Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato

Reporter gene activity under the regulation of the oil palm metallothionein-like gene promoter (MT3-A) was assessed in prokaryotes and transgenic tomato. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuroni...

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Main Author:	Kamaladini, Hossein
Format:	Thesis
Language:	English
Published:	2011
Subjects:	Functional analysis Oil palm Metallothionein
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spelling	my-upm-ir.195282014-06-30T07:27:40Z Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato 2011-07 Kamaladini, Hossein Reporter gene activity under the regulation of the oil palm metallothionein-like gene promoter (MT3-A) was assessed in prokaryotes and transgenic tomato. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuronidase (GUS) gene in pCAMBIA 1304 vector were produced. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS gene transcripts and enzyme activity in response to increasing concentrations of Cu2+ and Zn2+ in E. coli and A. tumefaciens. In E. coli, a high level of GUS activity driven by W1MT3-A and W2MT3-A was observed in treatment with 25μM Cu2+ resulting in an increase in the GUS mRNA level to 7.2 and 7.5 x 10-4 pmole/μL respectively, compared to the control (without Cu2+) at 5.1 x 10-4 pmole/μL. The application of different Zn2+ concentrations resulted in a strong decrease in the GUS activity and GUS mRNA level in E. coli and A. tumefaciens. The results of Agrobacterium-mediated transformation of tomato showed that MS medium containing 2 mg/L zeatin, 6 mg/L hygromycin and 300 mg/L of timentin was optimum to acquire the maximum putative shoot regeneration of tomato var. MT1. Histochemical GUS assay analysis of different tissues of transgenic tomato showed that W1MT3-A or the W2MT3-A only drove GUS expression in the reproductive tissues and organs, including the stamen, fruit as well as the seed coat compared to the controls. Based on the standard curves the concentrations of GUS cDNA driven by W1MT3-A and W2MT3-A fragments in fruits of independent lines of transgenic tomato (containing one copy number) were calculated to be 9.9 x 10-4 and 8.3 x 10-4 pmole/μL, respectively. In transgenic tomato fruits harboring the W1MT3-A, the highest level of GUS activity was observed (13.5-fold) in the independent line containing three copy number compared to the control (untransformed fruit). In the leaves of L1W1MT3-A seedlings, the highest and the lowest GUS activity was observed at 150 μM and 50 μM Cu2+, respectively compared to the control without Cu2+. Results of the Zn2+ and Fe2+application showed no increase in GUS activity driven by the MT3-A promoter in the leaves of L1W1MT3-A seedlings compared to the control. The highest level of GUS activity driven by W1MT3-A was observed in treatments with 100 μM ABA (4-fold), compared to the absence of ABA as control. In addition, the leaves of the L1W1MT-A seedlings under 14 days drought stress showed an increase (11.6-fold) in GUS activity compared to the untreated L1W1MT3-A seedlings as control. This is the first report that showed the oil palm MT3-A promoter is functional in prokaryotes and produced detectable GUS transcripts and enzyme activities. Furthermore, it showed that the MT3-A promoter is a tissue specific promoter in transgenic tomato and can be induced by abiotic stress such as drought and ABA or metal ions like Cu2+. Stressinducible activity of the oil palm MT3-A promoter may potentially be used in prokaryotic, monocot and dicot plants in genetic engineering for improving their metabolism, nutritional value and production of novel products. Functional analysis Oil palm Metallothionein 2011-07 Thesis http://psasir.upm.edu.my/id/eprint/19528/ phd doctoral Universiti Putra Malaysia Functional analysis Oil palm Metallothionein Faculty of Agriculture English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Functional analysis Oil palm Metallothionein
spellingShingle	Functional analysis Oil palm Metallothionein Kamaladini, Hossein Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
description	Reporter gene activity under the regulation of the oil palm metallothionein-like gene promoter (MT3-A) was assessed in prokaryotes and transgenic tomato. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuronidase (GUS) gene in pCAMBIA 1304 vector were produced. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS gene transcripts and enzyme activity in response to increasing concentrations of Cu2+ and Zn2+ in E. coli and A. tumefaciens. In E. coli, a high level of GUS activity driven by W1MT3-A and W2MT3-A was observed in treatment with 25μM Cu2+ resulting in an increase in the GUS mRNA level to 7.2 and 7.5 x 10-4 pmole/μL respectively, compared to the control (without Cu2+) at 5.1 x 10-4 pmole/μL. The application of different Zn2+ concentrations resulted in a strong decrease in the GUS activity and GUS mRNA level in E. coli and A. tumefaciens. The results of Agrobacterium-mediated transformation of tomato showed that MS medium containing 2 mg/L zeatin, 6 mg/L hygromycin and 300 mg/L of timentin was optimum to acquire the maximum putative shoot regeneration of tomato var. MT1. Histochemical GUS assay analysis of different tissues of transgenic tomato showed that W1MT3-A or the W2MT3-A only drove GUS expression in the reproductive tissues and organs, including the stamen, fruit as well as the seed coat compared to the controls. Based on the standard curves the concentrations of GUS cDNA driven by W1MT3-A and W2MT3-A fragments in fruits of independent lines of transgenic tomato (containing one copy number) were calculated to be 9.9 x 10-4 and 8.3 x 10-4 pmole/μL, respectively. In transgenic tomato fruits harboring the W1MT3-A, the highest level of GUS activity was observed (13.5-fold) in the independent line containing three copy number compared to the control (untransformed fruit). In the leaves of L1W1MT3-A seedlings, the highest and the lowest GUS activity was observed at 150 μM and 50 μM Cu2+, respectively compared to the control without Cu2+. Results of the Zn2+ and Fe2+application showed no increase in GUS activity driven by the MT3-A promoter in the leaves of L1W1MT3-A seedlings compared to the control. The highest level of GUS activity driven by W1MT3-A was observed in treatments with 100 μM ABA (4-fold), compared to the absence of ABA as control. In addition, the leaves of the L1W1MT-A seedlings under 14 days drought stress showed an increase (11.6-fold) in GUS activity compared to the untreated L1W1MT3-A seedlings as control. This is the first report that showed the oil palm MT3-A promoter is functional in prokaryotes and produced detectable GUS transcripts and enzyme activities. Furthermore, it showed that the MT3-A promoter is a tissue specific promoter in transgenic tomato and can be induced by abiotic stress such as drought and ABA or metal ions like Cu2+. Stressinducible activity of the oil palm MT3-A promoter may potentially be used in prokaryotic, monocot and dicot plants in genetic engineering for improving their metabolism, nutritional value and production of novel products.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Kamaladini, Hossein
author_facet	Kamaladini, Hossein
author_sort	Kamaladini, Hossein
title	Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
title_short	Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
title_full	Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
title_fullStr	Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
title_full_unstemmed	Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato
title_sort	functional analysis of oil palm metallothionein-like gene promoter (mt3-a) in bacteria and transgenic tomato
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Agriculture
publishDate	2011
_version_	1747811403709808640

Functional Analysis of Oil Palm Metallothionein-Like Gene Promoter (MT3-A) in Bacteria and Transgenic Tomato

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