Glucuronidase Gene:

Hikmat M. Masyab; Qutaiba S.  Al-Nema; Mozahim Q.  Al-Mallah

doi:10.14500/aro.11239

Hikmat M. Masyab Department of Biology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq http://orcid.org/0000-0002-7819-3637
Qutaiba S. Al-Nema Department of Biology, College Education for Pure Sciences, University of Mosul, Mosul, F.R Iraq https://orcid.org/0000-0003-3863-7990
Mozahim Q. Al-Mallah Department of MLT, Al-Hadbaa University College, Mosul, F..R. Iraq https://orcid.org/0009-0006-7093-3238

Keywords: Glucuronidase protocol, Gluconacetobacter diazotrophicus, Interaction, Nitrogen fixation, Spinach

Abstract

Gluconacetobacter diazotrophicus lives inside plant tissue cells in the form of colonies and excretes about half of the fixed nitrogen, which offers potential power that improves plant growth. The aim of this study is to find the interaction of glucuronidase (GUS)-labeled G. diazotrophicus with spinach seedlings and the detection of GUS genes using X-gluc dye (5-bromo-4-chloro-3-indolyl-β-D- glucuronic acid). The GUS protocol is used to detect GUS-labeled G. diazotrophicus in spinach seedling tissues by chemical detection using X-gluc dye. The results show that the spinach seedlings are successfully infected with GUS-labeled G. diazotrophicus , with the survival of the seedlings throughout their growth period and an improvement in the growth of pollinated seedlings. The outcomes of the microscopic inspection of the root slices reveal the presence of bacterial cells at the root tips and their concentration in the area of the cell walls of the peripheral cells. Furthermore, the findings of microscopic examinations of longitudinal sections for cotyledons show the presence of a number of bacteria within epidermal cell walls. This indicates that the determinants of the interaction between these bacteria and spinach seedlings are suitable for the expression of the gene responsible for the formation of the nitrogenase enzyme.

Downloads

Download data is not yet available.

Author Biographies

Hikmat M. Masyab, Department of Biology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Hikmat M. Masyab is an Assistant Prof at the Department of Biology, Faculty of Science and health, Koya
University. He got the B.Sc. degree in Biology, the M.Sc. degree in Biology/Botany and the Ph.D. degree in
Biotechnology. His research interests are in Biotechnology, Plant Tissue Culture and Genetic Engineering.

Qutaiba S. Al-Nema, Department of Biology, College Education for Pure Sciences, University of Mosul, Mosul, F.R Iraq

Qutaiba S. Al-Nema is a Professor at the Department of Biology, College Education for Pure Sciences, University of Mosul. He got the B.Sc. degree in Biology, the M.Sc. degree in Biology/Botany and the Ph.D. degree in Biotechnology. His research interests are in Biotechnology, Plant Tissue Culture and Genetic Engineering.

Mozahim Q. Al-Mallah, Department of MLT, Al-Hadbaa University College, Mosul, F..R. Iraq

Mozahim K. Al-Mallah is a Professor at the Department of Medical Laboratory Techniques, Alhadba University College, Mosul. He got the B.Sc. degree in Plant protection, the M.Sc. degree in Virology and the Ph.D. degree in Genetic Engineering. His research interests are in Biotechnology, Plant Tissue Culture and Genetic Engineering.

References

AL-Mallah, M.K., and Masyab, H.M., 2014. Expression of GUS and GFP reporter genes in transgenic hairy roots of tomato and potato plants via Agrobacterium rhizogenes mediated transformation. Australian Journal of Basic and Applied Sciences, 8(2), pp.234-239.

