Cloning Dof1 transcription factor in plant expression vector and its tranformation in wheat

Abstract

A single transcription factor modulates coordinated expression of a set of metabolites in a biochemical pathway, therefore, the use of transcription factors is an imperative strategy in generating plants with desirable traits. The earlier approaches of single-gene modulation were not highly effective because more than one enzymes are involved in a metabolic pathway and the expression of any single enzyme might be masked by concurrent mechanisms in order to maintain homoeostasis. The aim of the study was to develop transgenic wheat overexpressing Triticum aestivum Dof1 (TaDof1) transcription factor that affects the activity of multiple genes involved in nitrogen and carbon metabolisms that ultimately modulates nitrogen assimilation in plants. The TaDof1 was got synthesized, cloned under the control of CaMV35S promoter and terminated by Nos terminator in a binary expression vector pSB219 that carried phosphinothricin N-acetyltransferase cassette as a plant selection marker. The vector pSB219 with TaDof1 construct was transformed in AGL1 strain of Agrobacterium tumefaciens by electroporation method. The immature embryos of two elite wheat cultivars Galaxy and Faisalabd-2008 were excised and placed on callus induction medium (CIM) to initiate callus formation. One to two weeks old embryogenic calli were selected for infection with freshly grown culture of A. tumefaciens harboring TaDof1 cassette. The infected calli were shifted to co-cultivation medium containing 400 µM of acetosyringone. The calli were incubated on co-cultivatoin medium for 48 hours for TDNA delivery into plant genome. In order to suppress the overgrowth of bacterial cells, calli were placed on callus induction medium containing antibiotic timentin (160 mg/L) for 3-4 weeks. To induce shoot formation, the calli were shifted to regeneration (MSK) medium supplemented with kinetin hormone (1 mg/L). The plants were subjected to first round of selection in which they were shifted to regeneration (MSK) medium containing phosphinothricin (2 mg/L) for 2 weeks. The survived plants were transferred to MSO medium for 2 weeks for root formation. The second round of selection was done in which the plants were transferred to regeneration (MSK) medium containing 3 mg/ L of phosphinothricin for another 2 weeks. After selection, the plants showing resistance against phosphinothricin were transferred to MSO medium until long and healthy roots were developed. The plants having healthy shoots and roots were transferred to a mixture of peat moss, vermiculite and perlite (ratio 2:1:1 respectively) in plastic pots. To confirm the integration of complete TaDof1 cassette in plant genome, gene junction PCR was performed on the putative transgenic plants. Out of 31 positive events, only 8 plants possessed complete TaDof1 cassette while the rest of plants had truncated constructs. The seeds of T0 plants were sown in pots that were kept under controlled conditions to obtain T1 transgenic wheat lines. To induce the expression of TaDof1, six selected T1 wheat lines were subjected to nitrogen stress. Total RNA was isolated after 2 and 4 weeks of nitrogen stress in order to quantify the expression of TaDof1 and other related genes. The results of quantitative RT-PCR revealed that only a few transgenic lines showed upregulation of TaDof1 and related genes after 2 weeks of nitrogen stress. The increase in expression levels of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase (PK) genes ranged from 0.33-0.80 fold in transgenic Faisalabad-2008 and 0.33-17 fold in transgenic Galaxy. However, a substantial rise in the expression of TaDof1 and the genes affected by TaDof1 was observed after 4 weeks of nitrogen stress. The expression profiles of the genes CS, ICDH, PEPC and PK ranged from 0.4-88 fold in transgenic Faisalabad-2008 and 0.35464 fold in transgenic Galaxy. The maximum increase of 464 fold was recorded for ICDH gene expression after 4 weeks of stress. The effect of overexpression of TaDof1 on various agronomic, physiological and biochemical traits of T1 plants was also investigated. For morphological analysis, the data of plant height (cm), number of spikes plant-1, spike length (cm), number of grains spike-1 and seed weight (g) was recorded. Significantly increased values were observed in most of the selected T1 lines for agronomic traits. Under low-nitrogen conditions, all the selected T1 lines exhibited a remarkable increment in chlorophyll a, b and total chlorophyll contents as compared to wild type plants. For chlorophyll a, b and total chlorophyll, the maximum rise of 31 mg g-1FW, 40 mg g-1FW and 35 mg g-1FW respectively was observed in transgenic line of Galaxy (G4) as compared to control plants having 4 mg g-1FW, 6 mg g-1FW and 5 mg g-1FW of chlorophyll a, b and total chlorophyll contents respectively. For soluble protein content, 4 out of 6 T1 lines exhibited significantly increased values with transgenic line of Galaxy (G4) showing the highest value (12 mg g-1FW) in comparison with wild type plant (7 mg g-1FW). A considerable rise in soluble sugar content was recorded in all the T1 lines. The maximum rise of 9.9 mg g-1FW was recorded for transgenic line of Galaxy (G1) as compared to control plant (4.9 mg g-1FW). These findings clearly indicated that overexpression of Dof1 transcription factor not only enhances nitrogen assimilation in plants by modulating the pathway associated with nitrogen and carbon metabolisms but also improves plant growth. The results depict the potential of employing transcription factors in engineering plant metabolisms that pave the way for future characterization of Dof1 plants.