| Rice(Oryza sativa L.) is one of the most important food crops in the world. Low temperature stress is one of the important environmental factors that affect rice production, seriously affecting the rice yield, quality and geographic distribution in seedling and generation stages. Thus, illustrate the mechanism of cold tolerance in rice, and found that the main effect genes, cold tolerance in rice in order to cultivate cold resistant rice varieties by genetic engineering means, is one of the hotspots in the field of plant gene engineering research. Micro RNAs(mi RNAs) are approximate 21 nucleotide(nt) single-stranded RNAs that play important roles in plants biological processes by targeting m RNAs cleavage or translational repression. mi R156 k was reported to be involved in the regulation of plant growth and development. In our previous study, we found mi R156 k was down-regulated under cold stress by microarray. After that we use real-time PCR assay to verify the microarray results. So we speculated that mi R156 k plays a important role in response to cold stress in rice.In this study, we over-expression of mi R156 k was done with the Agrobacterium co-cultivation method to check their effect on cold response. Then, we treated the transgenic plant to cold stress, and analysis the function of transgenic plants to cold stress.The main results in this study are as follows:(1) Over-expression of mi R156 k and fouctional analysis in response to cold stress in riceWe constructed the pre-mi R156 k plant overexpression vectors and transformated for rice callus. Through PCR, Southern blotting detection, we finally obtained transgenic lines of mi R156 k.(2) Functional analysis of mi R156 k in response to cold stress in riceThen we treated the T2 mi R156 k overexpression rice with cold stress, the result shown that transgenic plants decreased the tolerance to cold stress at the very early development stage and the young seedling stage in rice. The chlorophyll content, proline content and the activity of SOD were also shown significant lower than wild type rice under cold stress.(3) The molecular mechanism of mi R156 k in cold stress responseWe further examined the expression patterns of cold signaling-related genes in both WT and OX plants, including Os01g22249, a ROS scavenging gene and Os P5 CS, a proline synthase gene. Our real-time PCR data indicated that expression of Os01g22249 and OsP5 CS was up-regulated in both WT and OX plants under cold stress. We also investigated the expression patterns of Osmi R156 k targeted 19 SPL genes predicted previously. We firstly analyzed their expression changes in response to cold stress, based on the rice mRNA microarray data carried ou t our lab. SPL3, SPL14 and SPL17 were differentially expressed in response to cold stress. Our real-time PCR data further confirmed that expression levels of SPL genes were induced by cold treatment. However, higher transcript levels were found in WT plants than in OX, especially at 9h post cold treatment. |
