Plant Mol Biol 54(1):71–84Įdwards K, Jonstone C, Thomson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Development 122:1567–1575ĭean G, Casson S, Lindsey K (2004) KNAT6 gene of Arabidopsis is expressed in roots and is required for correct lateral root formation. Plant Cell 8:1277–1289Ĭlark SE, Jacobsen SE, Levin JZ, Meyerowitz EM (1996) The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. Curr Genomics 12:1–14Ĭhuck G, Lincoln C, Hake S (1996) KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 18:1900–1907īolle C, Schneider A, Leister D (2011) Perspectives on systematic analyses of gene function in Arabidopsis thaliana: new tools, topics and trends. Development 119:823–831īelles-Boix E, Hamant O, Witiak SM, Morin H, Traas J, Pautot V (2006) KNAT6: An Arabidopsis homeobox gene involved in meristem activity and organ separation. Science 301:653–657īarton MK, Poethig SR (1993) Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the wild type and in the shoot meristemless mutant. Overall, the proposed procedure is highly effective for expression studies of KNOX genes in Arabidopsis mutants and will serve as a fundamental work protocol to open opportunities for genetic studies of genes involving insertional mutants in Arabidopsis.Īlonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. The use of different relative expression quantification produces a similar indication of expression level. Moreover, qRT-PCR was effective for transcript analysis among the knox mutant samples. However, no gene suppression was observed for the positively selected knat5 mutant. Surprisingly, the insertions resulted in strong repression of the respective KNOX genes. T-DNA insertion mutants for all Arabidopsis KNOX genes (except for knat4) were isolated based on kanamycin screening, phenotype selection, and PCR genotyping. The troubleshooting and challenges that might occur are also presented and discussed.
#Using serial cloner to see where tdna mutant should fall serial#
This protocol contains a reproducible and serial procedure containing detailed and step-by-step laboratory and field works covering all experiment steps from the screening of homozygous mutant lines to the KNOX expression analysis using qRT-PCR in a single paper. In the present study, we established a reproducible protocol that is important for genetic studies of KNOX genes using Arabidopsis insertional mutants.
In Arabidopsis, studies of KNOX genes especially among members of class II KNOX genes remain limited and functional data are largely lacking. KNOTTED1-like homeobox ( KNOX) genes serve important roles in meristem function and many developmental processes in all higher plants.
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