裂殖壶藻SlMYB118基因的克隆及生物信息学分析
Cloning and bioinformatics analysis of SlMYB118gene from Schizochytrium limacinum SR21
通讯作者: 薛婷(1988—),女,正高级实验师,博士,研究方向为海洋微藻培育和基因组生物信息学分析研究。E-mail:xueting@fjnu.edu.cn.代容春(1973—),女,副教授,硕士,研究方向为植物生理与分子生物学。E-mail:biodrc@fjnu.edu.cn
收稿日期: 2023-02-15
基金资助: |
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Received: 2023-02-15
作者简介 About authors
陈菁(1998—),女,硕士,研究方向为裂殖壶藻转录因子的研究。E-mail:1577134974@qq.com
裂殖壶藻是一种含有丰富DHA的海洋微藻。本课题组前期发现裂殖壶藻的SlMYB118基因与脂肪酸的合成密切相关,是潜在的DHA合成调控的转录因子。本文通过PCR的方法克隆裂殖壶藻的SlMYB118基因的cDNA全长序列,运用生物信息学的方法对其分析。结果预测SlMYB118蛋白质的等电点为8.75,分子量为84.81 kDa,定位于细胞核,且其具有106个潜在的磷酸化位点。SlMYB118基因的结合位点是探索转录调控机制、建立DHA合成相关转录调控网络的关键,本研究为后续确定其结合位点奠定了理论基础。
关键词:
Schizochytrium limacinum SR21 is a kind of marine microalgae rich in DHA.The research group that the author joined previously found that SlMYB118 gene of S.limacinum SR21 was closely related to fatty acid synthesis and was a potential transcription factor for DHA synthesis regulation.In this paper,the full-length cDNA sequence of SlMYB118 gene was cloned by PCR and analyzed by bioinformatics method.The results predicted that SlMYB118 protein had an isoelectric point of 8.75,a molecular weight of 84.81 kDa,was localized in the nucleus,and had 106 potential phosphorylation sites.This study laid the theoretical foundation for the subsequent identification of the binding site of SlMYB118 gene,which was a key issue for the transcriptional regulatory mechanism and the establishment of the transcriptional regulatory network related to DHA synthesis.
Keywords:
本文引用格式
陈菁, 苏永昌, 孙化淼, 陈由强, 薛婷, 代容春.
CHEN Jing, SU Yongchang, SUN Huamiao, CHEN Youqiang, XUE Ting, DAI Rongchun.
DHA(Docosahexaenoic acid),即二十二碳六烯酸,是一种多不饱和脂肪酸,在人体内无法自行合成,具有促进大脑发育、保护视网膜、降低甘油三酯含量、改善血管功能和治疗心脑血管疾病等重要作用[1⇓-3]。DHA的主要来源是深海鱼油和海洋微藻[4],其中鱼油是DHA的传统来源,但受鱼的品种、部位、季节、产地的影响而产生波动,DHA含量不稳定,此外鱼油生产工艺较为复杂,产品多具有腥味,且易受海洋污染物的影响[5⇓⇓-8];而可生成DHA的微藻能够弥补鱼油资源有限的缺陷,其培养具有不受季节、地域等条件的限制,培养周期短、产量高、不含有鱼腥味、脂肪酸组成和提取纯化工艺简单、不破坏生态环境的特点和优势,打破了传统鱼油应用的限制[9-10]。因此,利用海洋微藻生产DHA具有更加广阔的应用前景。
