岳陳陳 余旭亞 趙永騰 王惠萍
二丁基羥基甲苯對雨生紅球藻蝦青素和油脂積累的影響*
岳陳陳1余旭亞1趙永騰1王惠萍2①
(1. 昆明理工大學(xué)生命科學(xué)與技術(shù)學(xué)院 昆明 650500;2. 昆明市兒童醫(yī)院 昆明 650228)
以雨生紅球藻()為對象,研究高光照、缺氮條件下,外源添加不同濃度的二丁基羥基甲苯(Butylated hydroxytoluene, BHT)對雨生紅球藻生長、蝦青素積累、油脂合成、脂肪酸組成、碳水化合物、蛋白含量以及蝦青素和脂肪酸合成相關(guān)酶基因的影響。結(jié)果顯示,添加不同濃度的BHT后,2 mg/L BHT添加組蝦青素積累量為最高,顯著高于其他實(shí)驗(yàn)組和對照組(<0.05),達(dá)到31.66 mg/g,是對照組的1.87倍。油脂含量達(dá)45.56%,高于同期對照組(39.06%),脂肪酸組成變化不顯著。在此條件下,蝦青素合成關(guān)鍵酶基因和表達(dá)水平分別是對照組的5.19倍和2.04倍;脂肪酸合成關(guān)鍵酶基因和表達(dá)水平較對照組顯著提高(0.05),分別是對照組的4.56倍和3.02倍。與對照組相比,2 mg/L BHT添加組的碳水化合物和蛋白含量均呈下降趨勢。研究表明,在脅迫條件下,外源添加適量濃度的BHT能有效促進(jìn)雨生紅球藻中蝦青素的積累,同時(shí)提高了藻細(xì)胞內(nèi)的油脂含量。
雨生紅球藻;二丁基羥基甲苯;蝦青素;油脂;蝦青素合成基因;脂肪酸合成基因
蝦青素是一種紅色脂溶性酮式類胡蘿卜素,在水產(chǎn)養(yǎng)殖、生物醫(yī)藥、化妝品等領(lǐng)域應(yīng)用廣泛(陳超等, 2014; Higuera-Ciapara, 2006; Shah, 2016)。研究發(fā)現(xiàn),脅迫條件,如高光照、高鹽、缺氮等,能促進(jìn)微藻積累蝦青素(Chen, 2017; Gao, 2015; Liu, 2016; Zhekisheva, 2002)。雨生紅球藻()(綠藻門、團(tuán)藻目)是一種分布于世界各地的單細(xì)胞淡水微藻,由于其蝦青素含量高而被公認(rèn)為天然蝦青素生產(chǎn)的主要原料(何磊等, 2011; Lorenz, 1999; Rao, 2010),生產(chǎn)中,一般采用增加生物量和細(xì)胞內(nèi)蝦青素積累量的方法來提高蝦青素的產(chǎn)量。
脅迫條件下,雨生紅球藻除了積累蝦青素外,還大量積累脂肪酸和甘油三酯(Saha, 2013)。研究發(fā)現(xiàn),雨生紅球藻中約有95%的游離蝦青素會和脂肪酸發(fā)生酯化并儲存在富含三酰基甘油的胞質(zhì)脂質(zhì)體中(Holtin, 2009; Yuan, 2000; Chen, 2015)。蝦青素合成以丙酮酸和甘油醛-3-磷酸(GA3P)為起始物,經(jīng)1-脫氧-D-木酮糖-5-磷酸合成酶(DXS)催化,形成1-脫氧-D-木酮糖-5-磷酸,最后經(jīng)類胡蘿卜素酮酶(BKT)合成游離蝦青素(Estévez, 2001; Huang, 2016)。編碼細(xì)胞中酰基載體蛋白(ACP)的合成,脂肪酸合成的第一個循環(huán)是乙酰-CoA與丙二酸單酰-ACP在β-酮酰-ACP合酶(KAS)的催化下生成乙酰乙酰-?;d體蛋白,進(jìn)入下一步脂肪酸的合成 (張梅等, 2018)。二丁基羥基甲苯(BHT)是一種人工合成的酚類抗氧化劑,因其抗氧化性強(qiáng)、揮發(fā)性低、穩(wěn)定性高、耐用性好等優(yōu)點(diǎn)被廣泛應(yīng)用于化工、食品、醫(yī)藥等領(lǐng)域(趙磊等, 2016)。Nanou等(2010)研究發(fā)現(xiàn),外源添加BHT能提高真菌中β-胡蘿卜素的積累。而β-胡蘿卜素是蝦青素合成通路中重要的中間代謝產(chǎn)物,這為本研究將BHT用于雨生紅球藻誘導(dǎo)蝦青素積累提供了理論依據(jù)。
