使用BODIPY荧光染料快速测定微藻油脂含量方法

使用BODIPY505/515荧光染料,通过荧光分光光度法测定藻细胞中的油脂含量。结果表明：BODIPY505/515的最佳染色条件为二甲基亚砜（DMSO）体积分数2%,BODIPY505/515最终质量浓度0.25μg/mL,染色时间30min,染色温度35℃。在最佳染色条件下,微藻油脂含量与荧光强度呈线性相关（R2=0.976 4）。通过测定BODIPY505/515染色的不同种属微藻的荧光强度,应用该关系计算其油脂含量,与质量法测定的结果相比没有显著差异。该方法较为普适,比传统方法相比具有简便快捷,试样用量少的特点,与尼罗红荧光染料相比具有较窄的发射波谱范围,不会与微藻的自身荧光相互干扰,更适于过程监控及高含油藻株的筛选。     

温度和摄食对溪红点鲑幼鱼呼吸代谢的影响

在不同水温［(5.5±0.5) ℃、(8.5±0.5) ℃、(11.5±0.5) ℃、(14.5±0.5) ℃、(17.5±0.) ℃］条件下,分别测定了饱食和空腹状态下溪红点鲑幼鱼的耗氧率和排 氨率,分析了温度和摄食对溪红点鲑幼鱼呼吸代谢的影响.结果表明：饱食后,5个温度梯度组溪红点鲑幼鱼的耗氧率和排氨率均迅速上升,达最大值后缓慢下降,并逐渐恢复到初始水平;饱食状态下,溪红点鲑幼鱼耗氧率（OR）和排氨率（NR）与温度（t）的回归方程分别为OR=-0.0601t⁴+2.5542t³-39.256t²+276.26t-598.75(n=650,R²=1,4.5 ℃4+0.0826t³-1.2318t²+8.6186t-18.838(n=650,R²=1,4.5 ℃0.9738(n=650,R²=0.9974,4.5 ℃1.0896( n=650,R²=0.9977,4.5 ℃相似文献   

微囊藻和栅列藻的垂直迁移及生态学意义

本研究在自制沉降柱中,检测铜绿微囊藻(Microcystis aeroginosa)和斜生栅列藻(Scenedesmus obliquus)不同生长时期的细胞群体在不同光照条件下的垂直迁移。本文首次提出藻密度衰减系数(K)的概念、拟合方法和生态学意义,并根据等细胞密度沉降面的沉降速率来计算细胞沉降速率(Vc),以这两个参数来衡量细胞的沉降特性,对细胞垂直迁移研究的方法进行了的探索。结果表明,两种藻细胞群体在三个生长时期和四种光照条件的沉降特性表现已定的差异。栅列藻细胞密度的衰减系数(K)最小值为-0.0021cm^-1(上浮),最大值为0.0065cm^-1(下沉);微囊藻的K最小值为-0.0029cm^-1(上浮),最大值为0.0036cm^-1(下沉)。两种藻沉降曲线的指数拟合的R²大多在0.6以上,说明K值是一个较好的衡量细胞群体沉降特性的参数,细胞密度与深度的关系较好地服从指数规律。细胞沉降速度V的最大值(4.681cm·h^-1,下沉)出现在栅列藻的指数期,最大上浮速率-1.790cm·h^-1,出现在微囊藻的稳定期。本文得到的活体藻细胞群体的垂直迁移特性差异的实验数据,说明该实验方法的适用性和两种藻沉降特性差异的可检测性。本文讨论了水华优势种形成与藻细胞群体迁移特性之间的关系。     