Boddey, R.M., Urquiaga, S., Alves, B.J.R., and Reis, V., 2003. Endophytic nitrogen fixation in sugarcane: Present knowledge and future applications. Plant and Soil, 252, pp.139-149. DOI: https://doi.org/10.1023/A:1024152126541

Chiu, W., Peters, G.A., Levieille, G., Still, P.C., Cousins, S., Osborne, B., and Elhai J., 2005. Nitrogen deprivation stimulates symbiotic gland development in Gunnera manicata. Plant Physiology, 139, pp.224-230. DOI: https://doi.org/10.1104/pp.105.064931

Cocking, E.C., Stone, P.J., and Davey, M.R., 2006. Intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus. In Vitro Cellular and Developmental Biology-Plant, 42, pp.74-82. DOI: https://doi.org/10.1079/IVP2005716

Dietz, B.R., 2022. Genetic Manipulations in Known Endophytes Furthering the Application as a Biofertilizer. M. Sc. Thesis. University of Minnesota, Minnesota, U.S.A.

Dong, Z., Zelmer, C.D., Canny, M.J., McCully, M.E., Luit, B., Pan, B., Faustino, R.S., Pierce, G.N., and Vessey, J.K., 2002. Evidence for protection of nitrogenase from O(2) by colony structure in the aerobic diazotroph Gluconacetobacter diazotrophicus. Microbiology (Reading), 148, pp.2293-2298. DOI: https://doi.org/10.1099/00221287-148-8-2293

Duca, D., Lorv, J., Patten, C.L., Rose, D., and Glick, B.R., 2014. Indole-3-acetic acid in plant-microbe interactions. Antonie van Leeuwenhoek, 106, pp.85-125. DOI: https://doi.org/10.1007/s10482-013-0095-y

Eskin, N., Vessey, K., and Tian, L., 2014. Research progress and perspectives of nitrogen fixing bacterium, Gluconacetobacter diazotrophicus, in monocot plants. International Journal of Agronomy, 2014, p.208383. DOI: https://doi.org/10.1155/2014/208383

Furness, D., Fenech, M., Dekker, G., Khong, T.Y., Roberts, C., and Hague, W., 2013. Folate, Vitamin B12, Vitamin B6 and homocysteine: Impact on pregnancy outcome. Maternal and Child Nutrition, 9(2), pp.155-166. DOI: https://doi.org/10.1111/j.1740-8709.2011.00364.x

Grillo-Puertas, M., Delaporte-Quintana, P., Pedraza, R.O., and Rapisarda, V.A., 2018. Intracellular polyphosphate levels in Gluconacetobacter diazotrophicusaffect tolerance to abiotic stressors and biofilm formation. Microbes and Environment, 33(4), pp.440-445. DOI: https://doi.org/10.1264/jsme2.ME18044

Kong, Q., Li, J., Wang, S., Feng, X., and Shou, H., 2023. Combination of hairy root and whole-plant transformation protocols to achieve efficient CRISPR/Cas9 genome editing in soybean. Plants (Basel), 12, p.1017. DOI: https://doi.org/10.3390/plants12051017

Lambrecht, M., Okon, Y., Vande Broek, A., and Vanderleyden, J., 2000. Indole-3-acetic acid: A reciprocal signalling molecule in bacteria-plant interactions. Trends Microbiology, 8, pp.298-300. DOI: https://doi.org/10.1016/S0966-842X(00)01732-7

Masyab, H.M., 2018. Expression patterns of GUS gene in five different strains of Arabidopsis thaliana plants. In: International Conference on Pure and Applied Sciences (ICPAS 2018), pp.59-63. DOI: https://doi.org/10.14500/icpas2018.bph24

Medeiros, A.F.A., Polidoro, J.C., and Reis, V.M., 2006. Nitrogen source effect on Gluconacetobacter diazotrophicus colonization of sugarcane (Saccharumspp.). Plant and Soil, 279(1-2), pp.141-152. DOI: https://doi.org/10.1007/s11104-005-0551-1

Miki, B., 2008. Marker genes and promoters. In: Stewart, C.N., editor. Plant Biotechnology and Genetics: Principles, Techniques, and Applications. John Wiley and Sons, Ltd., New Jersey, U.S.A. DOI: https://doi.org/10.1002/9780470282014.ch9

Mohammed, A.A.H., and Masyab, H.M., 2020. Genetic transformation of Nigella sativa L. Plants with Agrobacterium rhizogenes 35S GUS R1000 and estimation of thymoquinone level in transformed hairy roots cultures. Plant Archives, 20(Suppl 1), pp.3649-3652.

