Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Paper Details

Research Paper 01/04/2018
Views (233) Download (23)

Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Mohamad Al-Hajaj, Zainab Al-Dallee
Int. J. Biosci.12( 4), 312-329, April 2018.
Certificate: IJB 2018 [Generate Certificate]


Agrobacterium is broad used, after suitable modified, to be the utmost efficient vector for gene transport into plant cells. This study was carried out to produced Agrobacterium tumefaciens LBA4404 strain contains binary vector has human GBA  gene to became ready for  transforming any desired plant to expression glucocerebrosidase. Recombinant glucocerebrosidase is current treatment for gaucher disease which is lysosomal storage disease caused by mutations in the encode for glucocerebrosidase. In this study, Human whole blood was pivotal source of RNA by using GENEzol™ TriRNA Pure Kit, and the Hu-GBA gene was amplified by designed special primers,GBA gene was introduced inthe plant expression vector pCAMBIA1304 beneath the domination of the cauliflower mosaic virus 35S promoter and grated recombinant pcambia1304-GBA vector having GBA gene by restriction and ligation methods.Calcium chloride heat-shock transformation is used to introduce pcambia1304-GBA into E. coli DH5α and plantation on LB agar medium contains kanamycin 50 mg/l. The transformed colonies were proven by colony PCR and amplification GBA gene from isolated recombinant pcambia1304-GBA. The recombinant isolation plasmid was transferred to Agrobacterium tumefaciens LBA4404 using modified freezing-thaw method, transformation Agrobacterium colonies were enhanced by colony PCR. Results demonstrated that The1561bp-GBA gene was amplified from human total blood RNA which was confirmed by sequencing the PCR product which gives 100% identified withHomo sapiens glucosylceramidase beta (GBA), transcript variant 1, mRNA NM_000157.3 Gene Bank and colony PCR assured that Agrobacterium tumefaciens LBA4404 carried Hu-GBA gene.


AlexanderR, Johnson A, Thorpe J. 2003 Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions. Nucleic Acids Research 31(12), 3208-3216.

African Biosafety Network of Expertise (ABNE). 2010. Plant Transformation Using Agrobacterium tumefaciens Available at

Arets JM, Yasothan U, Kirkpatrick P. 2010 Velaglucerase alfa. Nature Reviews Drug Discover 9(11), 837-838.

Azhakanandam K. (eds.). 2015. Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants.

Barampuram S, Zhang Z. 2011“Recent advances in plant transformation,” in Plant Chromosome Engineering: Methods and Protocols, Methods in Molecular Biology,(701) ed. Birchler. J. A (Berlin: Springer), 1–35.

Barton NW. 1991. Replacement therapy for inherited enzyme deficiency-macrophage-targeted glucocerebrosidase for Gaucher`s disease. The New England Journal of Medicine 324(21). 1464-1470.

Belchetz PE, Crawley JC, Braidman IP, Gregoriadis G. 1977 Treatment of Gaucher`s disease with liposome-entrapped glucocerebroside: beta- glucosidase. Lancet 2(8029). 116-117.

Behnoush S, Mokhtar J, Hamid R, Mehdi M. 2014. Cloning Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill) Jundishapur Journal of Natural Pharmaceutical Products 9(1), 9-15.

Benfey PN, Ren L, Chua NH. 1990a Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. Euro (pean Molecular Biology Organization Journal. (9),1677–1684.

Bergmans HE, Van IM, Hoekstra WP. 1981. Transformation in Escherichia coli: stages in the process. Journal of Bacteriology.(146), 564-57.

Beulter E, Dale GL, Guinto DE, Kuhl W. 1977.Enzyme replacement therapy in Gaucher`s disease: Preliminary clinical trial of a new enzyme preparation. Proceedings of the National Academy of Sciences 74(10), 4620-4623.

Beutler E, Grabowski GA. 2001.Gaucher disease. In: The Metabolic and Molecular Bases of Inherited Disease, 8th Edition (Scriver CR, Beaud et al, Sly WS, Valle D, eds),  3635-3668. New York: McGraw Hill.

Birch RG. 1997. Plant transformation: problems and strategies for practical application. Annual Review of Plant Physiology and Plant Molecular Biology (48), 297–326.

Birnboim HC. 1992 Extraction of high molecular weight RNA and DNA from cultured mammalian cells. Methods Enzymol (216).154-60.

