浮萍合成生物学研究进展

随着浮萍基因组和转录组数据的不断获得,以及高效稳定的浮萍遗传转化和基因编辑体系的建立,以浮萍为底盘的表达系统已经成功表达了多种外源蛋白,浮萍有望成为植物合成生物学的研究热点。现简要介绍浮萍的基本信息,对浮萍科植物的基因组和转录组信息、遗传表达体系、浮萍合成生物学使能技术和合成的外源蛋白进行概述,同时简要总结浮萍合成生物学的优势,对浮萍合成生物学未来的发展前景进行展望。     

浮萍相关研究的几方面重要进展

浮萍科是最简单的单子叶水生漂浮植物之一,已被广泛用于植物生理学、遗传学、生态学研究以及水体污染检测等。近些年来,浮萍在植物衰老机理、植物生物反应器和除草剂筛选等研究方面取得了令人瞩目的进展。结合本实验室的研究工作,对近年来的相关文献进行了综述。     

饲喂浮萍对黄粉虫生长发育及繁殖的影响

为探索浮萍Lemna minor生物质利用新途径,解决浮萍在污水中引起的二次污染现象,本研究利用黄粉虫Tenebrio molitor L.食性杂,易饲养,可转化利用多种有机生物质资源的的特性,将浮萍烘干、磨碎后,与麦麸配制成50%浮萍∶50%麦麸,60%浮萍∶40%麦麸,70%浮萍∶30%麦麸,80%浮萍∶20%麦麸不同比例的混合饲料(加水使饲料含水量保持为18%)来饲养黄粉虫幼虫(以纯麦麸为对照组);同时以浮萍替代豆饼作为添加料,按照白菜∶麦麸∶浮萍=10∶4∶1做成混合饲料饲养成虫,来研究浮萍对黄粉虫生长发育及繁殖的影响。结果显示:与纯麦麸相比,幼虫试验中各处理组的生物量增长率、饲料利用率均降低,死亡率增高,而饲喂"50%浮萍∶50%麦麸"饲料的黄粉虫幼虫的各指标与对照组差异不显著,为最佳浮萍饲料配比;在成虫试验中,以浮萍为添加料的成虫单雌产卵量为586.33±6.84粒,成虫寿命为105.5±1.43 d,均显著高于以豆饼为添加料的对照组。本研究对利用黄粉虫转化处理浮萍的效果做出了初步评价,即黄粉虫取食转化浮萍是可行的,为浮萍的生物质利用提供参考。     

浮萍光周期诱导的探讨──与日本青萍6746对比试验

浮萍从野外生长到室内无菌培养,能在较大的时间范围内用于浮萍的灭菌,以０．０４％ＨｇＣｌ２的灭菌效果最佳。在相同的培养条件下,浮萍比日本青萍６７４６的繁殖和营养生长速度略快,但浮萍对光周期反应不敏感。     

介绍稀脉浮萍6746(Lemna per pusilla Torr.6746 Lemnapau cicostata Hegelm 6746)及膨胀浮萍(Lemna gibba L.)

“稀脉浮萍6746”和“膨胀浮萍”都是当前研究高等植物开花生理的一种重要试验材料。1981年日本著名植物生理学者古谷雅树来中国讲学时曾赠给我国一些原种。目前在北京大学生物学系等单位有原种保存,有些单位已相继利用它们开展了一些高等植物开花生理的研究工作。     

稀脉浮萍花芽发育过程中的“群体效应”（简报）

稀脉浮萍群体的“单独化”会抑制稀脉浮萍成熟花的开花。增加培养基体积可稍微降低稀脉浮萍的成熟花开花百分率。培养过开花稀脉浮萍的“开花旧培养基”可部分逆转“单独化”的处理效果，而非开花旧培养基则否。     

不同光温条件下紫背浮萍叶肉细胞和叶绿体超微结构的研究

以浮萍科紫萍属紫背浮萍为试验材料,利用透射电子显微镜研究了紫背浮萍叶肉细胞和叶绿体的超微结构。结果发现:强光下,紫背浮萍叶肉细胞内的叶绿体数明显增加;弱光时虽然叶绿体数减少,但叶绿体基粒片层结构变厚,增加了光合作用反应面积。与25℃适温条件相比,10℃低温和35℃高温下,紫背浮萍细胞均出现一定程度的逆境胁迫作用。表现在低温时部分叶绿体皱缩成带状,叶肉细胞之间出现较大空隙;高温时叶绿体外膜溶解,基质外渗,脂质小球数量增多。紫背浮萍通过改变细胞内部形态结构的方式来适应不同光强和温度环境条件,对逆境光温条件具有一定的耐受性。     

