Cloning And Primary Functional Study Of Drought Tolerance Relate MiRNAs In Rice

Rice is one of the most important food crops in China, and it is the major water user in agriculture which itself is the major water user in Chinese economy. However, China is also a state with very poor amount of available water resources per capital, drought stress has become an important factor limiting rice yield in China. Therefore, breeding for drought resistance in rice, improvement of drought resistance ability in rice through application of new biotechnowledge in rice is significantly important.Micro RNAs are a class of endogenous non-coding single-stranded small molecule RNA about 22 nucleotides long. Widely distributed in the plant genome, miRNA is a kind of negative regulators of gene expression, which regulates gene expression at the level of transcription and post-transcription. Its target genes coding signaling proteins, enzymes, transcription factors, mainly involve in many biological processes, including development, metabolism, stress resistance and so on. Studies have shown that a miRNA is usually targeting more than one target genes, even with a variety of biological processes, and therefore, transformation of miRNA into plant genomes may be more practically important than traditional insertion of functional genes. If the key miRNA in drought tolerance responsce is uncovered, a single gene insertion into rice genome could economically regulate gene expressons of many drought related genes, which will hopefully create good new drought-resistant transgenic lines. Therefore, miRNA research has important biological and economic significance.This study mainly cloned the four potentially drought tolerance or response related miRNAs screened from the previous study in our laboratory, they are osa-miR171a、osa-miR397b、osa-miR398a、osa-miR408,carried out genetic trasformation of these osa-miRNAs into rice genome to explore the possible role of these miRNAs in drought responses in rice plant. Major findings are as follows:1. To the four drought-response related candidate osa-miRNAs, the over expression vectors had been constructed and transformed into rice plant cv.Nipponbare.As a result, many transformant lines had been obtained.2. The target genes of the four candidate osa-miRNAs had been predicted through bioinformatic method.3. Expression analysis at seedling stage showed that four candidate osa-miRNAs showed up-regulated expressions under drought stress at varying degrees. 4. The T1 generation seedlings of osa-miR408 and osa-miR397 transformants showed drought toleramce to a certain extent, under drouhgt stress by water withholding.5. For osa-miR408, a number of other studies had been conducted:a. analysis of the promoter region revealed that its promoter region contains multiple drought stress-related cis-acting elements; b. in the TO genereation plants of osa-miR408 transformants, real-time PCR analysis of the four predicted target genes discovered that three target genes showed negative regulation; c. two target genes (LOC_Os03g15340.1 and LOC_Os08g37670.1) had been selected from the three negative regulated target genes, over-expression vectors and targeting site mutated vectors were constructed and transformed into rice variety Nipponbare resulting in a large amount of transgenic plants with over-expressed target genes.Through this study and previous results, we believe that miRNA, copper balance and drought stress are in close linkage.