目前,裂殖壶藻的研究还停留在结合DHA合成途径菌株性能的改善、发酵过程的调节以及油脂的提取和纯化[13]。而近年来,植物转录因子的研究已成为热点之一,调控脂肪酸合成和油脂积累的转录因子更是备受青睐。转录因子并不单独作用,而是形成代谢调控网络,共同发挥调控脂肪酸合成的作用,这些转录因子分属各个家族,包括AP2家族、B3家族、DOF家族、MYB家族、HAP3/CBP和CHD3等[14]。研究报道,拟南芥MYB118基因是与种子发育时胚胎建成、脂肪酸从头合成和代谢及油脂积累相关的关键转录因子,过表达MYB118基因能够正调控一系列与脂肪酸合成、糖酵解途径相关的基因,从而调控种子中脂肪酸的生物合成和油脂的积累[15],但该基因在其他物种中有关脂肪酸生物合成的调控研究尚未见报道。为了挖掘裂殖壶藻DHA合成相关的转录因子,本课题组前期利用氮源胁迫下不同培养时期的细胞RNA-Seq数据,通过加权基因共表达网络分析(Weighted gene co-expression network analysis,WGCNA)方法构建网络,寻找到1个与DHA含量特异相关的模块,且该模块中的关键基因[包含SlMYB118基因(GenBank登录号:OQ784160.1)]与脂肪酸的合成相关,由此推断该模块中SlMYB118基因是潜在的裂殖壶藻 DHA合成调控的转录因子[16]。因此,本文对裂殖壶藻的SlMYB118基因进行研究,经PCR实验过程获取该基因的cDNA全长,并对SlMYB118基因进行蛋白质理化性质分析,预测蛋白质的亲/疏水性、保守结构域、结构预测以及系统进化等生物信息,为后续确定SlMYB118基因的结合位点在转录调控机制研究、DHA合成相关转录调控网络的建立奠定了理论基础。
1 材料与方法
1.1 实验材料
1.1.1 藻种
裂殖壶藻购买于美国马里兰洲洛克菲勒的美国模式培养物集存库(American type culture collection,ATCC)。
1.1.2 试剂
TransZol Up Plus RNA Kit、TransScript One-Step gDNA Removal and cDNA synthesis SuperMix反转录试剂盒、Trans5α Chemically Competent Cell、EasyPure© Plasmid MiniPrep Kit、X-gal(20 mg/mL)、IPTG(500 mmol/L)购自北京全式金生物技术有限公司;TIANgel Maxi Purification Kit购自天根生化科技(北京)有限公司;克隆载体pMD19-T、DNA Marker购自Takara公司;Gloria Nova HS2X Master Mix、琼脂粉购于ABclonal公司;蛋白胨、酵母粉、氨苄青霉素钠购自北京索莱宝科技有限公司;葡萄糖购自西陇科学股份有限公司。
1.1.3 培养基
采用YPD培养基培养裂殖壶藻,配方为1 L海水中加入5 g葡萄糖、1 g蛋白胨和1 g酵母粉,于115℃高压灭菌锅中灭菌20 min,pH为5。
1.1.4 设备
灭菌锅[施都凯仪器设备(上海)有限公司]、新型迷你型恒温培养振荡器(上海智城分析仪器制造有限公司)、全自动样品快速研磨仪(上海净信实业发展有限公司)、小型台式冷冻离心机(德国艾本德股份公司)、DYY-5型稳压稳流电泳仪(北京市六一仪器厂)、Mini PCR仪(美国Applied Biosystems)、凝胶成像仪(美国BIO-RAD公司)、电热恒温水槽(上海恒一科技有限公司)、电热恒温培养箱(福州科远贸易有限公司)。
1.2 实验方法
1.2.1 裂殖壶藻总RNA的提取
取2 mL、密度为5.58×107个/mL、处于对数生长期的藻液接种,加入装有100 mL YPD培养基的锥形瓶中,振荡培养箱的温度设置为28°C、转速设置为200 r/min。培养至对数生长期时,取50 mL裂殖壶藻,利用离心机8 000 r/min离心5 min。将藻泥装入1.5 mL离心管中,用液氮速冻,取出后加入TransZol Up试剂1 mL,然后加入直径为4 mm的钢珠,利用自动研磨仪进行研磨,参数设置为60 Hz、3 min。之后的步骤按照TransZol Up Plus RNA Kit的说明书进行提取,提取结束后,利用2.0%琼脂糖凝胶电泳验证该RNA的质量,以及利用NanoDrop-2000分光光度仪检测RNA的浓度。利用所提的RNA为模板,遵循反转录试剂盒的要求,合成cDNA的第一链,并保存于-20°C以备用。
1.2.2 SlMYB118基因的扩增