利用稻田养萍诱集控制福寿螺危害的效应研究

利用自制的“T 形槽”进行实验室试验, 研究了福寿螺(Pomacea canaliculata)对浮萍(Lemna minor)和不同日龄水稻(Oryza sativa)的偏好选择, 结果发现, 无论水稻日龄是15 d, 还是23 d 和30 d, 均有60%以上的福寿螺选择取食浮萍, 且与福寿螺个体大小和选择取食的时间没有明显关联。在模拟稻田生境中, 引入浮萍3 d 后, 水稻受福寿螺的啃食危害明显减少, 水稻存活率高出对照组10%以上, 且30 日龄水稻秧苗的存活率明显高于15 日龄处理。在此基础上, 分析了不同大小福寿螺与浮萍摄入量的关系。这一结果可为浮萍的合理投放提供参考。研究表明, 利用稻田放养浮萍诱集福寿螺取食, 可以减少福寿螺对水稻的危害。     

基于人工培养的浮萍快速无性繁殖营养条件研究

为探究并优化浮萍人工培养技术, 研究以广布种紫萍(Spirodela polyrrhiza)和青萍(Lemna minor)为主要研究对象, 探索两种浮萍植物在不同营养水平的Hoagland和Hunter培养液中的鲜重、叶状体数的生长变化状况。结果表明: (1)紫萍和青萍鲜重在Hoagland培养液的不同营养水平下先增加后减少, 而在Hunter营养液中呈持续增长趋势; 两种浮萍的鲜重最大相对增长率(RGR)分别为0.11和0.18, 鲜重受浮萍品种和培养基类型及其不同营养水平影响显著(P<0.05), 以青萍在Hunter原液培养基下的无性繁殖产生的生物量最高。(2)紫萍和青萍叶状体数在Hoagland培养液的不同营养水平下先增加后减少, 而同鲜重一样在Hunter营养液中呈不断增长趋势; 两种浮萍的叶状体最大相对增长率分别为0.14和0.19, 叶状体生长的RGR变化同样受浮萍品种和培养基类型及营养水平影响显著(P<0.05), 以青萍在Hunter原液的营养环境下收获的叶状体数最高。(3)两种浮萍在Hoagland和Hunter营养液的不同营养水平下鲜重/叶状体比呈下降趋势, 表明两种浮萍在适应不同营养时优先繁殖子代叶状体, 以扩大种的适合度。研究认为Hunter原液可作为广布种青萍的最优培养条件, 可实现短时间内收获较大浮萍鲜生物量和叶状体数, 为进一步资源化利用提供原材料。     

浮萍植物在水体净化中的研究及展望

水体污染是当前亟须解决的全球性问题。利用植物对水体进行修复独具环境友好性和费用低廉性,近年受到广泛关注。浮萍作为一种新型环境及能源生物,在水体污染治理和能源生产方面具有巨大潜力。近年来,浮萍在重金属吸附、有机物去除和克藻研究方面取得了一定进展。紫萍的基因测序工作已经完成,组培和转化工作的进展也为通过基因工程手段选育污水净化能力强的浮萍提供了基础。综述了浮萍在水体净化方面的最新进展。     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

Die Wasserlinse – eine unterschätzte Vielzweckpflanze

Tiny, inconspicuous duckweeds (Lemnaceae) are often considered a nuisance, due to their propensity to rapidly cover surfaces of water bodies. However, their pronounced growth potential, together with their particular morphological, clonal and genomic properties and their easy cultivation make them valuable model plants for studies, and have established them as indicator plants in the field of ecotoxicology. Duckweeds can make a valuable contribution to wastewater remediation, and the biomass that their growth thereby provides can be of value in many ways, including energy generation, fuel production and use as fertilizer and animal food. Duckweeds grown under controlled conditions can contribute to human nutrition and serve as biofactories for heterologous protein expression. The versatile usage potential of duckweeds has not been thoroughly exploited, as it could and should be in the future.     