Brady RO, Kanfer JN, Shaprio D. 1965 Metabolism of glucocerebrosidase II. Evidence of an enzymatic deficiency in Gaucher`s disease. Biochemical and Biophysical Research Communications (18),221-225.

Brumshtein B. 2010 Characterization of gene activated human acid beta glucosidase: Crystal structure, glycan composition, and internalization into macrophages. Glycobiology 20(1),24-32.

Chang, Angela Y, Chau, Vivian WY, Landas, Julius A, Pang. 2017. Preparation of calcium competent Escherichia coli and heat-shock transformation, Journal of Experimental Microbiology and Immunology (1), 22-25.

CIMMYT.2005 Laboratory Protocols: CIMMYT Applied Molecular Genetics Laboratory. Third Edition. Mexico, D.F.: CIMMYT

Dagert M, Ehrlich S. 1979 Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells, Gene, 6(1), 23-28.

Dale G, Beutler E. 1976 Enzyme Replacement therapy in Gaucher`s disease: a rapid, high-yield method for purification of glucocerebrosidase. Proceedings of the National Academy of Sciences 73(12), 1464-1470.

DeLucia A, M, Six D, A, Caughlan RE, Gee P, Hunt I, Lam JS, Dean CR. 2011. Lipopolysaccharide (LPS) Inner Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1. Mbio. 2:e00142-11.

Doll RE, Bruce A, smith FI.1995. Regulation of the human acid beta- glucosidase promoter in multiple cell types. Biochimicaet Biophysica Acta 1261(1), 57-67.

Elbein AD, Tropea JE, Mitchell M, Kaushal GP. 1990 Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase .Journal of Biological Chemistry265(26), 15599-15605.

Furbish FS, Blair HE, Shiloach J, Pentchev PG and Brady RO. 1977 Enzyme replacement therapy in Gaucher`s disease: large- scale purification off glucocerebrosidase suitable for human administration. Proceedings of the National Academy of Sciences USA 74(8), 3560-3563.

Furbish FS, Steer CJ, Krett NI, and Barranger JA. 1981 Uptake and distribution of placental glucocerebrosidase in the rat hepatic cells and effects of sequential deglycosylation. Biochimicaet Biophsica Acta 673 (4), 425-434.

GelvinSB. 2003 Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” tool. Microbiology and Molecular Biology Reviews. (67), 16–37.

Goldman DP, Clarke AE, Garber AM. 1992 Creating the costliest orphan. The Orphan Drug Act in the development of Ceredase. International Journal of Technology Assessment in Health Care 8(4), 583-597.

Grabowski GA. 2008. Phenotype, diagnosis, and treatment of Gaucher’s disease. Lancet; (372), 1263– 1271.

Hajdukiewicz P, Svab Z, Maliga P. 1994 the small, versatile pPZP family of agrobacterium binary vectors for plant transformation. Plant Molecular Biology. (25): 989-994. Doi:10.1104/pp.107.113001.

Hiroaki I, Hiroshi N,  Hiroto O. 1990 High efficiency transformation of    Escherichia coli with plasmidGene. (96)1, 23-28

Hollack CE. 2012 An evidence- based review of the potential benefit  of taliglucerase alfa in the treatment of patients with Gaucher`s disease. Core evidence(7), 15-20.

Holsters M, De Waele D, DepickerA, Messens E, van Montagu M, Schell J. 1978 Transfection and transformation of Agrobacterium tumefaciens. Molecular and General Genetics MGG (163), 181-187.

Horowitz M, Wilder S, Horowitz Z, Reiner O, Gelbart T, Beutler E. 1989. The human glucocerebrosidase gene and pseudogene: structure and evolution. Genomics; (4),87–96. [PubMed: 2914709]. 

Jang C, Magnuson T. 2013 A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli.Plos One.8(2), e57075.

Jenkins C, Ling CL,Ciesielczuk HL, Lockwood J, Hopkins S, Mchugh TD, Gillespie SH, Kibbler CC, Jenkins C. 2012 Detection and Identification of Bacteria in Clinical Samples by 16s rRNA    Gene Sequencing: Comparison of two Different Approaches in clinical Practice. Journal of Medical Microbiology. 61(4), 483-88.

Juliana B, Ligia M, Ana M, Adriana F, Solange M, Isaias R, Paulo L. 2012 Generation of a Chinese Hamster Ovary Cell Line Producting Recombinant Human Glucocerebrosidase. Journal of Biomedicine and Biotechnology 2012 (2012).

