Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana
Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana
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DOI：
10.1007/s13205-021-02750-7
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发表时间：
2021-03-26
期刊：
影响因子：
2.8
通讯作者：
Sahoo, Lingaraj
中科院分区：
文献类型：
--
作者：
Sharma, Prabin Kumar;Goud, Vaibhab V.;Sahoo, Lingaraj
绿色产油微藻小球藻（Chlorella sorokiniana）是一种高产的小球藻属物种，并且是用于生产生物燃料、营养品和重组治疗性蛋白质的潜在宿主。缺乏稳定高效的遗传转化体系是该物种改良的主要瓶颈。本文首次报道了一种高效、稳定的根癌农杆菌介导的转化体系。sorokiniana。共培养C. Sorokiniana细胞（λ（680）处的光密度=1.0）与细胞密度为OD 600 =0.6的农杆菌在BG 11琼脂培养基上（pH5.6）中，在25 +/-2 ℃下在黑暗中培养3天，导致显著更高的转化效率（每10（6）个细胞220 +/-5潮霉素抗性菌落）。在含有30 mg/L潮霉素的BG 11液体培养基上初步选择转化细胞，然后在含有75 mg/L潮霉素的BG 11琼脂培养基上选择同质转化体。PCR分析证实了hptII的存在，并且virG扩增的缺乏排除了转化的微藻细胞中的农杆菌污染。Southern杂交证实hpt Ⅱ基因已整合到C. sorokiniana。qRT-PCR和Western blot分析证实了hptII和GUS基因在转基因细胞系中的表达。发现转化细胞的比生长速率、生物量倍增时间、PSII活性和脂肪酸谱与野生型未转化细胞相似，清楚地表明转基因表达不影响生长和基本代谢过程。该协议可以促进未来生产生物燃料，类胡萝卜素，营养品和治疗性蛋白质的机会。
The green oleaginous microalgae, Chlorella sorokiniana, is a highly productive Chlorella species and a potential host for the production of biofuel, nutraceuticals, and recombinant therapeutic proteins. The lack of a stable and efficient genetic transformation system is the major bottleneck in improving this species. We report an efficient and stable Agrobacterium tumefaciens-mediated transformation system for the first time in C. sorokiniana. Cocultivation of C. sorokiniana cells (optical density at lambda (680)=1.0) with Agrobacterium at a cell density of OD600=0.6, on BG11 agar medium (pH 5.6) supplemented with 100 mu M of acetosyringone, for three days at 25 +/- 2 degrees C in the dark, resulted in significantly higher transformation efficiency (220 +/- 5 hygromycin-resistant colonies per 10(6) cells). Transformed cells primarily selected on BG11 liquid medium with 30 mg/L hygromycin followed by selecting homogenous transformants on BG11 agar medium with 75 mg/L hygromycin. PCR analysis confirmed the presence of hptII, and the absence of virG amplification ruled out the Agrobacterium contamination in transformed microalgal cells. Southern hybridization confirmed the integration of the hptII gene into the genome of C. sorokiniana. The qRT-PCR and Western blot analyses confirmed hptII and GUS gene expression in the transgenic cell lines. The specific growth rate, biomass doubling time, PSII activity, and fatty-acid profile of transformed cells were found similar to wild-type untransformed cells, clearly indicating the growth and basic metabolic processes not compromised by transgene expression. This protocol can facilitate opportunities for future production of biofuel, carotenoids, nutraceuticals, and therapeutic proteins.