Murashige, T., and Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiologia Plantarum, 15, pp.473-497. DOI: https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Muthukumarasamy, R., Revathi, G., and Loganathan, P., 2002. Effect of inorganic N on the population, in vitro colonization and morphology of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus). Plant and Soil, 243, pp.91-102. DOI: https://doi.org/10.1023/A:1019963928947

Parniske, M., 2000. Intracellular accommodation of microbes by plants: A common developmental program for symbiosis and disease. Current Opinion in Plant Biology, 3, pp.320-328. DOI: https://doi.org/10.1016/S1369-5266(00)00088-1

Rasheed, J.H., 2014. GUS-gene as a visual marker for Gluconacetobacter diazotrophicus co-cultivated with carrot plantlets. International Journal of Science and Technology, 3, pp.776-779.

Rocafull, Y.R., Badia, M.J., Garciea, M.O., Álvarez, B.D., and Sánchez, J.R., 2016. Isolation and characterization of Gluconacetobacter diazotrophicus strains. Cultivos Tropicales, 37(1), pp.34-39.

Rodriguez, M.V., Tano, J., Ansaldi, N., Carrau, A., Srebot, M.S., Ferreira, V., Martínez, M.L., Cortadi, A.A., Siri, M.I., and Orellano, E.G., 2019. Anatomical and biochemical changes induced by Gluconacetobacter diazotrophicus stand up for Arabidopsis thaliana seedlings from Ralstonia solanacearum infection. Frontiers in Plant Science, 10, p.1618. DOI: https://doi.org/10.3389/fpls.2019.01618

Ruhullah, M., Rahman, M.S., Sarafat, A.M.D., Mohsin G.M., and Khalekuzzaman, M., 2017. Establishment of agrobacterium mediated genetic transformation in popular rice cultivar BRRI dhan-29 through reporter GUS gene expression system in Bangladesh. FPI, 1(2), pp.67-75.

Sebring, R.L., Duiker, S.W., Berghage, R.D., Regan, J.M., Lambert, J.D., andBryant, R.B., 2022. Gluconacetobacter diazotrophicus inoculation of two lettuce cultivars affects leaf and root growth under hydroponic conditions. Applied Sciences, 12, p.1585. DOI: https://doi.org/10.3390/app12031585

Sevilla, M., and Kennedy, C., 2000. Genetic analysis of nitrogen fixation and plant growth stimulating properties of Acetobacter diazotrophicus, an endophyte of sugarcane. In: Triplett, E.W., editor. Prokaryotic Nitrogen Fixation: A Model System for Analysis of a Biological Process. Scientific Press, Wymondham, UK, Horizon, pp.737-760.

Shojaei, T.R., Salari, V., Ramazan, D., Ehyaei, M., Gharechahi, J., and Chaleshtori, R.M., 2010. The effect of plant growth regulators, explants and cultivars on spinach (Spinacia oleracea L.) Tissue culture. African Journal of Biotechnology, 9(27), pp.4179-4185.

Varghese, F., Kabasakal, B.V., Cotton, C.A.R., Schumacher, J., Rutherford, A.W., Fantuzzi, A., and Murray, J.W., 2019. A low-potential terminal oxidase associated with the iron-only Nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii. Journal of Biological Chemistry, 294(24), pp.9367-9376. DOI: https://doi.org/10.1074/jbc.RA118.007285

Xiong, A.S., Peng, R.H., Zhuang, J., Chen, J.M., Zhang, B., Zhang, J., and Yao, Q.H., 2011. A thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants. PLoS One, 6(11), e26773. DOI: https://doi.org/10.1371/journal.pone.0026773

Glucuronidase Gene

A Strong Evidence of a Novel Interaction of Glucuronidase-labeled Gluconacetobacter diazotrophicus with Spinach, Spinacia oleracea L. Seedlings

Abstract

Downloads

Author Biographies

References