Chemie  |  Biochemie  |  Medizin


Joel Tan, 2004 | Ottenbach , ZH


The formation of gametes is crucial in the life cycle of sexually reproducing organisms. In plants, the gametes are formed from reprogrammed somatic cells. The female germ cells are generated in the female gametophyte. Whilst the basic principles related to the morphology of the female gametophyte are known, the underlying molecular processes are less understood. The RK domain (RKD) transcription factors are involved in egg cell formation; however, their precise functions remain unknown. When the AtRKD1 and AtRKD2 genes are ectopically expressed in Arabidopsis thaliana, tissue proliferation and the expression of an egg-cell marker are induced. Understanding how these genes control egg cell development might enable plant propagation through seeds without fertilisation or recombination, maintaining hybrid vigour in hybrid crops. To better understand the role of the RKD genes in female gametophyte development, four different RKD transcription factors were knocked down in A. thaliana, employing a knockdown strategy with artificial microRNAs that are expressed under a late-stage egg cell-specific promoter. The knockdown transformation lines showed no abnormal egg cell phenotypes. A phenotype was observed in a single transformation line, likely caused by a side-effect of T-DNA integration that can induce chromosomal translocations.


Can any egg-cell-specific phenotypes be induced through a triple knockdown of the RKD genes in Arabidopsis thaliana? Can any conclusion be drawn with respect to what role the RKD transcription factors play in the later stages of egg cell development?


To acquire transgenic A. thaliana plants, which have downregulated RKD transcription factors, the following steps were necessary: plasmids holding the amiRNA sequences were propagated in E. coli via heat-shock insertion. Subsequently, Sanger sequencing was performed on the products of the miniprep to check if the desired plasmids were indeed assembled as intended. Next, A. tumefaciens bacteria were transformed with the plasmids, after which the inflorescences of two wild-type A. thaliana plants were transformed. The seeds, which subsequently developed from those two plants were selected and 48 T1 plants were transferred onto soil. After three weeks on soil, DNA was extracted from the 48 plants, and PCR was done on them to screen for transformants, verified with electrophoresis. In the last step, seed set analysis and gametophyte analysis of the transformants were conducted.


15 out of 48 plants were doubly verified to be transformants through herbicide selection and genotyping PCR. Seeds of each of the 15 transformant plants were screened for abnormalities, which might have been caused by the amiRNA knockdown. Only one line showed a high frequency of underdeveloped seeds. This particular line had an abortion rate of roughly 50%, both for pollen grains and female gametophytes. The remaining fourteen lines could not be differentiated from the wild-type phenotype.


Only one line showed a female gametophyte abortion phenotype, which is an unexpected result. Some degree of female gametophyte abortion should have been present in all transformation lines if the amiRNAs would have had the desired effect. The abortion of half of all pollen grains and half of all female gametophytes in the outlier line points towards a frequent side-effect of transformation called chromosomal translocation. As a consequence of this phenomenon, the chromosomes may encounter difficulties pairing up during meiosis, and severe genetic deficiencies can arise in gametes carrying translocations, leading to gamete abortion. It is possible that downregulating three out of the five RKD genes might not suffice to induce a female gametophyte phenotype due to functional redundancy.


It is likely that there is a functional redundancy amongst the five RKD genes; however, their exact functions still remain obscure. This means that apomixis technology is still out of reach and requires further research. The following steps could be taken to continue working with these plants: Quantitative reverse transcription PCR could be used to determine the level of expression of the amiRNAs in the plants. Given satisfying amiRNA expression levels, plants from both types of lines could be crossed to generate plants with a quadruple knockdown against four out of five RKD genes. An additional construct might even be introduced to downregulate all five genes. If all of this yields ambiguous results, it is always possible to start afresh and attempt alternative methods, such as the CRISPR-Cas method.



Würdigung durch den Experten

Stefan Grob

Joel Tan hat eine sehr ausführliche und gewissenhaft geschriebene Arbeit eingereicht. Dafür hat er über einen längeren Zeitraum im Labor von Prof. Grossniklaus (UZH) Experimente durchgeführt, welche leider nicht die gewünschten Ergebnisse geliefert haben. Trotzdem hat Joel Tan aus den ihm zur Verfügung stehenden Daten das Maximum herausgeholt und die Resultate professionell vorgestellt und diskutiert. Darüber hinaus konnte er sich substantiell in die komplexe Materie vertiefen und weiterführende Fragen aufbringen. Die sprachliche Qualität der Arbeit ist klar überdurchschnittlich.


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