气温和降雨量对中国湿地生态系统CO2交换的影响

湿地由于具有较高的初级生产力以及较低的有机质降解速率而成为缓解全球变暖的潜在有效碳汇.虽然近年来中国湿地生态系统CO₂交换过程及其影响机制研究取得了一系列进展,但尚缺乏对数据进行系统性整合分析.基于29篇文献的数据,对中国21个典型湿地植被净生态系统CO₂交换(NEE)、生态系统呼吸(R_eco)、总初级生产力(GPP)、NEE的光响应参数以及R_eco的温度响应参数进行整合分析,并探讨了这些指标对温度与降雨的响应.结果表明： 年尺度上,气温和降雨量对NEE(R²=50%,R²=57% )、GPP(R²=60%,R²=50%)和R_eco(R²=44%,R²=50%)均有显著影响(P<0.05).生长季尺度上,NEE (R²=50%)、GPP (R²=36%)和R_eco(R²=19%)与气温呈显著相关(P<0.05);同时NEE(R²=33%)和GPP(R²=25%)也与降雨量呈显著相关(P<0.05),但R_eco与降雨量的相关关系不显著(P>0.05).生长季降雨量与最大光合速率(A_max)之间呈显著相关 (P<0.01),但与表观量子产率(α)、白天生态系统呼吸速率(R_eco,day)无显著相关(P>0.05).生长季气温对α、A_max和R_{eco, day}均无显著影响(P>0.05).生态系统基础呼吸速率(R_ref)与降雨量无显著相关(P>0.05),但是生态系统呼吸的温度敏感系数(Q₁₀)与降雨量呈显著的线性负相关(P<0.05),同时气温对Q₁₀(R²=0.35)、R_ref(R²=0.46)均产生显著影响(P<0.05).     

气温和降雨量对中国湿地生态系统CO2交换的影响

湿地由于具有较高的初级生产力以及较低的有机质降解速率而成为缓解全球变暖的潜在有效碳汇.虽然近年来中国湿地生态系统CO₂交换过程及其影响机制研究取得了一系列进展,但尚缺乏对数据进行系统性整合分析.基于29篇文献的数据,对中国21个典型湿地植被净生态系统CO₂交换(NEE)、生态系统呼吸(R_eco)、总初级生产力(GPP)、NEE的光响应参数以及R_eco的温度响应参数进行整合分析,并探讨了这些指标对温度与降雨的响应.结果表明： 年尺度上,气温和降雨量对NEE(R²=50%,R²=57% )、GPP(R²=60%,R²=50%)和R_eco(R²=44%,R²=50%)均有显著影响(P<0.05).生长季尺度上,NEE (R²=50%)、GPP (R²=36%)和R_eco(R²=19%)与气温呈显著相关(P<0.05);同时NEE(R²=33%)和GPP(R²=25%)也与降雨量呈显著相关(P<0.05),但R_eco与降雨量的相关关系不显著(P>0.05).生长季降雨量与最大光合速率(A_max)之间呈显著相关 (P<0.01),但与表观量子产率(α)、白天生态系统呼吸速率(R_eco,day)无显著相关(P>0.05).生长季气温对α、A_max和R_{eco, day}均无显著影响(P>0.05).生态系统基础呼吸速率(R_ref)与降雨量无显著相关(P>0.05),但是生态系统呼吸的温度敏感系数(Q₁₀)与降雨量呈显著的线性负相关(P<0.05),同时气温对Q₁₀(R²=0.35)、R_ref(R²=0.46)均产生显著影响(P<0.05).     

尼罗红荧光优化法快速检测微拟球藻细胞内油脂含量

【目的】利用尼罗红荧光染色法快速检测微拟球藻细胞内的油脂含量。【方法】系统性地调整激发波长与发射波长,确定最佳二甲基亚砜(DMSO)浓度、尼罗红终浓度、染色时间和细胞密度的范围,比较重量法和Triolein标准品的油脂含量分别与荧光强度之间的关系。【结果】分析得出了尼罗红荧光强度与微拟球藻细胞油脂含量的关系,确定优化后染色条件为:二甲基亚砜浓度为5%,尼罗红终浓度为1 mg/L,染色时间为6 min,且细胞密度为(0.5-3.0)×10~6 cells/m L的范围内,激发波长和发射波长分别为515 nm和570 nm。重量法和Triolein标准品的油脂含量分别与尼罗红染色荧光强度之间的关联性,表明尼罗红荧光染色方法可以用来快速准确地检测细胞内的油脂含量,且油脂含量与荧光强度之间正相关,相关系数R~2为0.997 3。尼罗红染色优化后油脂的检测下限达到2μg,大大减少了测定油脂含量所需细胞量。【结论】针对不同种属系统性地确认了荧光激发波长和发射波长,并优化验证得到了最佳尼罗红荧光染色条件,可以快速准确地检测微量微拟球藻细胞内的油脂含量,便于大规模筛选高产油突变藻株。     

