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

Guanhua Yuan,Yulin Cui,Song Qin,Xiaoying Xu,Wei Zhang,Tianzhong Liu,Wenlei Zhang

doi:10.3389/fmicb.2019.01749

Abstract

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.

Highlights

Astaxanthin (3, 3 -dihydroxy-ß-carotene-4, 4 -dione) is the strongest antioxidant in nature and has been widely used as an additive in health care products and cosmetics (Han et al, 2013)
There must be some operational details in H. pluvialis transformation waiting to be disclosed and more efficient genetic toolboxes are still needed for the establishment of its efficient genetic engineering system
It was generally thought that the utilization of those endogenous ones could greatly facilitate the integration and expression of foreign genes in the host genome (Radakovits et al, 2010)

Summary

Introduction

Astaxanthin (3, 3 -dihydroxy-ß-carotene-4, 4 -dione) is the strongest antioxidant in nature and has been widely used as an additive in health care products and cosmetics (Han et al, 2013). H. pluvialis cultivation has been commercialized and the edibility of its whole-cell biomass has been approved by the FDA (Panis and Carreon, 2016) It is considered as a good biological producer for recombinant pharmaceutical proteins (Saei et al, 2012). An agrobacterium-mediated method was once proposed to transform H. pluvialis, which was based on a hptII gene driven by a plant-general CaMV35S promoter (Kathiresan and Sarada, 2009; Kathiresan et al, 2009). This method was not repeated successfully by the other researchers (Sharon-Gojman et al, 2015), and similar failed attempts may have been encountered. There must be some operational details in H. pluvialis transformation waiting to be disclosed and more efficient genetic toolboxes are still needed for the establishment of its efficient genetic engineering system

Methods

Results

Conclusion