Regulation Effect Of MiR398 In Panax Ginseng Under The High Salt And High Copper Stress

Micro RNAs(mi RNAs) are a class of non-coding RNAs of 22 nucleotides that regulate gene expression. Mi R398 directly linked with stress regulatory networks by targeting Cu/Zn superoxide dismutase(CSD) in higher plants. Reactive oxygen species(ROS) is inevitable byproduct of photosynthesis and respiration in plant, which is a hurt for plant development. Salinity stresses, heavy metal, cold damage, mechanical damage and other stresses can induce photosynthetic electron transport chain and respiratory chain to produce excess ROS. CSD is the major superoxide dismutase converting ROS into H2O2 and O2. Salinity and heavy metals stresses are the main factors affecting the growth of ginseng. In this experiment, we studied relative expression levels of mi R398 and target gene CSD in Panax ginseng. We build mi R398 sponge element to achieve adsorption of mi R398 by introducing plant genome. Then us lift silencing of the target gene CSD and increase the relative expression level of CSD gene. Thus the above works enhance the ability of plants to withstand salinity and heavy metal stress. Specific experiment results are as follows:1. Total RNA was extracted by CTAB method. The c DNA was synthesized by RT-primer, and the PCR product link with clone vector p MD18-T. Then the p MD18-T was transformated into E.coli JM109. The mi R398 sequence was obtained by sequencing extracted plasmid. The mi R398 sequence was submitted to Gen Bank, and the accession number is KM922596;2. In the salinity and high copper stress conditions, the relative expression levels of pg-mi R398 and its target genes CSD changed. Under stress conditions, the relative expression level of mi R398 significantly decreased, and the relationship between mi R398 and stress response in Pg was determined; the expression level of CSD gene increased under stress conditions, and the negative regulatory relationship between mi R398 and CSD gene in Pg was verified;3. pBI121-398 S expression vector was constructed, in order to achieve specific "adsorption" of mi R398. The p BI121-398 S expression vector was transformated into A4 engineering bacteria after sequencing, to obtain transgenic Pg hairy roots hairy-p BI121-398S;4. Through real-time quantitative PCR, we detected the relative expression level of mi R398 and target gene CSD in transgenic ginseng hairy roots hairy-p BI121-398 S. The mi R398 relative expression level was significantly decreased by 40%, the relative expression level of target gene CSD was improved, which verified the mi R398 sponge "adsorption" effection;5. The growth rate of transgenic hairy roots hairy-p BI121-398 S is higher than that of the control; Relative level of total ginsenosides of transgenic Pg hairy roots is higher than that of control by 9% analyzed with the vanillin colorimetric determination.