Isolation And Identification Of Microrna In Oryza Sativa And Brassica Napus Under Cd Stress And Functional Characterization Of MiR395

MicroRNA (miRNA) is a novel class of short, endogenous non-coding small RNAs that regulate gene post-transcriptional expression in bot plants and animals. Thus far, a growing number of miRNA have been isolated and characterized from a variety of plant species by bioinformatical and experimental methods and deposited in the major miRNA database (http://www.sanger.ac.uk/). Cadmium has become one of the major sources of toxic heavy metal pollution in agricultural lands. Accumulation of Cd by plants may disrupt many cellular functions. However, plants may also regulate their adaptation to heavy mental stress, e.g. gene expression for sulfate and GSH assimilation are up-regulated. Although a number of plant miRNA have been isolated, many of other miRNAs, particularly those in response to heavy stresses and non-conserved miRNAs remain to be identified.As a cereal grain, rice is the most important staple food for a large part of the world human population. The rice genome sequencing was completed in 2002, which brings basic foundation to analysis of miRNA data. To identify novel heavy metal stress-regulated miRNA, we constructed a library of sRNAs from Oryza sativa that were exposed to 80μM CdCl2. Sequencing of the clones and subsequent analysis revealed 19 new miRNA representing 6 families and have no sequence conservation in any other plant species. We also cloned 10 miRNAs that were previously reported in Oryza sativa. Nine of them are conserved miRNA in plants. There were 56 new other endogenous siRNAs identified from the library. We predicted 34 potential target for the 19 new miRNAs. Transcript analyses revealed that 6 new and 10 known miRNAs showed different gene expression under Cd, Cu and Al stresses. miR602 was up-regulated and its target XET was down-regulated. Also, miR604 and its target gene LTP was differentially regulated in expression under the metal stress. Brassica napus was the third leading source of vegetable oil in the world in 2000. In this study, we identified 5 miRNA from a small RNA library which was constructed under sulfur deficiency and Cd stress. Two new ATP sulfurylase genes, BnAPS3 (FJ626851) and BnAPS4 (FJ626850), were cloned. Both genes along with BnSultr2;1 and BnAPSl were identified as the targets of miR395. Analysis with 5’RACE and transformation of miR395d into B. napus confirmed that all of them were the authentic targets of miR395. Our results support the importance of miRNA in regulating plant responses to abiotic stresses and suggest that identification of a comprehensive set of miRNA would facilitate our understanding of regulatory mechanisms for plant tolerance to sulfate-deficiency and heavy metal stress. The transformants display a phenotype of shorten or no trichomes on the surface of leaf and stem at the early stage and a delayed transition from a juvenile to an adult stage of vegetative development.There are 6 miR395 loci in Arabidopsis thaliana, controlling 4 differernt sulfate assimilation gene. To get insights into the regulatory role of miR395, we constructed transgenic Arabidopsis thaliana plants over-expressing pre-miR395c and pre-miR395e. We also constructed target mimicry mutants in SLIM1 to silence miR395 expression. Analysis of transformants revealed that miR395 blocked all of its targets expression. Correspondingly, when the activity of miR395 was reduced in Mimic395, the target mRNA levels were increased by sulfur deficiency. Further, analysis of GFP fluorescence revealed that the SULTR2;1PRO::SULTR2;1mutant:GFP is more active than SULTR2;1PRO::SULTR2;1:GFP lines during sulfur deficiency. These results suggest that miR395 induced cleavage of SULTR2;1.