Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus

Endophytes, by definition, are organisms including fungi that live in close association with living plant tissues. Since no plants without endophytic fungi have been found to date, it is considered that endophytic fungi in plants are a common and general phenomenon. However, very few microorganisms...

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Main Author:	Shamsuddin, Raba’atun Adawiyah
Format:	Thesis
Language:	English
Published:	2010
Subjects:	Molecular cloning Endophytic fungi Plant cells and tissues
Online Access:	http://psasir.upm.edu.my/id/eprint/113971/1/113971.pdf
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spelling	my-upm-ir.1139712024-12-04T08:10:49Z Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus 2010-08 Shamsuddin, Raba’atun Adawiyah Endophytes, by definition, are organisms including fungi that live in close association with living plant tissues. Since no plants without endophytic fungi have been found to date, it is considered that endophytic fungi in plants are a common and general phenomenon. However, very few microorganisms living in plants were reported to be inulin degraders. Inulin occurs as a reserve carbohydrate in plants. Depolymerization of inulin involves the action of inulinase enzymes, isolated from microorganisms including fungi. In this study, the endophytes producing inulinase were isolated from plant species in Serdang area. Thus, the fungal isolates were screened for inulin degrading enzymes. The results were evaluated in terms of substrate consumption and cell growth. All fungal isolates were able to degrade inulin and, among these, two isolates, named Asf1 and Onf1, were selected as the best inulinase producers. Genomic DNA of all isolates were extracted and amplified by polymerase chain reaction (PCR). A 1341 bp DNA fragment containing inulinase gene was successfully amplified from Asf1 fungal isolate and was named as inu2 gene in this study. Based on the morphological characteristics, Asf1 fungal isolate display closely-related to that genus of Aspergillus. The 18S ribosomal DNA sequence analysis revealed their high similarity of 99% to those of Aspergillus species. According to the neighbor-joining phylogenetic tree, Asf1 fungal isolate was shown to be closest to the species from the genus Aspergillus. The complete sequence designated Asf1 inu2 gene, was successfully obtained via rapid-amplification of cDNA ends-polymerase chain reaction (RACE-PCR). A 2.3 kb DNA fragment encoding endoinulinase, inu2, from Asf1 fungal isolate includes an open reading frame of 1,552 bp with calculated molecular weight of 55954.1 Da and signal peptide sequence of 23 amino acids. The deduced amino acid sequence of the Asf1 Inu2 showed 97%, 96%, 69% and 22% identities to that of A. ficuum inu2, A. niger inuB, P. purpurogenum and K. marxianus, respectively. A phylogenetic analysis showed that fungal endo- and exo-inulinases have indepently evolved the respective hydrolytic activities toward terminal and internal β-(2→1)-fructofuranosidic linkages in inulin. Molecular cloning Endophytic fungi Plant cells and tissues 2010-08 Thesis http://psasir.upm.edu.my/id/eprint/113971/ http://psasir.upm.edu.my/id/eprint/113971/1/113971.pdf text en public http://ethesis.upm.edu.my/id/eprint/18027 masters Universiti Putra Malaysia Molecular cloning Endophytic fungi Plant cells and tissues
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Molecular cloning Endophytic fungi Plant cells and tissues
spellingShingle	Molecular cloning Endophytic fungi Plant cells and tissues Shamsuddin, Raba’atun Adawiyah Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
description	Endophytes, by definition, are organisms including fungi that live in close association with living plant tissues. Since no plants without endophytic fungi have been found to date, it is considered that endophytic fungi in plants are a common and general phenomenon. However, very few microorganisms living in plants were reported to be inulin degraders. Inulin occurs as a reserve carbohydrate in plants. Depolymerization of inulin involves the action of inulinase enzymes, isolated from microorganisms including fungi. In this study, the endophytes producing inulinase were isolated from plant species in Serdang area. Thus, the fungal isolates were screened for inulin degrading enzymes. The results were evaluated in terms of substrate consumption and cell growth. All fungal isolates were able to degrade inulin and, among these, two isolates, named Asf1 and Onf1, were selected as the best inulinase producers. Genomic DNA of all isolates were extracted and amplified by polymerase chain reaction (PCR). A 1341 bp DNA fragment containing inulinase gene was successfully amplified from Asf1 fungal isolate and was named as inu2 gene in this study. Based on the morphological characteristics, Asf1 fungal isolate display closely-related to that genus of Aspergillus. The 18S ribosomal DNA sequence analysis revealed their high similarity of 99% to those of Aspergillus species. According to the neighbor-joining phylogenetic tree, Asf1 fungal isolate was shown to be closest to the species from the genus Aspergillus. The complete sequence designated Asf1 inu2 gene, was successfully obtained via rapid-amplification of cDNA ends-polymerase chain reaction (RACE-PCR). A 2.3 kb DNA fragment encoding endoinulinase, inu2, from Asf1 fungal isolate includes an open reading frame of 1,552 bp with calculated molecular weight of 55954.1 Da and signal peptide sequence of 23 amino acids. The deduced amino acid sequence of the Asf1 Inu2 showed 97%, 96%, 69% and 22% identities to that of A. ficuum inu2, A. niger inuB, P. purpurogenum and K. marxianus, respectively. A phylogenetic analysis showed that fungal endo- and exo-inulinases have indepently evolved the respective hydrolytic activities toward terminal and internal β-(2→1)-fructofuranosidic linkages in inulin.
format	Thesis
qualification_level	Master's degree
author	Shamsuddin, Raba’atun Adawiyah
author_facet	Shamsuddin, Raba’atun Adawiyah
author_sort	Shamsuddin, Raba’atun Adawiyah
title	Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
title_short	Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
title_full	Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
title_fullStr	Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
title_full_unstemmed	Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
title_sort	molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus
granting_institution	Universiti Putra Malaysia
publishDate	2010
url	http://psasir.upm.edu.my/id/eprint/113971/1/113971.pdf
_version_	1818586172856729600

Molecular cloning and sequence analysis of an inulinase gene from an endophytic fungus

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