Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology

Recently, artificial microRNA (amiRNA) technology has been widely used as a tool for creating loss-of-function mutants, especially in studies involving functional profiling, as it is able to silence genes or gene families in a specific manner. Artificial microRNA is derived by replacing the native m...

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المؤلف الرئيسي:	Noor Hydayaty Md. Yusuf
التنسيق:	أطروحة
اللغة:	English English
منشور في:	2017
الموضوعات:	QK900-989 Plant ecology
الوصول للمادة أونلاين:	https://eprints.ums.edu.my/id/eprint/37815/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/37815/2/FULLTEXT.pdf
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id	my-ums-ep.37815
record_format	uketd_dc
spelling	my-ums-ep.378152023-12-06T07:32:46Z Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology 2017 Noor Hydayaty Md. Yusuf QK900-989 Plant ecology Recently, artificial microRNA (amiRNA) technology has been widely used as a tool for creating loss-of-function mutants, especially in studies involving functional profiling, as it is able to silence genes or gene families in a specific manner. Artificial microRNA is derived by replacing the native mature miRNA duplex from an endogenous precursor miRNA (pre-miRNA) with synthetic ones. Like mature miRNA, amiRNA is designed with an ability to bind complementarily to its target _gene. The aim of the study was to develop amiRNAs using different backbones, which can then be used to silence the endogenous pineapple microRNA MIR535 family. In order to find the efficient amiRNA silencing in pineapple, stems of precursors were modified, as this will affect their processing efficiency by endogenous miRNA biogenesis. And, in order to silence the MIR535family, amiRNA was designed with the ability to bind to this mature region, as this increase the probability of it targeting more than one miR535 member. The amiRNAs were developed from newly discovered pre-miRNA from pineapple, and previously identified ones from Arabidopsis thaliana and 0ryza sativa. The first step involved the identification of the pre-miRNAs from pineapple transcriptomic libraries through in silico analysis. The amiRNAs were then designed to target theMIR535family, and subsequently inserted into precursors, and synthesized. The sequences of the expression cassette (promoter, enhancer, and terminator) were then fused into it, before transforming it into the plant expression vector, pCambia1303. Transgenes were then inserted into pineapple callus (MD2 hybrid) through Agrobacterium mediated transformation. Transgenic lines developed were used for expression profiling of amiRNAs and miR535's through stem-loop RT-qPCR. Three precursors found from pineapple (pre-miR156, pre-miR399, pre-miR2673) were modified (to have 20nt and S0nt stem) and used to carry amiRNA, together with the precursors of A. thaliana (pre-miR319) and 0. sativa (pre-miR528). Here, transgenic lines which have been inserted with these precursors showed the presence of amiRNA. Two pineapple precursors were found to be highly efficient in expressing amiRNA i.e. pre-miR156-50nt stem (Cq value of 20), followed by pre-miR2673 (Cq value of 24.4). The precursors from A. thaliana and 0. sativa were also found to be functional in pineapple, each with the Cq values of 20.8 and 23.8, respectively. Next, the ability of this amiRNA to silence the target gene (mature miR535b) was observed. In conjunction with the expression of amiRNA in transgenic callus, the expressions of target gene was found downregulated, with the highest silencing rate was by amiRNA produced from pre-miR156-50nt and pre-miR319. Also, the expressions of other mature miR535's were also quantified, and were found downregulated. In conclusion, the amiRNA technology was successfully developed for pineapple evidenced by the creation of loss-of-function mutant intheMIR535 family. The pineapple endogenous precursor was found capable to serve as backbone for amiRNA technology in pineapple. This study suggests that targeting 'common region' when designing amiRNA results in the silencing of several genes of the same family at the same time. Now, two highly efficient amiRNA precursors, pre-miR156 and pre-miR319 can be utilized in gene silencing- programs in pineapple. 2017 Thesis https://eprints.ums.edu.my/id/eprint/37815/ https://eprints.ums.edu.my/id/eprint/37815/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/37815/2/FULLTEXT.pdf text en validuser dphil doctoral Universiti Malaysia Sabah Institut Penyelidikan Bioteknologi
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	QK900-989 Plant ecology
