Ecological genetics

From October 3 to October 5, 2023, the Third International Conference “Genetically modified organism: the history, achievements, social and environmental risks” was held at St. Petersburg State University as part of the implementation of the Program for the creation and development of a world-class Scientific Center “Agricultural Technologies for the Future. ”This issue is dedicated to the 300^th anniversary of St. Petersburg State University and presents materials from selected conference reports.

BACKGROUND: Identification of target genes responsible for visible phenotypic effect may contribute to the development of transgene-free bioengineering strategies and application of crop varieties with edited genome. CAPRICE (CPC) is a single-repeat R3 MYB transcription factor, involved in anthocyanin biosynthesis and trichome formation. It is assumed that CPC controls the expression of Dihydroflavonol-4-reductase (DFR), a key gene of anthocyanin biosynthesis.

AIM: The aim of the study was to determine whether knockout of the CPC gene using CRISPR/Cas9 results in visible anthocyanin accumulation.

MATERIALS AND METHODS: Three guide RNAs were designed to excise a MYB domain from the CPC gene of Arabidopsis thaliana. Anthocyanin content and expression of CPC and DFR genes were studied in edited plants.

RESULTS: The expected 662 bp deletion was detected in 2,7% of glufosinate-resistant plants, however none of the mutations were homozygous. Four edited lines were studied in four generations. An upregulation of the DFR gene was observed in edited lines, however CPC gene expression, anthocyanin content and trichome development were not significantly different from those in control plants. Moreover, in A. thaliana pigmentation did not directly depend on DFR or CPC gene expression.

CONCLUSIONS: Our results suggest that CPC gene is involved in regulation of DFR gene expression and anthocyanin biosynthesis pathway, however in case of mutations plants might utilize other transcription factors to maintain homeostasis. Therefore, CPC gene is not a suitable target for CRISPR/Cas studies in Arabidopsis.

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown.

AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish.

MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2^–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2).

RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis.

CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

Mitogen-activated protein kinases (MAPKs) play an important role as intracellular regulators of signal pathways in plants. Some MAPKs have been shown to be induced in the roots of legume plants during symbiosis with nitrogen-fixing rhizobial bacteria. One of such signal regulators, MAPK6, was shown to be involved in the development of symbiosis between Pisum sativum L. pea plants and rhizobia. Using genetic engineering approaches, we overexpressed the MAPK6 gene in transgenic roots, that resulted in an increase in the number of nodules and the biomass of pea plants. New approaches for genome editing of pea plants have been designed using the CRISPR/Cas9 prime-editing technique, when the MAPK6 gene was used as a target. We have analyzed the transgenic roots of pea transformants and observed the presence of the gene encoding Cas9 the pegRNA sequence in the genome of transformants. Therefore, the possibility of using genetic engineering methods to obtain plants with increased efficiency of symbiosis will be investigated in future experiments.

Редакция сожалеет, что в опубликованной версии тезиса доклада произведения инициал отчества автора М. Лебедевой был указан как А вместо В. Имя автора — Лебедева Марина Валерьевна. Редакция уверена, что допущенная ошибка не могла существенно повлиять на восприятие произведения и интерпретацию информации читателями. В электронной форме на сайте журнала ошибка исправлена, файл статьи и выпуска обновлены.