藻类基因工程的研究技术及方法

本文从藻类外源基因转移的载体系统、藻类基因的克隆、藻类的遗传转化三方面详细介绍了藻类基因工程的研究技术及方法,综述了藻类基因工程的研究进展,并对藻类基因工程的前景作了展望     

杜氏盐藻基因工程研究现状及应用前景

杜氏盐藻(简称盐藻)作为一种新型生物反应器,成为藻类基因工程的研究热点之一.利用基因工程手段对盐藻进行遗传重组改造以生产外源性物质是目前研究的重要领域.从盐藻相关基因的克隆、cDNA文库的建立、基因组文库的构建、筛选标记的确立和外源基因的表达等五个方面概述了国内外盐藻基因工程的研究进展,并对基因工程技术在盐藻深入研究中的应用作了前景展望.     

杜氏盐藻（Dunaliella salina）基因工程的研究现状及应用前景

杜氏盐藻（简称盐藻）作为新型的生物反应器,成为藻类基因工程的研究热点之一。利用基因工程手段对盐藻进行遗传改造以生产外源物质是目前研究的重要领域。本文从盐藻相关基因的克隆、cDNA文库的建立、基因组文库的构建、筛选标记的确立和外源基因的表达等五个方面全面概述了国内外盐藻基因工程的研究进展,特别是对最新的研究结果进行了综述,并对基因工程技术在盐藻深入研究中的应用做了前景展望。     

杜氏盐藻分子生物学最新进展及展望

杜氏盐藻是一种无细胞壁的单细胞双鞭毛真核藻类,是一种十分重要的藻类资源。过去对杜氏盐藻的研究多集中在形态学、耐盐机理及β-胡萝卜素等方面,近年来,随着藻类基因工程的快速发展,本研究课题组及国内外在杜藻盐藻分子生物学方面做了大量工作,现就杜氏盐藻在这一领域的研究进展进行综述,主要是重要功能基因的克隆与分析、杜氏盐藻调控序列的研究以及杜氏盐藻作为宿主表达外源基因等。     

叶绿体基因工程研究进展

叶绿体是植物细胞和真核藻类执行光合作用的重要细胞器,在叶绿体中表达外源基因比在细胞核中表达具有一些独特优势。叶绿体基因工程涉及叶绿体的基因组特征、转化系统的优点、转化过程及方法等方面,叶绿体基因工程在提高植物光合效率、改良植物特性、生产生物药物及改善植物代谢途径等方面已得到应用。尽管叶绿体基因工程还存在同质化难度高、标记基因转化效率较低、宿主种类偏少等问题,但作为外源基因在高等植物中表达的良好平台其仍然具有广阔的发展和应用前景。     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

维生素对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

近年来分子遗传学和基因工程研究证实 ,大肠杆菌的载体和启动子往往可以适用于蓝藻 ,尤其是单细胞蓝藻的转基因 ,这使得蓝藻基因工程得到了较快的发展 ,利用藻类为宿主的基因产物的生产也受到日益关注 ,因此微藻的培养受到广泛重视。刘凤龙等通过三亲结合转移方式将人ＴＮＦ α基因转入鱼腥藻 71 2 0中并得到表达[1 ] ,但转基因鱼腥藻高效培养方面需进一步研究。混合营养培养方式因具有缩短培养周期、实现细胞高密度培养等优点 ,已成为微藻培养新技术。藻类生长除了需要合适的营养源外 ,许多生长因子对生长具有较大影响 ,本文对维生素Ｂ1 、…     

微藻育种与培养研究概况

微藻类的天然产物具有广泛的遗传变异和潜在的经济价值,但是这些天然产物的获取还依赖于品种的改良和适宜的培养条件。本文概括性地论述了微藻的遗传育种和优化培养。它包括微藻类的遗传多样性、选择育种、诱变育种、细胞融合、基因工程以及固定化培养和生物反应器的应用。这些生物技术为微藻类的利用开辟了广阔的前景。     

DHA合成途径中关键酶基因在毕赤酵母中的表达研究

DHA（二十二碳六烯酸）是人体所需的一种非常重要的多不饱和脂肪酸,主要存在于深海鱼类和海洋微藻类生物中。由于高等植物自身不能合成DHA,因此通过基因工程方法在作物(如玉米)中表达DHA,将会成为最健康的DHA来源,同时也能够减轻人类对海洋资源的破坏。从深海藻类中挑选了5个DHA合成途径中的关键酶基因,分别是Δ4脱饱和酶基因（D4）、Δ5脱饱和酶基因（D5）、Δ6脱饱和酶基因（D6）、C18延长酶基因（E18）和C20延长酶基因（E20）。密码子优化并合成这5个基因,为确定优化后的核苷酸序列是否能在真核生物中正确表达,以pPIC9K为基础载体连入目的基因转化到毕赤酵母GS115 (His4+MUts)中进行表达分析。结果表明,这些基因在甲醇诱导96 h后蛋白表达量最大,Western Blot结果表明表达产物为目的蛋白。该结果为进一步培育能够自身合成DHA的作物奠定了基础。     

获得重组杀蚊藻类的生产、销售权

美国Cyanotech公司从美国Memphis大学获得了生产销售用基因工程制造的杀死蚊子幼虫的蓝藻植物的世界性独占权。该大学的Edward Stevens,Jr.使用编码来自Bacillus thuringiensis var. israelensis(BTi)的杀虫蛋白的基因性状转化蓝藻植物Synechococcus。BTi是特异地作用于蚊虫及黑色昆虫(等)。蓝藻植物将成为蚊子幼虫的食物源,将包被来自BTi基因的藻类喷洒在池内,不给环境带来巨大影响又能控制蚊虫的     

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

The epoxy‐xanthophylls antheraxanthin and violaxanthin are key precursors of light‐harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP‐deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.     

