Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants

Transgenic plants offer advantages for the manufacture of recombinant proteins with terminal mannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and for the development of alternative expression systems to produce recombinant lysosomal enzymes. In the...

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Autor principal:	Zainab Tuama Aldahlee
Formato:	article
Lenguaje:	AR EN
Publicado:	College of Science for Women, University of Baghdad 2021
Materias:	signal peptide, Hu-GBA gene, pCAMBIA1304 vector, Soybean, Agrobacterium- mediated transformation Science Q
Acceso en línea:	https://doaj.org/article/58fdce38dc394de9bd0306fe4b1e83d2
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spelling	oai:doaj.org-article:58fdce38dc394de9bd0306fe4b1e83d22021-12-04T16:13:01ZDesigning Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants10.21123/bsj.2021.18.4.11732078-86652411-7986https://doaj.org/article/58fdce38dc394de9bd0306fe4b1e83d22021-12-01T00:00:00Zhttps://bsj.uobaghdad.edu.iq/index.php/BSJ/article/view/4401https://doaj.org/toc/2078-8665https://doaj.org/toc/2411-7986 Transgenic plants offer advantages for the manufacture of recombinant proteins with terminal mannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and for the development of alternative expression systems to produce recombinant lysosomal enzymes. In the present study the sequence of the natural cDNA encoding for the human lysosomal enzyme glucocerebrosidase (GCD) was modified to enhance its expression in soybean plants. The glucocerebrosidase gene signal peptide was substituted with that signal peptide for the Arabidopsis thaliana basic endochitinase gene to support the co-translational translocation into the endoplasmic reticulum (ER), and the storage vacuole. So, targeting signal from tobacco chitinase A, to facilitate GCD trafficking from the ER to the storage vacuole, appropriate primers were designed containing both an ER and vacuolar targeting signals, (VTS). Those primers were used for PCR amplification of the human GBA gene (Hu-GBA) gene from constructed PGEM-GBA plasmid which was cloned in the plant expression vector pCAMBIA1304. The resulted construct was transported in Agrobacterium tumefaciens strain LBA4404 and was used for transformation of cotyledon explants. After 5-day of seedling, cotyledons were cut and used as explants. After infection and co-cultivation, hygromycin B was added in selection media as a selective agent for the transformants cotyledons. The presence of the Hu-GBA transgene in the genomes of transgenic plants was determined by polymerase chain reaction PCR as a band of size1587 bp. The GBA mRNA expression in modified soybean was detected by qRT-PCR compared with control GBA mRNA. Zainab Tuama AldahleeCollege of Science for Women, University of Baghdadarticlesignal peptide, Hu-GBA gene, pCAMBIA1304 vector, Soybean, Agrobacterium- mediated transformationScienceQARENBaghdad Science Journal, Vol 18, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	AR EN
topic	signal peptide, Hu-GBA gene, pCAMBIA1304 vector, Soybean, Agrobacterium- mediated transformation Science Q
spellingShingle	signal peptide, Hu-GBA gene, pCAMBIA1304 vector, Soybean, Agrobacterium- mediated transformation Science Q Zainab Tuama Aldahlee Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
description	Transgenic plants offer advantages for the manufacture of recombinant proteins with terminal mannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and for the development of alternative expression systems to produce recombinant lysosomal enzymes. In the present study the sequence of the natural cDNA encoding for the human lysosomal enzyme glucocerebrosidase (GCD) was modified to enhance its expression in soybean plants. The glucocerebrosidase gene signal peptide was substituted with that signal peptide for the Arabidopsis thaliana basic endochitinase gene to support the co-translational translocation into the endoplasmic reticulum (ER), and the storage vacuole. So, targeting signal from tobacco chitinase A, to facilitate GCD trafficking from the ER to the storage vacuole, appropriate primers were designed containing both an ER and vacuolar targeting signals, (VTS). Those primers were used for PCR amplification of the human GBA gene (Hu-GBA) gene from constructed PGEM-GBA plasmid which was cloned in the plant expression vector pCAMBIA1304. The resulted construct was transported in Agrobacterium tumefaciens strain LBA4404 and was used for transformation of cotyledon explants. After 5-day of seedling, cotyledons were cut and used as explants. After infection and co-cultivation, hygromycin B was added in selection media as a selective agent for the transformants cotyledons. The presence of the Hu-GBA transgene in the genomes of transgenic plants was determined by polymerase chain reaction PCR as a band of size1587 bp. The GBA mRNA expression in modified soybean was detected by qRT-PCR compared with control GBA mRNA.
format	article
author	Zainab Tuama Aldahlee
author_facet	Zainab Tuama Aldahlee
author_sort	Zainab Tuama Aldahlee
title	Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
title_short	Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
title_full	Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
title_fullStr	Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
title_full_unstemmed	Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
title_sort	designing primers with a plant signal peptide to enhance the expression of gba1 in transgenic soybean plants
publisher	College of Science for Women, University of Baghdad
publishDate	2021
url	https://doaj.org/article/58fdce38dc394de9bd0306fe4b1e83d2
work_keys_str_mv	AT zainabtuamaaldahlee designingprimerswithaplantsignalpeptidetoenhancetheexpressionofgba1intransgenicsoybeanplants
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Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants

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