Study On Function Of MiRNA164 And Its Target Gene Of Potato

Micro RNAs(mi RNAs) are a class of endogenous small molecule RNAs, which regulate gene expression on post-transcriptional level. NAC transcription factors play variety of biological functions on signal transduction network in plant resistance to adverse circumstances, which can be induced to express by adversity stresses such as drought and salt. Research showed that mi RNA164/NAC involved in the regulation of plant growth and development as well as the resistance to biotic and abiotic stress tolerance in model plant Arabidopsis thaliana. However, there was no report about the study on the expression pattern of mi RNA164/NAC in potato. In this research, we used potato known mi RNA164 sequence and online tool ps RNATarget to predict its target genes, through the bioinformatics analysis to designing specific primers to construct the plant expression vector, to obtain the transgenic potato plants by the Agrobacterium mediated genetic transformation. In order to further validate mi RNA164/NAC regulation mode, the potato ami RNA164 expression vector was constructed using artificial mi RNA(ami RNA) technique, and transformed to obtain the transgenic potato plants, through the changes of morphological characteristics and q RT-PCR to analyze the NAC transcription factor gene expression quantity for further validation of mi RNA164/NAC regulation mode. The research results are as follows:1. Three target genes of mi RNA164 were obtained through the online predict tools ps RNATarget. It was found that the three target genes are all belong to the NAM subfamily of NAC transcription factors family, and gene structure are highly conservative by bioinformatics analysis.2. Specific primers were designed to clone NAC262 transcription factor gene and construct plant expression vector, which was driven by the Ca MV 35 S promoter. The potato cultivar ??Kenxin NO.3?? was as the materials for genetic transformation and obtained 8 transgenic potato plants.3. The potato ami RNA164 expression vector p CPB121-mi R164 was constructed driven by the Ca MV 35 S promoter and The potato cultivar ??Gannong NO.2?? was as the materials for genetic transformation and obtained 10 transgenic potato plants.4. After the transgenic plants were treated by PEG 6000, and the relative expressional levels of NAC262 gene were analyzed using the q RT-PCR techniques and the result showed that NAC262 gene expression demonstrated a trend of rising from 0 to 8 h under 20% PEG stress, and when the stress reached 8 h, NAC262 gene expression reached the maximum amount, while NAC262 gene expression demonstrated a trend of decreasing from 8 to 32 h,. Overall, the relative expressional levels of NAC262 gene in root was higher than stem/leaf tissue., The NAC262 gene expression in the transgenic potato plant lines T1 and T2 was significantly lower than the untransformed potato variety ??Gannong NO.2?? plants under PEG stress, while the expression of NAC262 gene in the transgenic potato plant lines T3 and T4 was significantly higher than the untransformed potato variety ??Kenxin NO.3?? plants.5. Five transgenic potato plant lines were choosed randomly to calculate and measure the number and length of lateral roots and found thatthe lateral root number of T1 and T2 transformed with mi RNA164 decreased significantly compared with the untransformed controls, but the root length had no significant change. While the lateral root number of T3 and T4 transformed with NAC262 increased significantly, the root length was shortened, while leaf and stem was thicker than the untransformed plants.In summary, mi RNA164 can mediate NAC262 transcription factor to regulate the development of lateral root in potato and the expression pattern was similar to Arabidopsis thaliana. The mi RNA164/NAC expression pattern was induced by PEG stress and involved in plant response to drought stress resistance.