利用SnapGene软件设计引物。依据本实验室建立的裂殖壶藻基因组数据库中的SlMYB118基因序列来设计一对引物:上游(F)、下游(R)(表1),下划线为EcoRⅠ、SalⅠ酶切位点,并且送到福州尚亚生物技术有限公司进行合成。
表1 SlMYB118基因引物序列
Tab.1
基因Gene | 引物序列(5’→3’) Primer sequence | 退火温度/°C Annealing temperature |
---|---|---|
SlMYB118 | F:CGGAATTCATGTTTGTGGAAGATGGG | 60 |
R:GCGTCGACTCAATCTAAGAGAATGGA |
以cDNA为模板扩增SlMYB118基因,采用Gloria Nova HS2X Master Mix,以F和R为引物进行PCR扩增。PCR反应体系50 μL:cDNA模板2 μL,F、R引物各1 μL,Gloria Nova HS2X Master Mix 25 μL,ddH2O 21 μL。在PCR仪中,首先94°C预变性5 min;其次94°C变性30 s,60°C退火30 s,72°C延伸1 min,35个循环;再72°C终延伸7 min;最终4°C保存。利用1.0%琼脂糖凝胶电泳对PCR产物进行检测并分离,之后利用DNA凝胶回收试剂盒回收目的片段。
1.2.3 目的基因的克隆及测序
在16°C连接仪中,将回收的PCR产物与pMD19-T载体过夜连接,连接产物转化至Trans5α Chemically Competent Cell,并涂布于蓝白斑筛选平板培养基上,在37°C培养箱中倒置、过夜培养。挑单菌落进行培养,菌液进行抽提质粒验证并送至杭州擎科生物技术有限公司进行测序。
1.2.4 裂殖壶藻SlMYB118蛋白质的生物信息学分析
裂殖壶藻SlMYB118蛋白质的生物信息学内容如表2所示。
表2 蛋白质结构与功能预测的相关软件网站
Tab.2
种类Class | 软件与网址Software and URLs |
---|---|
理化性质Physicochemical properties | ProtParam tool( |
疏水性Hydrophobicity | ProtScale( |
保守结构域Conservative structural domain | NCBI Batch CD-search( |
跨膜结构Transmembrane structure | DeepTMHMM( |
亚细胞定位Subcellular localization | Hum-mPLoc 2.0(www.csbio.sjtu.edu.cn/bioinf/hum-multi-2/) |
信号肽Signal peptide | SignalP-5.0( |
磷酸化位点Phosphorylation sites | NetPhos-3.1( |
二级结构Secondary structure | SOPMA( |
三级结构Three-level structure | SWISS-MODEL( |
同源序列查找Homologous sequence search | NCBI的Protein BLAST ( |
系统进化树System evolution tree | MEGA11 |
2 结果与分析
2.1 裂殖壶藻总RNA的提取
图1
图1
裂殖壶藻总RNA的提取
注:1、2、3为裂殖壶藻的总RNA。
Fig.1
Extraction of total RNA from S. limacinum SR21
Note:1, 2, 3 were the total RNA of S.limacinum SR21.
2.2 以裂殖壶藻cDNA为模板的PCR扩增结果
以裂殖壶藻cDNA为模板进行PCR扩增,经琼脂糖凝胶电泳检测,与DL 2000 marker进行对比,可见扩增片段约为2 000 bp,与目的片段大小吻合(图2)。
图2
图2
SIMYB118基因的PCR产物电泳图
注:M为DL 2000 marker;1为cDNA扩增。
Fig.2
Electrophoresis of the PCRproduct of SIMYB118 gene
Notes: M was DL 2000 marker;1 was cDNA amplification.
2.3 pMD19-T克隆目的片段的检测结果
图3
图3
PMD-SIMYB118质粒电泳图
注:M为Supercoiled ladder;1为PMD-SlMYB118质粒。
Fig.3
Electrophoresis of PMD- SIMYB118 plasmid
Notes:M was Supercoiled ladder; 1 was the PMD-SlMYB118 plasmid.
