Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

Abstract

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions (Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT ® 3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

“…In addition, the use of the GUS reporter gene is also a problem, since we have already confirmed that the GUS reporter gene is not functional in red algal cells by histochemical analysis and enzymatic activity test (see below). This is supported by previous reports indicating background levels of the GUS enzymatic activity in red and brown macroalgal cells when the GUS reporter gene was driven by CaMV 35S and endogenous beta-tubulin promoters Gong et al, 2005 …”

Seaweed protoplasts: status, biotechnological perspectives and needs

“…However, the SV40 promoter reported to be more efficient than CaMV35S in expression of above mentioned genes in Laminaria. Endogenous tubulin promoter has also been used to determine its transcription promoting abilities for foreign gene gusA in protoplasts of Porphyra yezoensis using vector pATubGUS (Gong et al 2007). For the first time, the Expressed sequence tags (ESTs) from protoplasts and thalli have been used to identify the genes involved in cell wall regeneration and stress responses in Chondrus crispus and Laminaria digitata (Collen et al 2006;Roeder et al 2005).…”

Current Advances in Seaweed Transformation

“…To date, studies have been mainly focused on Porphyra species because of their economical values. As shown in Table 1, expression of the CaMV 35S-GUS gene was previously observed in P. miniata, P. tenera and P. yezoensis [37][38][39][40][41][42], all of which were performed by electoroporation using protoplasts. Kuang et al [38] also tested the particle bombardment of the CaMV 35S-GUS gene in P. yezoensis and got positive results.…”