| miRNAs are small, non-coding regulatory RNAs which come from endogenous eukaryoticgenome and play important regulatory roles in plant growth and development. To date, thereare a few reports about miRNAs in fruit tree, such as apple and grape. The sequencesconservation of miRNAs will lay a sound foundation for studying the effect of miRNAs onnon-model plants whose genomes are unknown. Despite the widespread cultivation ofmicropropagated strawberry plants, several problems are encountered in the use. One of themost serious of these problems was the epigenetic variation. Until now, there is no report onmiRNAs in strawberry and the relationship between miRNAs expression and epigeneticvariation. In this paper, species and their conservation of miRNAs, and the effects to miRNAexpression of organs, propagated manners, development stages, etc, were completely studiedby using cultivated strawberries as materials. The miRNAs with expressed difference werefound and their probable functions in strawberry growth and development were analyzed. Themain results were as follows:1. On the base of extracting high quality small RNAs (less than 150 bases) with themodified CTAB method, 74 miRNAs were checked in strawberry plants by gene chip. Therewere 47 miRNAs whose intensity value of fluorescent signals maintained from 400 to 1500and 27 miRNAs beyond 1500. Among them, the 46 miRNAs from miRBase represented 21miRNAs gene families, miR159 represented the largest miRNA family in F. ananassa, whichhad seven members. There were five members for miR169 family in F. ananassa, miR160,miR164, miR166 and miR167 all had four members, miR156, miR168 and miR171 all hadtwo members. Other 12 miRNA families including miR157, miR162, miR165, miR170,miR172, miR390, miR391, miR393, miR394, miR396, miR399 and miR535, only had onemember.2. According to the miRBase Registry, the 21 miRNA families checked in strawberrywere compared with other nine plants, such as Arabidopsis, rice, populus and so on. Fifteenfamiles (miR157, miR159, miR162, miR165, miR166, miR167, miR169, miR171, miR172,miR390, miR391, miR393, miR394, miR396 and miR399) were also detected in A. thaliana,and twelve families of them (the exceptions were miR157, miR165 and miR391) werecompletely conserved in rice. Three rice miRNA families (miR156, miR170 and miR535)were also present in strawberry. And 15(miR157, miR159, miR160, miR164, miR166,miR167, miR169, miR170, miR171, miR172, miR390, miR393, miR394, miR396 andmiR399) of these familes were also detected in P. trichocarpa. Among them, four miRNAfamilies (miR159, miR166, miR167 and miR169) deeply conserved in many plants fromeudicots to monocots, even to gymnosperms.3. Using small RNAs (less than 150 bases) as templates, the conserved miRNAs ofstrawberry were detected by three kinds of RT-PCR methods, including using 3' adaptor, 5'and 3' adaptor and stem-loop primers. The results showed that for fifteen conserved miRNAs,seven miRNAs were identified by using 3' adaptor, only one miRNA was successfullyidentified with the method of 5' adaptor and 3' adaptor, while fifteen miRNAs were allidentified by using stem-loop primers and which had high sensitivity. Three kinds of templates, total nucleic acid, total RNA and small RNAs were used to compare the effect onidentification of miRNAs with stem-loop primers. No difference was found in PCR fragmentsby using different nucleic acids as templates. Because the extraction of total nucleic acid wasquite simple and rapid, the suitable method for qualitative identification of the conservedmiRNAs from plants with RT-PCR was to use stem-loop primers and total nucleic acid astemplate. While the suitable method for quantitative study of miRNAs with RT-PCR was touse stem-loop primers and total RNA as template.4. The technology system of real time RT-PCR for quantitative study of miRNAs withstem loop RT-PCR and TaqMan probe was established.5. The expressed difference of 74 miRNAs between conventionally propagatedstrawberry plants (CS) and micropropagated strawberry plants (MS) was studied by usinggene chip. Four miRNAs, including miR535, miR390, miR169a and miR169d, displayedobvious expression difference between conventional and micropropagated strawberry palnts.Among these, miR535 and miR390 was two up-regulated genes and miR169a and miR169dwas two down-regulated genes. The ratio of conventionally propagated strawberryplant/micropropagated strawberry plant for miR535, miR390, miR169a and miR169d were2.6884, 2.2673, 0.2496 and 0.3814, respectively. Real-time RT-PCR applied to the twoup-regulated genes validated the microarray result.6. The difference expression of six miRNAs between root, receptacle, shoot tip and leafof strawberry was studied by real-time RT-PCR. When the relative quantitation of sixmiRNAs in strawberry leaf was as 1, the result showed that the highest expression inreceptacle was miR164, reaching 6.615, obviously beyond the quantitation in root (0.485),shoot tip (0.371) and leaf (1). While the expression of miR172 in shoot tip and receptaclewere 0.029and 0.020, lower than that in leaf. The lowest expression of miR172 was in root,only 0.0008454, the same as miR167, only 0.053. No obvious difference in expressions ofmiR156, miR159 and miR165 was found in four kinds of strawberry organs.7. The expressed difference of five miRNAs (miR156, miR172, miR164, miR167 andmiR159) with important functions in plant growth and development was compared between invitro and vivo, and different development stages of 'Toyonoka' and 'Allstar'. The resultshowed that he highest expression of miR156 was in in vitro, reaching 41.846 ('Toyonoka')and 35.782 ('Allstar'). The expressed quantitation in vivo (the first progeny ofmicropropagated strawberry plants) only had 0.999 ('Toyonoka') and 1.038 ('Allstar'). Theexpressed quantitation of miR156 reduced more than 30 times from in vitro to vivo. And noobvious difference was found in different adult development stages. While the expression ofmiR172 was very low ('Toyonoka': 0.040 and 'Allstar': 0.068) in in vitro, but increased morethan 8 times in vivo (MS1). And no obvious difference was found in different adult stages.8. The expression of miR169 could be up-regulated by tissue culture condition, andmiR390 and miR535 probably played an important role in epigenetic variation ofmicropropagated strawberries. |
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