图4
图4
SIMYB118基因的cDNA全长及氨基酸序列
Fig.4
Full length and amino acid sequence of cDNA of SIMYB118 gene
2.4 裂殖壶藻SlMYB118基因的生物信息学分析
2.4.1 裂殖壶藻SlMYB118蛋白质的理化性质
经Bioedit软件分析,裂殖壶藻SlMYB118基因的cDNA序列全长为2 280 bp,其中G+C含量为53.11%,A+T含量为46.89%。使用ProtParam Tool分析,SlMYB118蛋白质分子量为84.81 kDa,分子式为C3601H5604N1194O1155S22,理论等电点(pI)为8.75。该蛋白质的不稳定指数为70.54(<40为稳定蛋白质),推测其为不稳定蛋白质。预测该蛋白质在大肠杆菌中表达的半衰期大于10 h,在酵母中表达的半衰期大于20 h,而在哺乳动物网状细胞中体外培养表达的半衰期为30 h。SlMYB118蛋白质由20种基本氨基酸组成,氨基酸残基较多的是谷氨酰胺(Gln,8.6%)、组氨酸(His,8.8%)和丝氨酸(Ser,12.9%),氨基酸残基较少是苯丙氨酸(Phe,1.7%)、酪氨酸(Tyr,1.4%)、色氨酸(Trp,1.1%)以及半胱氨酸(Cys,0.5%)。由表3可知,SlMYB118蛋白质带有负电荷的氨基酸72个(Asp+ Glu)、正电荷的氨基酸76个(Arg+ Lys)。
表3 SlMYB118蛋白质的氨基酸组成
Tab.3
氨基酸 Amino acid | 数量/个 Quantity/pcs | 百分比/% Percentage | 氨基酸 Amino acid | 数量/个 Quantity/pcs | 百分比/% Percentage |
---|---|---|---|---|---|
丙氨酸Ala | 61 | 8.0 | 赖氨酸Lys | 20 | 2.6 |
精氨酸Arg | 56 | 7.4 | 蛋氨酸Met | 18 | 2.4 |
天冬酰胺Asn | 40 | 5.3 | 苯丙氨酸Phe | 13 | 1.7 |
天冬氨酸Asp | 42 | 5.5 | 脯氨酸Pro | 60 | 7.9 |
半胱氨酸Cys | 4 | 0.5 | 丝氨酸Ser | 98 | 12.9 |
谷氨酰胺Gln | 65 | 8.6 | 苏氨酸Thr | 37 | 4.9 |
谷氨酸Glu | 30 | 4.0 | 色氨酸Trp | 8 | 1.1 |
甘氨酸Gly | 35 | 4.6 | 酪氨酸Tyr | 11 | 1.4 |
组氨酸His | 67 | 8.8 | 缬氨酸Val | 25 | 3.3 |
异亮氨酸Ile | 24 | 3.2 | 吡咯懒氨Pyl | 0 | 0.0 |
亮氨酸Leu | 45 | 5.9 | 硒半胱氨酸Sec | 0 | 0.0 |
2.4.2 亲/疏水性分析
经ProtScale在线程序分析,亲/疏水性最大值为1.667(位于第754位氨基酸处),最小值为-3.622(位于第190位氨基酸处),总平均疏水性为-1.087,同时SlMYB118蛋白质含有较多的亲水域,说明SlMYB118蛋白质为亲水性蛋白质(图5),其脂肪系数为53.04。
图5
2.4.3 结构预测
图6中,根据NCBI保守结构域数据库(CDD)的结果表明,该蛋白质具有Myb_DNA-binding结构域,同时该结构域同属于SANT超家族。SANT超家族是Myb超家族蛋白质,包括转录因子和mRNA剪接因子(转录/RNA加工修饰/细胞分裂和染色体分割),符合对该蛋白质的预期。DeepTMHMM预测SlMYB118蛋白质处于膜内,因此SlMYB118蛋白质不存在跨膜结构域;Hum-mPLoc 2.0预测该蛋白质定位于细胞核。经过SignalP5.0在线预测信号肽的可能性为0.001 2,显然小于阈值0.500 0,因此SlMYB118蛋白质不存在信号肽序列。SlMYB118蛋白质磷酸化位点由NetPhos3.1在线软件进行预测,结果如图7所示,当潜在磷酸化位点的阈值为0.5时,可能存在106个磷酸化位点在SlMYB118蛋白质中,其中包括3个酪氨酸(Tyr)位点、22个苏氨酸(Thr)位点及81个丝氨酸(Ser)位点。
图6
图7
图7
SIMYB118蛋白质磷酸化位点预测
Fig.7
Prediction of SIMYB118 protein phosphorylation site
2.4.4 二级和三级结构预测
图8
图8
SIMYB118蛋白质二级结构预测
Fig.8
Prediction of secondary structure of SIMYB118 protein
图9
2.4.5 系统进化树