Advances in genetic engineering of marine algae

Algae are a component of bait sources for animal aquaculture, and they produce abundant valuable compounds for the chemical industry and human health. With today's fast growing demand for algae biofuels and the profitable market for cosmetics and pharmaceuticals made from algal natural products, the genetic engineering of marine algae has been attracting increasing attention as a crucial systemic technology to address the challenge of the biomass feedstock supply for sustainable industrial applications and to modify the metabolic pathway for the more efficient production of high-value products. Nevertheless, to date, only a few marine algae species can be genetically manipulated. In this article, an updated account of the research progress in marine algal genomics is presented along with methods for transformation. In addition, vector construction and gene selection strategies are reviewed. Meanwhile, a review on the progress of bioreactor technologies for marine algae culture is also revisited.     

Photobioreactor engineering: Design and performance

This review summarizes the recent advances in high-density algal cultures in the field of algal biotechnology. Photobioreactor engineering for economical and effective utilization of algae and its products has made impressive and promising progress. Bioprocess engineers have expedited the design and the operation of algal cultivation systems. Many of them in use today are open systems due to cost considerations, and closed photobioreactors have recently attracted a considerable attention for the production of valuable biochemicals or for special applications. For high-density cultures, the optimization of environmental factors in the photobioreactors have been explored, including light delivery, CO₂ and O₂ gas transfer, medium supply, mixing and temperature. It is expected that further advanced photobioreactor engineering will enable the commercialization of noble algal products within the next decade.     

Retained duplicate genes in green alga Chlamydomonas reinhardtii tend to be stress responsive and experience frequent response gains

Background

Green algae belong to a group of photosynthetic organisms that occupy diverse habitats, are closely related to land plants, and have been studied as sources of food and biofuel. Although multiple green algal genomes are available, a global comparative study of algal gene families has not been carried out. To investigate how gene families and gene expression have evolved, particularly in the context of stress response that have been shown to correlate with gene family expansion in multiple eukaryotes, we characterized the expansion patterns of gene families in nine green algal species, and examined evolution of stress response among gene duplicates in Chlamydomonas reinhardtii.

Results

Substantial variation in domain family sizes exists among green algal species. Lineage-specific expansion of families occurred throughout the green algal lineage but inferred gene losses occurred more often than gene gains, suggesting a continuous reduction of algal gene repertoire. Retained duplicates tend to be involved in stress response, similar to land plant species. However, stress responsive genes tend to be pseudogenized as well. When comparing ancestral and extant gene stress response state, we found that response gains occur in 13% of duplicate gene branches, much higher than 6% in Arabidopsis thaliana.

Conclusion

The frequent gains of stress response among green algal duplicates potentially reflect a high rate of innovation, resulting in a species-specific gene repertoire that contributed to adaptive response to stress. This could be further explored towards deciphering the mechanism of stress response, and identifying suitable green algal species for oil production.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1335-5) contains supplementary material, which is available to authorized users.     

ALGAL TRANSGENICS IN THE GENOMIC ERA1

The last few years have witnessed significant advances in the field of algal genomics. Complete genome sequences from the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana have been published, the genomes for two more algae (Chlamydomonas reinhardtii and Ostreococcus tauri) are nearing completion, and several others are in progress or at the planning stage. In addition, large‐scale cDNA sequencing projects are being carried out for numerous algal species. This wealth of genome data is serving as a powerful catalyst for the development and application of recombinant techniques for these species. The data provide a rich resource of DNA elements such as promoters that can be used for transgene expression as well as an inventory of genes that are possible targets for genetic engineering programs aimed at manipulating algal metabolism. It is not surprising therefore that significant progress in the genetic engineering of eukaryotic algae is being made. Nuclear transformation of various microalgal species is now routine, and progress is being made on the transformation of macroalgae. Chloroplast transformation has been achieved for green, red, and euglenoid algae, and further success in organelle transformation is likely as the number of sequenced plastid, mitochondrial, and nucleomorph genomes continues to grow. Importantly, the commercial application of algal transgenics is beginning to be realized, and algal biotechnology companies are being established. Recent work has shown that recombinant proteins of therapeutic value can be produced in microalgal species, and it is now realistic to envisage the genetic engineering of commercially important species to improve production of valuable algal products. In this article we review the recent progress in algal transgenics and consider possible future developments now that phycology has entered the genomic era.     

Sustainable algal biorefineries: capitalizing on many benefits of GABA

We provide physiological and metabolic insights into the complex role of γ-aminobutyric acid (GABA) in fine-tuning algal metabolism to improve productivity. Genetic engineering strategies to improve algal GABA biosynthesis are also discussed. Our aim is to provide an understanding of how GABA can be used for cost-competitive algae-based biofuels and bioproducts.     

Effectiveness of flashing light for increasing photosynthetic efficiency of microalgal cultures over a critical cell density

Critical cell density (CCD), the maximum cell concentration without mutual shading in algal cultures, can be used as a new operating parameter for high-density algal cultures and for the application of the flashing light effect on illuminated algal cultures. CCD is a function of average cell volume and light illumination area. The CCD is thus proposed as an index of estimation of mutual shading in algal cultures. Where cell densities are below the CCD, all the cells in photobio-reactors can undergo photosysnthesis at their maximum rate. At cell densities over the CCD, mutual shading will occur and some cells in the illumination chamber cannot grow photoautotrophically. When the cell concentration is higher than the CCD, specific oxygen production rates under flashing light were higher than those under continuous light. The CCD was found to be a useful engineering parameter for the application of flashing light, particularly in high-density algal cultures.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.