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Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene

Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some p...

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Autores principales: Yuan, Guanhua, Xu, Xiaoying, Zhang, Wei, Zhang, Wenlei, Cui, Yulin, Qin, Song, Liu, Tianzhong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687776/
https://www.ncbi.nlm.nih.gov/pubmed/31428066
http://dx.doi.org/10.3389/fmicb.2019.01749
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author Yuan, Guanhua
Xu, Xiaoying
Zhang, Wei
Zhang, Wenlei
Cui, Yulin
Qin, Song
Liu, Tianzhong
author_facet Yuan, Guanhua
Xu, Xiaoying
Zhang, Wei
Zhang, Wenlei
Cui, Yulin
Qin, Song
Liu, Tianzhong
author_sort Yuan, Guanhua
collection PubMed
description Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10(–5) per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.
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spelling pubmed-66877762019-08-19 Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene Yuan, Guanhua Xu, Xiaoying Zhang, Wei Zhang, Wenlei Cui, Yulin Qin, Song Liu, Tianzhong Front Microbiol Microbiology Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10(–5) per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis. Frontiers Media S.A. 2019-08-02 /pmc/articles/PMC6687776/ /pubmed/31428066 http://dx.doi.org/10.3389/fmicb.2019.01749 Text en Copyright © 2019 Yuan, Xu, Zhang, Zhang, Cui, Qin and Liu. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Yuan, Guanhua
Xu, Xiaoying
Zhang, Wei
Zhang, Wenlei
Cui, Yulin
Qin, Song
Liu, Tianzhong
Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title_full Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title_fullStr Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title_full_unstemmed Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title_short Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
title_sort biolistic transformation of haematococcus pluvialis with constructs based on the flanking sequences of its endogenous alpha tubulin gene
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687776/
https://www.ncbi.nlm.nih.gov/pubmed/31428066
http://dx.doi.org/10.3389/fmicb.2019.01749
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