将SlMYB118蛋白质序列提交至NCBI-Blastp进行比对,结果显示裂殖壶藻SlMYB118蛋白质序列与Hondaea fermentalgiana MYB98蛋白质序列相似度最高,为80.57%;与H.fermentalgiana MYB44蛋白质序列的相似度次之(48.60%)。在MEGA11软件中,采取邻接法(Neighbor-joining algorithm)、bootstrap重复次数为1 000次(图10),将其中15种与裂殖壶藻相似度较高的MYB蛋白质序列与目的蛋白质构建系统发育进化树。结果显示裂殖壶藻SlMYB118蛋白质与H.fermentalgiana MYB98蛋白质聚为一支,亲缘关系最近。
图10
图10
不同物种MYB蛋白质的系统进化树分析
Fig.10
Phylogenetic tree analysis of MYB proteins in different species
3 讨论
MYB类转录因子家族是指含有MYB结构域的一类转录因子,是植物中较大转录因子的家族之一。MYB转录因子的共同特征是具有保守的MYB结构域由N端1-4个R基序组成,根据R基序重复的数目可以分为1R-MYB、R2R3-MYB、3R-MYB、4R-MYB等 4种类型[17]。裂殖壶藻SlMYB118蛋白质通过保守结构域分析,表明该蛋白质具有2个Myb_DNA-binding结构域,属于R2R3-MYB。在植物中,R2R3-MYB普遍数目较多,且发挥多种重要功能,包括形态建成、次级代谢调节和抗逆性等生理生化活动[18⇓⇓⇓⇓⇓-24]。如拟南芥AtMYB37、AtMYB38和 AtMYB84能够调控其侧分生组织的形成[18];月季RhMYB113c基因通过MYB-bHLH或MYB-bHLH-WD40复合物调节花青素苷的合成,从而影响月季红色花瓣的着色[19];三七PnMYB1基因能够调控三七皂苷生物合成途径中基因的表达,促进三七皂苷的合成[20];Ye Z M等[21]的研究表明,CitMYB21基因负调控柑橘类类黄酮生物合成;在拟南芥中,过表达荷花的NnMYB4基因,使其AtC3H、At4CL1、AtF5H、AtCOMT1等木质素合成关键酶的表达量下调,从而负调控植物木质素的合成[22];在干旱胁迫条件下,白羊草中的BiMYB142、BiMYB143和BiMYB35基因通过调控其靶基因的表达,增强了白羊草抗旱的能力[23];在低温胁迫下,番茄SlMYB102基因对6种外源激素的积累具有积极作用,推测其可能参与了这6种激素对抗寒性的调控通路[24]。本课题组前期利用氮源胁迫下不同培养时期的细胞RNA-Seq数据,通过WGCNA分析,证实SlMYB118基因与脂肪酸的合成相关,由此推测SlMYB118基因是裂殖壶藻DHA合成调控的潜在转录因子。本研究通过蛋白序列比对,表明SlMYB118蛋白序列与H.fermentalgiana MYB98蛋白质序列最为相似;系统进化树显示H.fermentalgiana的MYB98、MYB44及MYB118蛋白质序列与裂殖壶藻的SlMYB118蛋白质序列聚为一支,可见2个物种之间的亲缘关系较近。裂殖壶藻与H.fermentalgiana均为破囊壶菌科,这与NCBI分类数据库一致。
转录因子在动、植物的生长发育及其对外界环境的反应中起着重要的调控作用,一直以来都是研究内容之一,现也是生物学研究领域的热点。多种微藻中调控DHA的机理已被研究[25-26],而裂殖壶藻调控DHA的机理目前仅局限于在不同供氧条件下的转录组和基因表达的分析[27]。大量研究推测,裂殖壶藻多不饱和脂肪酸的合成可能存在2种途径:一是以乙酰辅酶A为起点,经过一系列的碳链延长和脱饱和反应得到多不饱和脂肪酸(FAS途径);二是以乙酰辅酶A为起点,不经过一系列的碳链延长酶和脱饱和酶反应,而是形成多酮基与多羟基的化合物(PKS途径)[28]。随着测序技术的不断发展,已开展利用基因组、转录组来揭示参与DHA合成途径的关键功能基因(如延长酶、去饱和酶、聚酮合成酶等),目前利用过量表达脂肪酸合成途径中的单个关键酶/酶的亚基基因,或是通过同时过量表达代谢途径中的多个酶基因的方法来提高DHA含量,但这些方法在一定程度上都无法突破脂肪酸合成通量的限制[29-30]。DHA合成受多个层面的调控,不仅涉及到脂肪酸合成途径相关功能基因的表达,也与转录因子的导向作用密切相关,但参与DHA合成的关键转录因子还不清楚,且该信号通路激活的下游基因仍未知,及其参与调控裂殖壶藻DHA合成在国内、外也少有研究。因此,挖掘调控DHA合成的关键转录因子,并鉴定其下游靶基因,解析其参与DHA合成调控的分子机制是高产DHA裂殖壶藻藻种构建和选育亟需解决的关键科学问题。本研究为后续对裂殖壶藻SlMYB118基因的结构、功能及其对脂肪酸合成调控的研究提供了依据。
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Omega-3 long-chain polyunsaturated fatty acids and fish oil supplementation during pregnancy:which evidence?
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藻类对温度胁迫响应机制的国内外研究进展
