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

• Published in: Frontiers in microbiology Vol. 10; p. 1749
• Main Authors: Yuan, Guanhua, Xu, Xiaoying, Zhang, Wei, Zhang, Wenlei, Cui, Yulin, Qin, Song, Liu, Tianzhong
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 02-08-2019
• Subjects: alpha tubulin; biolistic transformation; flanking sequence; Haematococcus pluvialis; Microbiology; selection marker; selection marker; biolistic transformation; Haematococcus pluvialis; alpha tubulin; flanking sequence
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2019.01749

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 (
) gene were cloned along with its encoding region in
, 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
promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of
. Stable resistant colonies with introduced
genes were obtained after bombardments of either
NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10
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
genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of
.