一株高脂土壤小球藻的分离鉴定及脂质分析

对采自山西省庞泉沟国家自然保护区的土壤中的藻种进行分离鉴定, 获得了一株优良的高脂绿藻。经显微形态观察鉴定, 该藻株的形态特征属于小球藻属Chlorella (Chlorellasp. PQG67)。进一步对其rbcL和18S rDNA基因序列进行分析并构建系统树, 结果表明基因序列与普通小球藻Ch. vulgaris同源并聚为一支, 确定其为一株普通小球藻Ch. vulgaris PQG67。在不同光照强度下连续培养后测定其油脂含量稳定在30%左右, 在不同NaCl浓度胁迫条件下可达40%以上, 并通过叶绿素荧光值测量探索该藻株生长趋势。通过傅立叶变换红外光谱图对其油脂积累过程分析, 显示该藻株脂类成分在1634/cm附近, 有vC=O伸缩振动谱带, 随着培养时间的延长, 脂质含量的相对强度也在增加。可见该藻株具有较高的生长速率及产油能力, 是一株具产业化应用潜力的优良产油藻株。     

长期施用有机肥和过磷酸钙对潮土有效磷积累与淋溶的影响

利用20年定位试验研究了施用化肥和有机肥对潮土耕层土壤有效磷（Olsen-P）含量与作物产量的关系及土壤Olsen-P积累和垂直移动规律的影响.结果表明：土壤Olsen-P含量在10～40 mg·kg^-1能保证小麦、玉米有较高的产量,土壤Olsen-P含量大于40 mg·kg^-1发生显著淋溶,轻壤质潮土Olsen-P发生淋溶的阈值为40 mg·kg^-1.连续施用化肥（NPK）和秸秆还田处理（SNPK）施磷量在77～90 kg·hm^-2,平均每100 kg P·hm^-2使耕层土壤Olsen-P提高0.63～0.72 mg·kg^-1,每年提高0.49～0.65 mg·kg^-1,达到淋失阈值需要45～60年.有机肥与化肥结合（MNPK、MNPK2和1.5 MNPK）,年施磷量为210 kg·hm^-2时,土壤Olsen-P（Y）与施肥年度（x）的关系为：Y_{1.5 MNPK}=4.506x+6.4464 (R²=0.8862),平均每年增加4.5 mg·kg^-1,连续施用8年可使耕层土壤Olsen-P达到淋失阈值;年施磷量为125和140 kg·hm^-2时,土壤Olsen-P与施肥年度的关系为：Y_MNPK2=2.4765x+13.563 (R²=0.9307)和Y_MNPK=3.1097x+6.9615 (R²=0.8562),平均每年增加2.47和3.1 mg·kg^-1,连续施用11年可使耕层土壤Olsen-P达到淋失阈值.有机无机肥结合处理土壤Olsen-P积累速度是化肥处理的3.5倍,过量施用有机肥增加了土壤Olsen-P的积累和淋失.     

干旱死亡叶片与自然凋落叶化学性质对比研究

为了解干旱死亡叶片与自然凋落叶化学性质的差异性,对云南元江干热河谷5种植物(鞍叶羊蹄甲、白皮乌口树、灰毛浆果楝、细基丸和九里香)干旱死亡叶片和自然凋落叶化学性质进行比较分析。结果表明,干旱死亡叶片和自然凋落叶在碳和养分化学特性上表现出较大变异,且不同树种间存在极显著差异(P<0.001)。与自然凋落叶相比,干旱死亡叶片具有较高的可溶性碳、C/N比和Mg含量,而木质素、半纤维素和N含量则较低。干旱死亡叶片与自然凋落叶间的碳(R²=0.56,P<0.01)、纤维素(R²=0.52,P<0.01)、半纤维素(R²=0.85,P<0.001)、单宁(R²=0.99,P<0.001)、木质素/N(R²=0.60,P<0.01)、C/N(R²=0.64,P<0.001)和氮含量(R²=0.85,P<0.001)呈显著正相关。因此,可根据自然凋落叶化学性质预测未来极端干旱条件下干旱死亡叶片的化学性质。     