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Electron microscopy may reveal structure of docosahexaenoic acid-rich oil with in Schizochytrium sp.
[J].Schizochytrium sp. is an algae-like microorganism utilized for commercial production of docosahexaenoic acid (DHA)-rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells of Schizochytrium sp. employing sample preparation by high-pressure freeze substitution suggests the presence of secondary and tertiary semicrystalline structures of triacylglycerols within the oil bodies in Schizochytrium sp. A fine secondary structure consisting of alternating light- and dark-staining bands was observed inside the oil bodies. Dark bands were 29 +/- 1 A in width, and light bands were 22 +/- 1 A in width. The tertiary (three-dimensional) structure may be a multilayered ribbon-like structure which appears coiled and interlaced within the oil body. In freeze-fracture photomicrographs, Schizochytrium oil bodies exhibited fracture planes with terraces averaging 52 +/- 7 A in height which could correspond to the combined width of two halves of two light bands and one dark band observed in the high-pressure freeze substitution photomicrographs. The results suggest that triacylglycerols within Schizochytrium sp. oil bodies may be organized in a triple chain-length structure. High-pressure freeze substitution electron micrographs of two other highly unsaturated oil-producing species of microalgae, Thraustochytrium sp. and Isochrysis galbana, also revealed this fine structure, whereas microalgae containing a higher proportion of saturated oil did not. The results suggest that the staining pattern is not an artifact of preparation and that the triple chain-length conformation of triacylglycerols in Schizochytrium sp. oil bodies may be caused by the unique fatty acid composition of the triacylglycerols.
Production of high yields of docosahexaenoic acid by Schizochytrium sp.strain SR21
[J].The culture conditions for high‐yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1‐palmitoyl‐2‐DHA‐PC and 1.2‐di‐DHA‐PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2‐DHA‐PC.