Wasserlinsen als Nutzpflanzen

Duckweeds as crop plants Members of the plant family Lemnaceae (duckweeds) are not only interesting because they represent the smallest flowering plants; they possess also the fastest rates of producing biomass. As aquatic plants, duckweed production is not in competition with other agricultural crops that require fertile land while the cultivation of duckweeds does not contribute to further eutrophication of surface water. Instead, they can be cultivated on municipal or agricultural waste water and remove the nutrients during their propagation and growth. Duckweeds can thus be used for cleaning of waste water and the resulting biomass can be valuable starting material for animal feeds and the production of biofuels. Research focusing on these goals has begun to transfer from research laboratories to pilot plants in different parts of the world, e.g. in New Jersey and North Carolina, USA; Chengdu, P. R. China; and Armidale, Australia.     

Chromium removal from water by three species of dyckweeds

The effects of exogenous chromium (Cr) on the growth of three species of duckweeds: Spirodela punctata (G.F.W. Meyer) Thomps., Spirodela polyrhiza (L.) Schleiden and Lem na gibba L., were examined at Cr concentrations ranging from 0.1 to 20 p.p.m. Using ⁵¹Cr radiotracer methodology, it was demonstrated in Experiment 1 that sorption was directly related to ambient Cr concentrations after 8 days of exposure. At and above 1.0 p.p.m. Cr, negative effects on growth were evident, with seasonal variations apparently also affecting Cr uptake and toxicity. Chrome tolerance, based on the percentage of control growth achieved by exposed plants, showed S. polyrhiza to be the least tolerant to Cr, while S. punctata and L. gibba were more tolerant at concentrations above 1.0 p.p.m. In Experiment 2, duckweeds grown in nutrient-rich water with an initial concentration of 10 p.p.m. exhibited substantial growth, with bioaccumulation of Cr increasing with time.The possible use of various types of biological treatment systems for the removal of Cr from wastewater is discussed.     

Survey of duckweed diversity in Lake Chao and total fatty acid,triacylglycerol, profiles of representative strains

Lemnaceae (duckweeds) are widely distributed aquatic flowering plants. Their high growth rate, starch content and suitability for bioremediation make them potential feedstock for biofuels. However, few natural duckweed resources have been investigated in China, and there is no information about total fatty acid (TFA) and triacylglycerol (TAG) composition of duckweeds from China. Here, the genetic diversity of a natural duckweed population collected from Lake Chao, China, was investigated using multilocus sequence typing (MLST). The 54 strains were categorised into four species in four genera, representing 12 distinct sequence types. Strains representing Lemna aequinoctialis and Spirodela polyrhiza were predominant. Interestingly, a surprisingly high degree of genetic diversification within L. aequinoctialis was observed. The four duckweed species revealed a uniform fatty acid composition, with three fatty acids, palmitic acid, linoleic acid and linolenic acid, accounting for more than 80% of the TFA. The TFA in biomass varied among species, ranging from 1.05% (of dry weight, DW) for L. punctata and S. polyrhiza to 1.62% for Wolffia globosa. The four duckweed species contained similar TAG contents, 0.02% mg·DW^?1. The fatty acid profiles of TAG were different from those of TFA, and also varied among the four species. The survey investigated the genetic diversity of duckweeds from Lake Chao, and provides an initial insight into TFA and TAG of four duckweed species, indicating that intraspecific and interspecific variations exist in the content and composition of both TFA and TAG in comparison with other studies.     

Large‐scale screening and characterisation of Lemna aequinoctialis and Spirodela polyrhiza strains for starch production

• Duckweed is considered a promising feedstock for bioethanol production due to its high biomass and starch production. Selection of duckweed strains with high starch accumulation is essential for application of duckweeds to bioethanol production. Geographic differentiation had a large influence on genetic diversity of duckweeds.
• Biomass production, starch content and starch amount in geographically isolated strains of 20 Lemna aequinoctialis and Spirodela polyrhiza were calculated to evaluate their potential for bioethanol production. The influence of different collection time, culture medium and NaCl concentration on starch accumulation of the best strains were analysed.
• The results showed that biomass production, starch content and starch production of duckweeds demonstrated clonal dependency. The best strain was L. aequinoctialis 6000, with biomass production of 15.38 ± 1.47 g m^?2, starch content of 28.68 ± 1.10% and starch production of 4.39 ± 0.25 g m^?2. Furthermore, starch content of L. aequinoctialis 6000 was highest after 8 h of light, tap water was the best medium for starch induction, and NaCl did not induce starch accumulation.
• This study suggests duckweed biomass production and starch production demonstrate clonal dependency, indicating that extensive clonal comparisons will be required to identify the most suitable isolates for duckweed selective breeding for bioethanol.

