Use of the gl1 Mutant & the CA-rop2 Transgenic Plants of Arabidopsis thaliana in the Biology Laboratory Course.
2006; National Institutes of Health; Volume: 68; Issue: 9 Linguagem: Inglês
10.1662/0002-7685(2006)68[e148
Autores Tópico(s)Plant Genetic and Mutation Studies
ResumoMost of the experiments described in genetics laboratory manuals present only one-time or short-term experiments for high school biology or college genetics courses. In order to deepen conceptual understanding of important genetics concepts, students need to be exposed to a real research environment. Furthermore, the inquiry-based, research-driven laboratory teaching plan is the most effective in nurturing budding scientists through the stimulation of their interest in biology and scientific reasoning. Therefore, the development of simple lab teaching materials and methods in which students have the opportunity to perform genetic analysis within a semester is in much demand. Based on several semesters of teaching an introductory genetics course for undergraduate students and of a Master level genetics course for high school teachers, the author believes that the use of the glabrous1 (gl1) mutants and the homozygous transgenic plants (CA1) in Arabidopsis thaliana is a simple and effective way of teaching genetics laboratory techniques and analytical skills at high school and undergraduate levels. The introduction of Arabidopsis in the laboratory course not only provides an alternative model organism to/or as a complement for Drosophila, but also enables the students to become familiar with modern genetic research in plants. Most importantly, this inquiry-based, research-driven teaching method allows the students to integrate botany and statistic analysis into genetics, and promotes students’ intellectual growth by fostering the interactions between individual students and between students and teachers. Arabidopsis has quickly emerged as the most widely-used model organism in plant genetic and developmental studies since the 1980s (Meinke et al., 1998). It has several advantages over pea and maize, two organisms frequently mentioned in high school or college genetics courses. Arabidopsis is a tiny weed (Figure 1A) that is a member of the mustard family (Cruciferae or Brassicaceae). It is a self-pollinating species that can also be cross-fertilized. Each plant has about 5,000 seeds, serving as genetic stocks that are easy to manage. It has a very short life cycle of about six to eight weeks for most of the commonly used ecotypes in the laboratory, such as Landsberg erecta (Ler), Columbia (Col) and Wassilewskija (Ws). Arabidopsis has a small genome (125 megabases, five pairs of chromosomes), a size similar to Drosophila but 20–30 folds smaller than pea and maize. The Arabidopsis genome has been almost completely sequenced (115.4 megabases) in 2000, with about 25,000 protein-encoding genes (Arabidopsis Genome Initiative, 2000). Furthermore, genetic transformation in Arabidopsis is rather easy and has become routine. Figure 1 Leaf morphology phenotypes of the gl1, CA1 and CA1; gl1 double mutant plants, and the illustration of the crossing procedure. (A) A 5-week-old Col wild-type plant. (B–E) Developing leaves for various genotypes. (B) A normal leaf from Col. (C) ... The gl1 mutant (Oppenheimer et al., 1991) and the CA1 transgenic plants that express the constitutively active form of ROP2 small GTPase transgene (designated CA-rop2; Li et al., 2001) have different phenotypes in leaf morphology. Importantly, their leaf morphology phenotypes are easy for the students to score, although CA1 has other phenotypes (Li et al., 2001). In contrast to the hairy (with trichomes) leaves with regular shapes of the wild-type (WT), Col (Figure 1B), gl1 has no trichomes (hairless, or smooth) on the leaf surface (Figure 1C) but still has trichomes on the leaf edge, and the CA1 leaves are serrated and narrower and have longer petioles (Figure 1D). GL1 is located on Chromosome 3 and encodes a member of the large family of MYB transcription factors (Oppenheimer et al., 1991). CA-rop2 was constructed by placing the G12V mutant form of ROP2 small GTPase gene under the control of the constitutively active CaMV35S promoter and this cassette was then transformed into the Arabidopsis Col plants (Li et al., 2001). The G12V mutation locks ROP2 GTPase in the GTP bound form, thus causing it to become constitutively active and consequently activate the downstream signaling events. In this semester-long laboratory exercise, Col, the gl1-1 allele (abbreviated as gl1 in this report) and the CA1 transgenic plant are used for pair-wise crossing and subsequent genetic analysis. The resulting F1 generations would give indications regarding the dominance and recessiveness of the respective phenotypes. The F2 generations are then used to confirm the inheritance patterns and determine the number of genes involved using Chi-square (χ2)analysis. The cross of gl1 and CA1 results in the double mutants, CA1 gl1 (Figure 1E). Interestingly, the F2 segregation from the cross of gl1 and CA1 indicates that the GL1 locus and the CA-rop2 transgene integration site are linked. Therefore, these materials provide the students an excellent opportunity to learn how to infer the linkage and calculate the genetic distance between these two loci.
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