Cloning And Functional Research Of MiR166 And Its Target Gene In Potato

MicroRNAs(miRNAs) are endogenous, small non-coding RNAs that are involved in post-transcriptional gene silencing. In plants, most miRNA recognized target mRNA sequence by near-perfect complementary way in the post-transcriptional level, which causing degradation of the target genes or inhibiting its translation, and ultimately to suppress the expression of target gene. miR166 is a class highly conserved miRNA in monocots and dicots plants and its targets are the HD-Zip? subfamily members which regulated apical meristem, vascular bundle, paraxial region lateral organs developmentally. In this paper, three target genes of stu-miR166 b was cloned from potato, then bioinformatics analysis and cleavage site for verification, study the tissue expression pattern and stress response of stu-miR166 b and its target genes; Using pRS300 plasmid as a template to construct plant expression vector contain miR166 b mature sequences, through Agrobacterium-mediated transformation into a test tube potato and obtained transgenic plants and further analysis phenotypic. The main results of this paper are as follows:1. There have four stu-miR166 precursor in the potato genome, transcribed sequence are not the same three mature body stu-miR166a5p?stu-miR166a3 p and stu-miR166 b. stu-miR166a5 p predicted target genes are some hormones and early dehydration protein. stu-miR166 b have five target genes PGSC0003DMT400030829(StHB14) ? PGSC0003DMT400054421(StHB14-like) ? PGSC0003DMT400020801(StREV) ? PGSC0003DMT400006735(StHB15) and PGSC0003DMT400068037(StHB15-like). According transcriptome sequence NCBI registration and RT-PCR technique, which three gene StHB14, StHB15 and StREV was cloned, respectively. Bioinformatics analysis showed StHB14?StHB15 and StREV are non-secreted proteins, them have unique characteristic of HD-Zip ? transcription factor. Through RLM-RACE is determined stu-miR166 b cutting target gene in the tenth to eleventh of complementary binding region.2. Expression analyses revealed that stu-miR166b/ StHB14?StHB15 and StREV gene was expressed in root, stem, leaf of potato, and stu-miR166 b with a relative higher expression level in root of potato. Conversely, StHB14, StREV expression abundance is lowest in root, StHB14 in stem lowest.3. qRT-PCR analysis showed that stu-miR166b/StHB14, StHB15 and StREV have different transcription patterns in two stress treatments of above and underground tissues. In addition to the aboveground parts of NaCl for 24 h and underground parts of PEG stress after 24 h stu-mi R166 b expression decreased, others stu-miR166 b expression levels rised. When aboveground under PEG and NaCl stress, StHB14 is unique expression level down-regulated genes throughout the stages. In the underground part, when subjected to PEG stress StREV expression level down, NaCl inhibited the expression StHB14.4. Designing primers of miR166 b by WMD3 platform, after a two-step PCR synthesized precursor sequence containing miR166 b mature sequence by pRS300 plasmid as a template. We constructed transgenic vector containing CaMV35 S promoter and then obtained four miR166b-overexpresed potato plants by Agrobacterium-mediated transformation. qRT-PCR analysis showed that the target mRNA expression levels decreased in varying degrees of 0.35-5 times. Phenotypic analysis showed that transgenic plants growth was inhibited.