Endogenously synthesized (-)-proto-quercitol and glycine betaine arc principal compatible solutes of Schizochytrium sp.strain S8 (ATCC20889) and three new isolates of phylogenetically related thraustochytrids
[J].We report that endogenously synthesized (-)-proto-quercitol (1D-1,3,4/2,5-cyclohexanepentol) and glycine betaine were the principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new osmotolerant isolates of thraustochytrids (strains T65, T66, and T67). The compatible solutes were identified and quantified by use of nuclear magnetic resonance spectroscopy, and their identity was confirmed by mass spectroscopy and measurement of the specific optical rotation. The cellular content of compatible solutes increased with increasing NaCl concentration of a defined medium. (-)-proto-Quercitol was the dominating solute at all NaCl concentrations tested (0.25 to 1.0 M), e.g., cells of S8 and T66 stressed with 1.0 M NaCl accumulated about 500 micromol (-)-proto-quercitol and 100 micromol glycine betaine per g dry weight. To our knowledge, (-)-proto-quercitol has previously been found only in eucalyptus. The 18S rRNA gene sequences of the four (-)-proto-quercitol-producing strains showed 99% identity, and they displayed the same fatty acid profile. The only polyunsaturated fatty acids accumulated were docosahexaenoic acid (78%) and docosapentaenoic acid (22%). A less osmotolerant isolate (strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304), did not contain (-)-proto-quercitol or glycine betaine. Thus, the level of osmotolerance and the osmolyte systems vary among thraustochytrids.
Docosahexaenoic acid production by Schizochytrium sp.:review and prospect
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MYB transcription repressors regulate plant secondary metabolism
[J].MYB transcription factors (TFs) belong to one of the largest and important gene families, which regulate development under changing environmental conditions, primary and secondary metabolism, and plant response to stresses (biotic and abiotic stresses). MYB repressors have a conserved N-terminal domain like other MYB TFs, but the C-terminal domain makes them structurally and functionally different from the rest. MYB repressors usually possess some repressive motifs, such as EAR (ethylene-responsive element binding factor-associated amphiphilic repression motif), SID (Sensitive to ABA and Drought 2 protein interact motif), and TLLLFR motifs, which contribute to their repression function through a variety of complex regulatory mechanisms. In this review, we summarize recent developments in research of MYB repressors and suggest directions to future research.
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MYB transcription factors in Arabidopsis
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Blind homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis
[J].In seed plants, shoot branching is initiated during postembryonic development by the formation of secondary meristems. These new meristems, which are established between the stem and leaf primordia, develop into vegetative branches or flowers. Thus, the number of axillary meristems has a major impact on plant architecture and reproductive success. This study describes the genetic control of axillary meristem formation in Arabidopsis thaliana by a group of three R2R3 Myb genes, which are homologous to the tomato (Solanum lycopersicum) Blind gene and were designated REGULATORS OF AXILLARY MERISTEMS (RAX). rax mutants show new phenotypes that are characterized by defects in lateral bud formation in overlapping zones along the shoot axis. RAX genes are partially redundant in function and allow a fine-tuning of secondary axis formation. As revealed by monitoring of SHOOT MERISTEMLESS transcript accumulation, the RAX genes control a very early step of axillary meristem initiation. The RAX1 and RAX3 expression domains specifically mark a cell group in the center of the leaf axil from which the axillary meristem develops. Double mutant combinations of lateral suppressor and rax1-3 as well as expression studies suggest that at least two pathways control the initiation of axillary meristems in Arabidopsis.