     

Cytokinin-induced growth in the duckweeds <Emphasis Type="Italic">Lemna gibba</Emphasis> and <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Duckweeds, quick-growing aquatic plants, have been recently recognized as promising hosts for the large-scale production of recombinant proteins and as an ideal biomass feedstock for biofuel production. These possible wide-spread industrial uses of duckweeds intensified research aimed at understanding the mechanisms that control duckweed growth. Here, we describe how the hormone cytokinin affects growth. We performed a number of standard cytokinin growth- and physiological-response assays using sterile-grown colonies of Lemna gibba and Spirodela polyrhiza. Similar to land plants, cytokinin inhibited root elongation in duckweeds. Surprisingly, and in contrast to land plants, cytokinin promoted growth of aerial organs in both duckweed species, suggesting that the cytokinin growth response fundamentally differs between aquatic and land plants.     

两种浮萍植物的叶绿体超微结构对模拟酸雨的敏感性

用蒸馏水和不同pH值(5.5,4.5,3.5和2.5)的模拟酸雨培养浮萍(Lemn aminorL.)和紫萍(Spirodela polyrrihiza(L.)Schleid)48h后,测定叶状体膜系统渗漏率,观察叶绿体超微结构的变化。结果表明,随酸雨pH的下降,两种浮萍叶状体的膜系统渗率增大,叶绿体超微结构受损。浮萍和紫萍对酸雨的敏感性和伤害时细胞与叶绿体形态变化显示一定的种间差别。浮萍的结构性损伤始于pH4.5,原生质体收缩,出现质壁分离,基粒结构混乱;pH3.5时叶绿体肿胀呈球状,片层结构破坏并出现许多空泡。紫萍在pH3.5时膜系统的外渗率仍较低,基粒和基质类囊体结构无明显改变;pH2.5时叶绿体结构才出现严重伤害,但未见明显的肿胀与质壁分离现象。因此认为在两种浮萍的共生水体中,紫萍对酸雨污染有较强的生存竞争能力,而浮萍则可用作pH<4.5水体的灵敏指示植物。     

Key to the determination of taxa of Lemnaceae: an update

Species of Lemnaceae have a high potential for fast biomass production, and this is increasingly gaining attention among researchers in basic plant sciences as well as among entrepreneurs for feed, food and energy production. Hence, the correct identification of the species being used for different duckweed research and applications is becoming indispensable. Here, we present an updated identification key based on morphological markers to the currently accepted 36 species of duckweeds, considering all taxonomic revisions since the publication of the previous key by E. Landolt in 1986. We also provide supplementary morphological characterization and the geographical occurrence of each species of Lemnaceae.     

Molecular phylogenetic relationships among Lemnaceae and Araceae using the chloroplast trnL-trnF intergenic spacer

We test competing hypotheses of relationships among Aroids (Araceae) and duckweeds (Lemnaceae) using sequences of the trnL-trnF spacer region of the chloroplast genome. Included in the analysis were 22 aroid genera including Pistia and five genera of Lemnaceae including the recently segregated genus Landoltia. Aponogeton was used as an outgroup to root the tree. A data set of 522 aligned nucleotides yielded maximum parsimony and maximum likelihood trees similar to those previously derived from restriction site data. Pistia and the Lemnaceae are placed in two separate and well-supported clades, suggesting at least two independent origins of the floating aquatic growth form within the aroid clade. Within the Lemnaceae there is only partial support for the paradigm of sequential morphological reduction, given that Wolffia is sister to Wolffiella+Lemna. As in the results of the restriction site analysis, pantropical Pistia is placed with Colocasia and Typhonium of southeastern Asia, indicative of Old World affinities. Branch lengths leading to duckweed terminal taxa are much longer relative to other ingroup taxa (including Pistia), evidently as a result of higher rates of nucleotide substitutions and insertion/deletion events. Morphological reduction within the duckweeds roughly correlates with accelerated chloroplast genome evolution.