spellingShingle	QK900-989 Plant ecology Noor Hydayaty Md. Yusuf Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
description	Recently, artificial microRNA (amiRNA) technology has been widely used as a tool for creating loss-of-function mutants, especially in studies involving functional profiling, as it is able to silence genes or gene families in a specific manner. Artificial microRNA is derived by replacing the native mature miRNA duplex from an endogenous precursor miRNA (pre-miRNA) with synthetic ones. Like mature miRNA, amiRNA is designed with an ability to bind complementarily to its target _gene. The aim of the study was to develop amiRNAs using different backbones, which can then be used to silence the endogenous pineapple microRNA MIR535 family. In order to find the efficient amiRNA silencing in pineapple, stems of precursors were modified, as this will affect their processing efficiency by endogenous miRNA biogenesis. And, in order to silence the MIR535family, amiRNA was designed with the ability to bind to this mature region, as this increase the probability of it targeting more than one miR535 member. The amiRNAs were developed from newly discovered pre-miRNA from pineapple, and previously identified ones from Arabidopsis thaliana and 0ryza sativa. The first step involved the identification of the pre-miRNAs from pineapple transcriptomic libraries through in silico analysis. The amiRNAs were then designed to target theMIR535family, and subsequently inserted into precursors, and synthesized. The sequences of the expression cassette (promoter, enhancer, and terminator) were then fused into it, before transforming it into the plant expression vector, pCambia1303. Transgenes were then inserted into pineapple callus (MD2 hybrid) through Agrobacterium mediated transformation. Transgenic lines developed were used for expression profiling of amiRNAs and miR535's through stem-loop RT-qPCR. Three precursors found from pineapple (pre-miR156, pre-miR399, pre-miR2673) were modified (to have 20nt and S0nt stem) and used to carry amiRNA, together with the precursors of A. thaliana (pre-miR319) and 0. sativa (pre-miR528). Here, transgenic lines which have been inserted with these precursors showed the presence of amiRNA. Two pineapple precursors were found to be highly efficient in expressing amiRNA i.e. pre-miR156-50nt stem (Cq value of 20), followed by pre-miR2673 (Cq value of 24.4). The precursors from A. thaliana and 0. sativa were also found to be functional in pineapple, each with the Cq values of 20.8 and 23.8, respectively. Next, the ability of this amiRNA to silence the target gene (mature miR535b) was observed. In conjunction with the expression of amiRNA in transgenic callus, the expressions of target gene was found downregulated, with the highest silencing rate was by amiRNA produced from pre-miR156-50nt and pre-miR319. Also, the expressions of other mature miR535's were also quantified, and were found downregulated. In conclusion, the amiRNA technology was successfully developed for pineapple evidenced by the creation of loss-of-function mutant intheMIR535 family. The pineapple endogenous precursor was found capable to serve as backbone for amiRNA technology in pineapple. This study suggests that targeting 'common region' when designing amiRNA results in the silencing of several genes of the same family at the same time. Now, two highly efficient amiRNA precursors, pre-miR156 and pre-miR319 can be utilized in gene silencing- programs in pineapple.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Noor Hydayaty Md. Yusuf
author_facet	Noor Hydayaty Md. Yusuf
author_sort	Noor Hydayaty Md. Yusuf
title	Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
title_short	Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
title_full	Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
title_fullStr	Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
title_full_unstemmed	Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology
title_sort	development of pineapple transgenic lines for the functional profiling of mir535 gene family through the use of artificial microrna technology
granting_institution	Universiti Malaysia Sabah
granting_department	Institut Penyelidikan Bioteknologi
publishDate	2017
url	https://eprints.ums.edu.my/id/eprint/37815/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/37815/2/FULLTEXT.pdf
_version_	1794022685116727296

Development of pineapple transgenic lines for the functional profiling of MIR535 gene family through the use of artificial microrna technology

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