Prediction Of Rice Cold Stress Related MiRNA Genes And Verification Of Their Relativity With Cold Stress

Low temperature and cold injury are the common foe of the rice production; they are the environmental factors give an impact to plant growth, development, and geographical distribution and adversely affect rice production. Using the bioengineering strategies to improve cold stress tolerance in rice has been an important aspect of modern agricultural research. While understanding the mechanisms by which rice response to cold stress, the type of rice gene expression in cold stress condition and screening the key genes from considerable cold stress-relative are of fundamental importance to biology.Conventional methods of molecular biology increase the plant resistance by introducing functional genes. This strategy already has been achieved some success, but introducing a single gene has been difficult for a substantial increase, in another hand, introducing multigene ofen arouse poor coordination, difficult operation and genetic instability problems. MicroRNAs (miRNA) are kind of small, single-stranded no-coding RNAs, often only 22nt long. MicroRNAs can regulate gene express of eukaryote. Some of which have been shown to play important regulatory roles during plant development and stress tolerant process. For exploiting the utilization potential of microRNA in the field of plant cold stress tolerant genetic engineering, our laboratory use the miRNA database and miRNA target gene prediction software find the miRNA target mRNAs of rice by bioinformatics method, and select the cold stress related target mRNAs according to the mRNA database and mRNA microarray database of rice. These target mRNAs are the rice cold stress related miRNA gene. This will provide opportunities to understand the miRNA genetic mechanisms underlying cold stress response of rice. We will obtain the new miRNA genes of significance to rice cold stress-resistance which may be used in rice cold stress genetic engineering field. The main results was summarized as follows:1. Predication of target mRNAs for rice miRNAsTotal known rice miRNAs are isolated from miRNA database, and total rice miRNAs' target genes are predicted by miRNA target prediction software. The total number of predicted target mRNAs is 2138.2. Generation of rice cold stress related target mRNAsDownload gene ontology database, and search it by cold stress related key word chosen according by cold stress response mechanism and literature to gain cold stress related GO term. Make BLASTx to miRNAs' targets in Uniprot protein database, gain correspond protein sequences, and extractive their GO term. Compare cold stress related GO term to gain cold stress related target genes. In another way, according by some investigators, CBF and F-box gene families generally play important roles in plant cold stress tolerance response, so we select CBF and F-box target genes too. Combine upward 2 method, we gain 19 rice cold stress related target mRNAs.3. Predication of rice cold stress related miRNAs according to their target mRNAsIf one miRNA's target mRNAs' function is related about cold stress, thus this miRNA gene may have some relationship to cold stress; if one miRNA's most target mRNAs' function is related about cold stress, thus this miRNA may be the key cold stress related miRNA. By this method, we gained 10 cold stress related miRNA gene families, in these miRNAs, there are 4 miRNAs concluded the key cold stress related miRNAs.4. Verification of rice cold stress related miRNAsWe had randomly chosen 2 miRNAs from predication cold stress related miRNAs in previous step, osa-miR393 and osa-miR396c. Total RNA was isolated from rice young plant treated by different cold time, and was reverse transcripted to template cDNA. The expression is analyzed by real-time PCR. The result suggests that these 2 miRNAs are cold stress related miRNAs, also proves our strategy of cold stress related miRNA is effective and feasible.