معرفي ژنهاي مرجع براي تجزيه و تحليل qRT-PCR در بافت هاي مختلف برخي از گونه هاي جنس Artemisia

چكيده انگليسي :

Validation of Reference Genes for Quantitative Real time PCR in Various Tissues of Some Artemisia Species Azam Moosavi moosavi azam@yahoo com a moosavi@ag iut ac ir Date of submission Januray 22 2013 Department of Agricultural Biotechnology Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi 1 M Talebi Assist Prof Supervisor mtalebi@cc iut ac ir 2 B E Sayed Tabatabaei Prof Supervisor sayedt@cc iut ac irAbstract Artemisia is a large diverse genus of aromatic plants with various medical and nutritional as well as landscapeusage Artemisia species are a source of various anti malarial cytotoxicity antibacterial and antifungal effects and antioxidant properties which are due to sesquiterpene compounds In recent decades A annua has beenattracted a great deal of attentions because of its artemisinin which is a sesquiterpene lactone and hasanti malarial effects The amount of artemisinin production varied in different species Recently artemisinin isused against a malaria pathogen Plasmodium flasiparum which has a multidrug resistance Unfortunately production of artemisinin in plants is very low and its chemical synthesis is very difficult and expensive Inrecent years many efforts have been done to increase the expression of genes and enzymes which are involvedin artemisinin biosynthesis In order to evaluate the effects of changed in expression of artemisinin producinggenes the real time polymerase chain reaction is used This technique is sensitive enough to measure the lowmRNA copy number The errors of RNA degradation possibility difference of RNA concentrations in differentsamples and also technical errors may be accrued during the real time PCR For control of these errors selectingan accurate method for normalization is important Among abundant methods of normalization usinghousekeeping genes as internal controls in the analysis of gene expression is in general The expression levels ofthese genes should be stable in tested tissues or cells and significant changes should not be occurred underexperimental conditions However the expression levels of these reference genes in different tissues and organsare different and for each genotype and tissue one or several suitable internal control genes should be selectedcarefully In this study the stability expression of seven genes includes act pal cpr 18S rRNA EFl UBQ andRPS9 were studied in different tissues of Artemisia species A annua A absinthium A sieberi A dracunculusand Artemisia sp RNA was extracted from leaf stem and root tissues and cDNAs were synthesized All dataobtained from real time PCR were analyzed by Finder Norm software to estimated suitable genes The resultsshowed that among the various tissues of the Artemisia species RPS9 gene is the most reliable internal controlgene Combination of UBQ and 18S rRNA genes was recognized the best combination for gene normalizationamong Artemisia species These results show that housekeeping genes are regulated completely different invarious kinds of plant species and they have different expression patterns Since a reference gene with a stableexpression in an organism may not be suitable for normalization of gene expression in other organisms ordifferent experimental conditions it is essential to varify its validity before application The analysis of thesegenes in different tissues also confirmed the stability of RPS9 gene and the combination of RPS9 and 18S rRNAgenes can be applied in different tissues of Artemisia genus The amount of artemisinin measured in severalspecies of Artemisia available in Iran was performed with a hollow fiber micro extraction method The resultsshowed that the level of artemisinin in leaves were much higher than stems and also the amount of this valuablematerial in A annua is more than other species Key word Artemisia Artemisinin Reference gene Normalization Real time PCR

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معرفي ژنهاي مرجع براي تجزيه و تحليل qRT-PCR در بافت هاي مختلف برخي از گونه هاي جنس Artemisia

آرتميزيا , آرتميزينين , نرمال سازي , PCR در زمان حقيقي