Biolistic transformation of Haematococcus pluvialis with constructs based on the flanking sequences of its endogenous alpha tubulin gene

Abstract

18The complete sequence information of the alpha tubulin (tub) genes was obtained from 19 both Haematococcus pluvialis NIES144 and SCCAP K0084. , Putative transcriptional 20 elements and polyadenylation signals were identified respectively in their 5' and 3' 21 flanking regions. Three selection cassettes of tub/aadA, tub/hyr and tub/ble with 3 22 different antibiotic-resistant genes fused between the 5' and 3' flanking sequences of the 23 tub gene were constructed and utilized for biolistic transformation of H.pluvialis. 24 Antibiotic resistant transformants were obtained in the bombardments with the tub/aadA 25 cassette in 2 strains. It was found that, the foreign tub/aadA DNA could be completely 26 transferred and inherited in their genome through non-homologous recombination. 27 Moreover, transcripts of the insert and spectinomycin resistance were identified. 28 Transformation efficiencies up to 3×10 -5 per μg DNA could be obtained in H.pluvialis 29 NIES144 or SCCAP K0084 through utilization of a culture with a high percentage of 30 flagellate cells and by optimizing bombarding protocol. The presented selection marker 31 and optimized transforming procedures in this report should strengthen the platform 32 technology for genetical manipulation and modification of H.pluvialis. 33 34 35 36 37 38 39 3 40 41 Astaxanthin (3, 3'-dihydroxy-ß-carotene-4, 4'-dione) is the strongest antioxidant in the 42 nature, and has been widely used as an additive in health care products and cosmetics [1]. 43 Haematococcus pluvialis has attracted great attention as its richest biological source. 44 Currently, H.pluvialis cultivation has been commercialized to produce astaxanthin 45 powder, which can be utilized as feedstocks or as additives in aquacultural, 46 pharmaceutical and nutraceutical products [2]. H.pluvialis has been considered as an 47 organism for the production of recombinant pharmaceutical proteins[3]. 48 Researches on H.pluvialis have taken place over several decades, mostly focused on 49 cell differentiation [4], astaxanthin metabolism[5], cultivation methods [6] and 50 astaxanthin extraction technology[7]. Continuous efforts were made to improve its 51 cellular growth and the level of astaxanthin accumulation through traditional mutagenesis 52 of H.pluvialis [8]; however there are no reports of phenotypic modification through 53 genetic engineering methods. To date, only 4 researchers have reported stable genetic 54 transformation of H.pluvialis. Steinbrenner & Gerhard Sandmann in 2006 [9] 55 transformed H.pluvialis with a mutated phytoene desaturase (pds) gene conferring 56 resistance to norflurazon, using a biolistic method. Sharon-Gojman et al. in 2015[10] 57 optimized this vector system by shortening the promoter sequence from 2000 base pairs 58 (bp) to 1000 bp. And an agrobacterium-mediated method using a plant-general plasmid 59 pCAMBIA1301 could transform H.pluvialis, with a foreign hptII gene been identified in 60 the transformants [11]. Successful chloroplast transformation was also achieved thr...