| Identification of the origin of Chinese medicine is a major issue in medical research, its purpose is to solve the traditional Chinese medicine "pros and cons" of the problem, the traditional Chinese medicine origin identification methods such as character identification, microscopic identification, physical and chemical identification, etc. Because of subjectivity, poor stability and repeatability from defects, molecular biology can be used as a new technique for the identification of molecular medicine.MicroRNA (miRNA) as an endogeny of plant origin, non-coded-protein small molecule RNA, it plays an important role in plant growth and development, organizational aspects of organ differentiation and anti-Stress. SNP markers as the third generation of molecular marker, it also plays an important role in molecular genetics, pharmacogenomics and molecular identification. Honeysuckle is a commonly used bulk herbs, it’s appointed as one of the expensive medicines by the State Council. It’s mainly produced in Shandong, Henan and other places. It’s widely cultivated in China. Honeysuckle has detoxify, cool wind-heat powder effect. In recent years, market demand continues to expand and the supply and demand have become increasingly prominent. Honeysuckle origin of more complex species, it’s not only affects the quality of medicine, but also adversely affect the efficacy. Evaluation of existing quality standards difficult overall quality of honeysuckle, they are better quality and authentic origin honeysuckle. It has become a major problem in Lonicera japonica origin authentication solving, and it’s for Lonicera japonica medicinal quality standards established to provide scientific methods and data.The research is first to set Lonicera japonica herbs as research material, by miRNA and SNP molecular biology ways, it systematic analysis of different origins and different varieties of Lonicera japonica. The results show:Lonicera japonica’s miRNA and its target genes in Lonicera japonica different environmental and genetic conditions showed different expression levels. Henan Lonicera japonica and other sources Lonicera japonica have appeared in different SNP site.This research was first to use Hiseq sequencing constructed Lonicera japonica three different samples of miRNA database, it use of bioinformatics to analyze the three miRNA database of relevant information, we found three database-specific sequences and consensus sequences and pairwise comparisons of the number of genetic differences, at the same time we predicted a Lonicera japonica genome miRNA and its target gene, construction of the miRNA precursor structure and by RT-PCR and RLM-RACE method wherein a part of the predicted miRNA target genes were verified, we found two miRNA target genes and three interaction and expression in three different samples having differences Lonicera japonica. It can be used as molecular markers have been applied for between three venues. In the RT-PCR process, the research was obtained by screening a stable Lonicera japonica in different habitats and species of miRNA expression in internal control, it may provide a reference for Lonicera japonica miRNA fluorescence quantitative experiments. Then the research to 15 Lonicera japonica origin transcriptome database SNP sites for the overall analysis and cluster analysis, and 67 randomly selected sites SNP initial screening, then add 36 pairs of primers were Miseq joint screening after sequencing. Through specific primers with mismatches screening, it adjust the annealing temperature and the type of enzyme used. Finally we obtained two specific primers, using a combination can be distinguished from the origin of Henan Lonicera japonica and Lonicera japonica form other regions. |
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