Analysis the function of R2R3 MYB transcription factor RhMYB113c on regulating anthocyanin synthesis in Rosa hybrida
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Biosynthesis ofPanax notoginseng saponins regulated by R2R3-MYB transcription factor PnMYB1
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Effect of R2R3-MYB transcription factor CitMYB21 on flavonoids biosynthesis in citrus
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Effect of Nelumbo nucifera R2R3-MYB transcription factor NnMYB4 on lignin biosynthesis in Arabidopsis thaliana
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Excavation of R2R3-MYB transcription factor and its response to drought stress in Bothriochloa ischaemum
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Effect of hormone-induced SlMYB102 transcription factor on physiological characteristics of tomato at low temperature stress
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Mechanisms of sodium-acetate-induced DHA accumulation in a DHA-producing microalga,Crypthecodinium sp.SUN
[J].Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that is critical for the intelligence and visual development of infants. Crypthecodinium is the first microalga approved by the Food and Drug Administration for DHA production, but its relatively high intracellular starch content restricts fatty acid accumulation. In this study, different carbon sources, including glucose (G), sodium acetate (S) and mixed carbon (M), were used to investigate the regulatory mechanisms of intracellular organic carbon distribution in Crypthecodinium sp. SUN. Results show that glucose favored cell growth and starch accumulation. Sodium acetate limited glucose utilization and starch accumulation but caused a significant increase in total fatty acid (TFA) accumulation and the DHA percentage. Thus, the DHA content in the S group was highest among three groups and reached a maximum (10.65% of DW) at 96 h that was 2.92-fold and 2.24-fold of that in the G and M groups, respectively. Comparative transcriptome analysis showed that rather than the expression of key genes in fatty acids biosynthesis, increased intracellular acetyl-CoA content appeared to be the key regulatory factor for TFA accumulation. Additionally, metabolome analysis showed that the accumulated DHA-rich metabolites of lipid biosynthesis might be the reason for the higher TFA content and DHA percentage of the S group. The present study provides valuable insights to guide further research in DHA production.
Metabolomics analysis reveals 6-benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytrium sp.
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Transcriptome and gene expression analysis of docosahexaenoic acid producer Schizochytrium sp.under different oxygen supply conditions
[J].Background: Schizochytrium sp. is a promising strain for the production of docosahexaenoic acid (DHA)-rich oil and biodiesel, and has been widely used in the food additive and bioenergy industries. Oxygen is a particularly important environmental factor for cell growth and DHA synthesis. In general, higher oxygen supply favors lipid accumulation, but could lead to a reduction of the DHA percentage in total fatty acids in Schizochytrium sp. To tackle this problem, it is essential to understand the mechanisms regulating the response of Schizochytrium sp. to oxygen. In this study, we aimed to explore the acclimatization of this DHA producer to different oxygen supply conditions by examining the transcriptome changes.Results: Two different fermentation processes, namely normal oxygen supply condition (shift agitation speeds from 400 rpm to 300 rpm) and high oxygen supply condition (constant agitation speeds: 400 rpm), were designed to study how the fermentation characteristics of Schizochytrium sp. HX-308 were affected by different oxygen supply conditions. The results indicated that high oxygen supply condition resulted in 49% and 37.5% improvement in the maximum cell dry weight (CDW) and total lipid concentration, respectively. However, the DHA percentage in total fatty acids decreased to 35%, which was 31.4% lower than that produced by normal oxygen supply condition. Moreover, transcriptome analysis was performed to explore the effect of the oxygen supply condition on genetic expression and metabolism. The results showed that glycolysis and pentose phosphate pathway metabolism-associated genes (hexokinase, phosphofructokinase, fructose-bisphosphate aldolase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were substantially upregulated in response to high oxygen supply, resulting in more NADPH was available for Schizochytrium. Specially, high oxygen supply condition also led to genes (Delta 6 desaturase, Delta 12 desaturase, FAS, ORFA, ORFB, and ORFC) involved in fatty acid biosynthesis upregulation. In addition, a transcriptional upregulation of catalase (CAT) became apparent under high oxygen supply condition, while superoxide dismutase (SOD) and ascorbate peroxidase (APX) were found to be down-regulated.Conclusions: This study is the first to investigate the differences of gene expression at different levels of oxygen availability in the DHA producer Schizochytrium. The results of transcriptome analyses indicated that high oxygen supply condition resulting in more NADPH and acetyl-CoA production for cell growth and lipid synthesis in Schizochytrium. Delta 12 desaturase and ORFC showed higher expression levels at high oxygen supply condition, which might be the key regulators for enhancing fatty acid biosynthesis in the future. These results enrich the current knowledge regarding genetic expression and provide important information to enhance DHA production in Schizochytrium sp.
Tropical marine macroalgae as potential sources of nutritionally important PUFAs
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Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes
[J].Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules. Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids. These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism. Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics. It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes.
Transcriptomic analysis of the regulation of lipid fraction migration and fatty acid biosynthesis in Schizochytrium sp.
[J].Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.
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