基于高光谱特征的雅氏落叶松尺蠖虫口密度估算

【目的】多年来,蒙古高原典型落叶松害虫雅氏落叶松尺蠖Eeannis jacobsoni发生频繁,使森林生态系统遭到严重破坏。虫口密度可直接描述森林虫害严重程度,进而及时、快速获得害虫虫口密度信息显得极为重要。本研究旨在依据雅氏落叶松尺蠖暴发区的落叶松光谱实测数据和虫口密度数据,构建基于高光谱特征的虫口密度估算方法。【方法】以蒙古国后杭爱省和肯特省4个地点雅氏落叶松尺蠖暴发区为试验区。首先从这4个试验区选取不同程度受害的110株西伯利亚落叶松Larix sibirica样本树,调查虫口密度和测量冠层光谱反射率;其次通过光谱反射率数据获得微分光谱反射率(differential spectral reflectance, DSR)和计算改进型光谱指数(modified spectral index, MSI);再次运用多项式曲线拟合法,分析DSR和MSI对虫口密度的敏感性;然后借助连续投影算法(successive projection algorithm, SPA)提取敏感DSR和MSI;最后利用敏感DSR和MSI,结合多项式回归(polynomial regression, PR)和支持向量机回归(support vector machine regression, SVMR)算法,建立雅氏落叶松尺蠖虫口密度估算模型,并评定了其精度。【结果】DSR的敏感波段主要在黄边和红边波段内,其中572 nm的敏感性最显著(R²=0.5821,P<0.001),MSI的最敏感指数为TVI(R²=0.5386,P<0.001);TVI(R²_CV=0.6323,RMSE_CV=0.1513)比DSR₅₇₂(R²_CV=0.5581,RMSE_CV=0.1649)估算精度高,而多个DSR(R²=0.7309,RMSE_CV =0.1347)比多个MSI(R²_CV=0.6537,RMSE_CV=0.1453)更有估算潜力,其中SVMR模型性能始终优于PR模型,说明SVMR更加适用于虫口密度估算。【结论】MSI和DSR可作为虫口密度估算的敏感指标,多项式曲线拟合法能够挖掘MSI和DSR对虫口密度的敏感性;SPA是虫口密度敏感光谱特征提取的一种有效方法,其提取的DSR敏感指标和MSI敏感指数充分捕捉了针叶叶绿素吸收特征、水分吸收特征以及针叶细胞受损引起的反射特征。该研究不仅为利用航空航天遥感监测森林害虫虫口密度提供实验理论基础,而且为森林虫害遥感监测拓展了新途径。     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique

A staining protocol for rapid in situ detection of neutral lipid using flow cytometry in combination with Nile red staining was optimized. Staining efficiency was tested in terms of fluorescence intensity (% grandparent) in varied concentrations of Nile red and dimethyl sulfoxide (DMSO), with variable incubation period, temperature and pH level. The improved method was tested using two microalgae: Chlorella ellipsoidea and Chlorococcum infusionum. Maximum staining efficiency was recorded with a concentration of 5 μg mL−1 Nile red and 40 % DMSO in a 15-min incubation at 40 °C for both taxa (pH 6.5). The forward (FSC) and side scatter (SSC) two-dimensional dot plot showed highly scattered cells containing neutral lipid. The coefficient of variation, standard deviation, mean and median values were determined for quantification of neutral lipid. We also applied this modified method to detect the elevated level of neutral lipid in nitrate (NaNO3)-depleted cells; the efficiency of this technique was justified indicating a prominent 3- to 4-fold increase in neutral lipid in treated cells. Confocal images of stained cells also revealed accumulation of high levels of neutral lipid in treated microalgal cells.     

Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).     

Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24-0.47 microg/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5-10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2-5000 microg-lipid/ml-broth.     

山西地区高脂微藻的分离筛选

分别从山西省汾河流域、运城盐湖等水体采集水样,使用微挑法、平板涂布法对其中的微藻进行了分离纯化,并对分离得到的29株微藻和购买的3株微藻,进行了高脂藻株的筛选。结果表明：采用干重法对32株微藻的生长量进行测定,其干重介于48.9～422.2 mg/L之间;采用氯仿甲醇法对32株微藻的总脂含量进行测定,其总脂含量介于5.4%DW～30.1%DW之间;采用尼罗红荧光染色法对32株微藻的中性脂相对含量进行测定,其单位体积的荧光值介于4.1～181.5之间。综合评价藻株的总脂产率,最终筛选到山西省NY017盐生杜氏藻（Dunaliella salina）、NY023线形菱形藻（Nitzschia linearis）以及NY025谷皮菱形藻（Nitzschia palea）为高脂藻株,油脂产率分别为3.25、3.03、2.11 mg.L-1.d-1,具有生产生物柴油的潜力。     

尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究

【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。     

Determining intracellular lipid content of different oleaginous yeasts by one simple and accurate Nile Red fluorescent method

Abstract

A simple and accurate Nile Red fluorescent method was built to evaluate the lipid content of three different oleaginous yeasts by one standard curve. The staining of cells can be observed clearly by laser scanning confocal microscope, showing that Nile Red can enter into the cells of oleaginous yeasts easily. A series of conditions such as pretreating temperature, cell suspension concentration (OD₆₀₀), staining time, Nile Red concentration and the type of suspension solvent were learnt systematically to obtain the optimal process parameters for Nile Red staining. After optimization, the fitting curve of Nile Red fluorescent method was established under suitable conditions (pretreating temperature: 50?°C, OD₆₀₀: 1.0; staining time: 5?mins; Nile Red concentration: 1.0?μg/mL; suspension solvent: PBS) and it had a suitable correlation coefficient (R² = 0.95) for lipid content measurement of different oleaginous yeasts. By this study, the possibility of lipid content determination of different oleaginous yeasts by one fitting curve can be proven and this will improve the efficiency of researches related to microbial lipid production.     

尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂

研究一种快速准确测定微藻中中性脂的方法。湛江等鞭金藻是一种中性脂含量高且具有开发潜力的能源微藻。以湛江等鞭金藻为实验对象,首先优化尼罗红染色的条件。当二甲基亚砜体积分数为2.0%、尼罗红质量浓度为1.00μg/m L、细胞密度为1.0×106个/m L、激发波长为480 nm、检测波长为580 nm时,优化的染色时间为10min。其次测定了背景荧光对检测的影响。结果表明,在不同细胞状态下,背景荧光强度大约是微藻内荧光强度的20%左右,可以忽略。最后比较了尼罗红荧光法和重量法。结果表明,荧光强度与中性脂含量的相关系数R2=0.946 8,虽然两者相关性并不十分高,但作为一种快速测定微藻中中性脂的方法,尼罗红荧光法依然是研究微藻培养过程中中性脂含量变化的有效方法。     

Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.     

低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

为了建立一个高效的高产油微藻诱变育种流程,微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中,首先利用低场核磁共振技术,建立了直接检测干藻粉和培养液中小球藻油脂含量的方法,其信号强度与细胞中油脂含量存在特异的线性关系,干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99,说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比,具有快速、简便和准确等优点。但其通量不及尼罗红染色法,因此,我们开发了将尼罗红染色法用于初筛,低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法,并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株,然后利用低场核磁共振技术复筛得到9株高产油性能的藻株,其中一株甘油三酯含量超过20%,比原始藻株提高1倍,培养168 h后培养液油脂浓度达到5 g/L,证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。