the 4 randomly selected miRNAs were tissue-specific, suggesting that miRNAs may play an important role in banana development. Future work to characterize the miRNAs＇ targets and their mutual relationship is necessary to fully elucidate the functions of miRNAs in the development and physiology of bananas （Musa spp.）.