Kazan K, Manners JM. 2008. Jasmonate signaling :Toward an integrated view. Plant  Physiology 146(4),1459-1468.

Liu I, Liu M, Shergill K. 2006 the Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia coli DH5α. Journal of Experimental Microbiology and Immunology (9), 81-85. 

Lulu K, Xia Z, Xuefan G,Huiwen Z. 2017 Successful newborn screening for gaucher disease using fluorometric assay in China. Journal of Human Genetics (62), 763–768.

Nasanin E, Hamid R, Mohammad A, Mohammad R. 2012. Cloning, Transformation And Expression Of Human Gamma Inrerferon Genne In Tomato (Lycopersicon Esculentum Mill.) Biotechnology & Biotechnological Equipment, 26(2), 2925-2929.

Oltmanns H, Frame B, Lee LY, Johnson S, Li B, Wang K, Gelvin SB. 2010 Generation of backbone-free, low transgene copy plantsby launching T-DNA from the Agrobacterium chromosome. Plant Physiology (152), 1158–1166.

Pastores GM, Patel MJ, Firooznia H. 2000. Bone and joint complications related to gaucher disease. Current rheumatology reports (2): 175–180.

Ponce E, Witte DP, Hung A,  Grabowski G,A. 2001 Temporal and spatial expression of murine acid beta-glucosidase mRNA. Molecular Genetic and Metabolism 74(4), 426-434.

Rasha A. 2013 Cloning abiotic stress associated genes and Agrobacterium Tumefaciens mediated transformation of selected tropical Maize. A thesis submitted in fulfillment of the requirement for the degree of doctor of philosophy (Biotechnology) in school of pure and applied sciences of Kenyatta University.

Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning: ALaboratory Manual, 2nd ed. (Irwin N, Ford N, Nolan C, Ferguson M & Ochler M, eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sambrook J, Russell. 2001. Molecular cloning: Alabrotoray Manual. (NY: cold spring: cold spring habor press). Plant Cell Reports, (13),61-72.

Sanger M, Daubert S, Goodman RM. 1990 Characteristics of a strong promoter from figwort mosaic virus: comparison with analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter. Plant Molecular Biology. (14), 433–443.

Shaaltiel Y. 2007 Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gusher’s disease using a plant cell system. Plant Biotechnology Journal 5(5), 579-590.

Shahrzad A, Mohammad H, Hamid R, Hamid G, Khalil A.2012. Cloning, Transformation and Expression of Human Interferon α2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi) Jundishapur Journal of Natural Pharmaceutical Products 7(3), 111-116.

Shibat D, Liu YG. 2000.Agrobacterium-mediated plant transformation with large DNA fragments. Trends in Plant Science. 5(8), 354–357.

Sood P, Bhattacharya  A,   Sood A.  2011 Problems and possibilities of monocot transformation Biology Plantarum 55 (1), 1-15, 2011.

Sorge J, West C, Westwood B, Beutler E. 1985 Molecular cloning and nucleotide sequence of human glucocerebrosidasec DNA. Proceedings of the National Academy of Sciences of the United States of America, (82), 7279-93.

Sripriya R, Sangeetha M, Parameswari C, Veluthambi B, VeluthambiK.2011. Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector. Plant science(180),766–774.

Stahl PD, Rodman JS, Miller MJ, Schlesinger PH. 1978. Evidence for receptor mediated binding of glycoproteins, glycoconiugates, and lysosomal glycosidases by alveolar macrophages. Proceedings of the National Academy of Sciences of the United States of America 75(3), 1399-1403.

Takasaki S. 1984 Structure of the N- asparagine-linked oligosaccharide units of human placental beta-glucocerebrosidase. Journal of Biology Chemistry 259(16), 10112-10117.

Thorpe SR, Fiddler MB, Desnick RJ. 1974 Enzyme therapy IV. A method for determining the in vivo fate of bovine beta-glucuronidase in beta- glucuronidase deficient mice. Biochemical Biophysical Research Communication 61(4),1464-1470.

Traynor K. 2012 Taliglucerase alfa approved for Gusher’s disease. American Journal of Health-System Pharmacy 69(12), 1009.

Veluthambi K, Jayaswal RK, Gelvin SB. 1987 Virulence   genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proceedings of the National Academy of Sciences of the United States of America (84), 1881–1885.

Vincze E, Bowra S. 2006 Transformation of Rhizobia with Broad Host-range plasmids by using a freeze-Thaw method, Applied and environmental microbiology 72(3), 2290–2293.