Analysis On The Functions Of MiR168 Promoter And The MicroRNAs Regulation During Red Coloration Of Apple Skin

Plant micro RNAs(miRNAs) are non-coding small RNAs which are widely distributed in plant genomes and can regulate gene expression at the post transcriptional level by degrading or repressing targeted gene m RNAs, playing important roles in regulating plant growth, development and stress responses.(1)miR168 is highly conservation in many plant species, which can indirect control of all miRNA by regulating the expression of target gene AGO1. However, wether the function of miR168 in different plant species is conservation has not been reported. In this study, grape, poplar and Arabidopsis as test materials, we analyze number,position,function of their miR168 promoter cis-element and find changes in miR168 promoter function of grape and model plant.(2)Bagging influences the acuumulation of anthocyanin and regulates anthocyanin biosynthesis at transcription level. As a result,bagging can promote the fruit appearance quality.However, it has not been reported if bagging play roles in the regulatory mechanism of anthocyanin biosynthesis at post-transcriptional level.The bagged and unbagges ‘Granny Smith’ apple peel were used as experimental materials in this study and analysis of the different expressed miRNAs caused by bagging. These work lay important foundation for further researches on fruit coloration.The main results in this study as follows:(1)The results of the alignment of miRNA families showed the nucleotide sequences of the miRNAs, miR477, miR482 and miR3627, in same family were variable. And the conversation of them was less than the conserved miRNAs in plants,miR168, miR156 and miR172, especially miR168 was highly conserved.(2)Four miR168 promoters’ sequences with the 2-2.3kb length were isolated from grapes, poplar and Arabidopsis genome, respectively. We obtained p Vit Vin MIR168,p Pop Tri MIR168 A, p Pop Tri MIR168 B and p Ara Tha MIR168 A. The distribution of cis-element G-box in miR168 promoters of different plant species was different, analyzed by using Plant CARE software.The p Vit Vin MIR168 and p Pop Tri MIR168 A sequence contained two G-box conserved elements, only a G-box element in the p Pop Tri MIR168 B and three G-box in p Ath AramiR168 A.(3)GUS staining experiments with transgenic plant of different miR168 promoters show that p Vit VinmiR168:GUS are specific expressed in emerging leaf, emerging leafprimodia, blade tip of the cotyledons, vasculature of the cotyledons, lateral root tip, and primary root tip. Both p Vit VinmiR168:GUS and p Pop Tri MIR168A:GUS constructs produced the same expression patterns, except for emerging leaf which instead of hypocotyl/root junction. The expression of p Pop Tri MIR168 B was localized in emerging leaf primodia,primary root tip, lateral root primodia and hypocotyl/root junction. The expression of p Ara Ath MIR168 A was localized in emerging leaf, blade tip, vasculature of the cotyledons,emerging leaf primodia. Results showed similar expression patterns were observed for p Vit VinmiR168 and p Pop Tri MIR168 A.(4)The vector of complementary phenotype, vector p Ara AthmiR168A:miR168 m and vetor AGO1 m, were successfully constructed. The deficient phenotype of T1 generation transgenic plants with AGO1 mutants were mostly recovered to a wild-type phenotype, only9% transgenic plants were not completely recovery.Three set of phenotypic complementation vectors, consisted of vector AGO1 m and p Vit Vin MIR168:athmiR168 m,p Pop Tri MIR168A:athmiR168 m,p Pop Tri MIR168B:athmiR168 m, were constructed, respectively. After co-transformation, the positive transgenic plants were obtained. Analysis of ability to recover deficient phenotypem causing the AGO1 m in miR168 promoter from different plant species, the results showed that the function of p Vit VinmiR168 and Pop TrimiR168 A were similar, but different from Pop TrimiR168 B and p Ara AthmiR168 A.(5) Small RNA libraries were constructed from peels of ‘Granny Smith’ apples subjected to bagging followed by sunlight re-exposure treatments and from peels of apples without any bagging treatments. 201 known miRNAs belonging to 43 miRNA families and220 novel miRNAs were identified via high-throughput sequencing. After bag removal, some miRNAs were found to be differentially expressed after debagging. The expression levels of twenty-one miRNAs after bag removal 6h were significantly down-regulated compared to those of bag removal 0h, except miR399 d and the expression levels of three miRNAs were up-regulated. After annotated some target genes of high expressed miRNA, we found the miRNAs target gene were transcription factors, heat response factors, and antioxidant enzyme system et al. They might play important roles in photo-protective response and adaptation to high light stress after bag removal.(6)To further explore the effect of debagging on miRNAs regulating the expression of anthocyanin regulatory genes, four miRNAs and their target genes regulating anthocyanin accumulation, mdm-miR156, mdm-miR828, mdm-miR858 and miR5072, were compared between green cultivar ‘Granny Smith’ and red cultivar ‘Starkrimson’. Results showed that the differential expression levels of mdm-miR828 and mdm-miR858 and their target genesregulated anthocyanin contents in both apple cultivars, while the expression levels of mdm-miR156 and its target gene SPL only affected anthocyanin accumulation in ‘Granny Smith’, and the expression levels of miR5072 and its target gene Md ANR affected anthocyanin accumulation in ‘Starkrimson’.