本研究通過在培養(yǎng)基中添加不同濃度BHT,考察其對雨生紅球藻生長、蝦青素含量、油脂含量、脂肪酸組分、碳水化合物、蛋白含量等生理生化指標(biāo)及對蝦青素、脂肪酸合成相關(guān)酶基因表達(dá)的影響,優(yōu)化BHT添加濃度,為其改善雨生紅球藻中蝦青素和油脂積累的工藝提供依據(jù)。
雨生紅球藻分離、篩選自云南省瀘沽湖,本實(shí)驗(yàn)室保存。BHT購自生工生物工程(上海)股份有限公司;Trizol、逆轉(zhuǎn)錄試劑盒、引物(、、和)和熒光定量試劑盒購自上海碧云天生物技術(shù)有限公司;甲醇、DMSO、KOH、丙酮和葡萄糖等均為分析純。
1730R高速冷凍離心機(jī),丹麥Labogene Scanspeed公司;Ultrospec 2100pro紫外可見分光光度計(jì),美國General Electric公司;FD5-12冷凍干燥機(jī),西盟國際集團(tuán);熒光定量PCR儀,美國Bio-Rad公司。
1.3.1 雨生紅球藻的培養(yǎng) 以Bold’s Basal Medium (BBM)(Ebrahimian, 2014)為基礎(chǔ)培養(yǎng)基,將雨生紅球藻接種到容積為3 L的鼓泡式光生物反應(yīng)器中,2800 lx持續(xù)光照,培養(yǎng)溫度為(25±1)℃,連續(xù)通入0.1 vvm的無菌空氣,培養(yǎng)15 d(此時(shí)生物量約為7.0×105cells/ml)。
1.3.2 BHT誘導(dǎo) 將BHT溶于無水乙醇,制成質(zhì)量濃度為3.5 g/L的BHT母液,備用。3000 r/min離心5 min,收集上述的藻細(xì)胞,用無菌水洗3次,除去培養(yǎng)基,重懸于不含氮源的BBM培養(yǎng)基中,加入BHT母液,使得培養(yǎng)基中BHT的質(zhì)量濃度分別為0、1、2和3 mg/L(保持各組的乙醇添加量相同)。培養(yǎng)條件為:12000 lx持續(xù)光照,培養(yǎng)溫度為(28±1)℃,連續(xù)通入0.04 vvm的無菌空氣,培養(yǎng)15 d,隔天取樣,測定各生理生化指標(biāo)。
1.3.3 藻細(xì)胞生物量和蝦青素含量的測定 選用改進(jìn)過的方法測定誘導(dǎo)培養(yǎng)基中雨生紅球藻蝦青素的含量(Boussiba, 1991)。隔天定時(shí)取5 ml誘導(dǎo)培養(yǎng)基中的藻液,5000 r/min離心3 min,棄上清液,收集藻細(xì)胞。加入2 ml質(zhì)量分?jǐn)?shù)為5%的KOH和體積分?jǐn)?shù)為30%的甲醇混合液,置于65℃水浴15 min以破壞葉綠素,3500 r/min離心棄上清液,收集沉淀,水洗2次以洗去沉淀中殘余的葉綠素,離心收集沉淀,加入5 ml的DMSO,混勻后利用超聲破壁,反復(fù)抽提至藻體發(fā)白,5000 r/min離心3 min,取上清液,于490 nm波長下測定吸光度490 nm。按以下公式計(jì)算蝦青素質(zhì)量濃度:
蝦青素質(zhì)量濃度(mg/L)=(4.5×490 nm×a)/b
式中,a為DMSO體積(ml);b為藻液體積(ml)。
此外,每隔1 d定期取10 ml誘導(dǎo)培養(yǎng)基中的藻液,離心收集細(xì)胞,冷凍,干燥,稱重,細(xì)胞生物量和蝦青素含量按以下公式計(jì)算:
細(xì)胞生物量(g/L)=藻粉干重/藻液體積
蝦青素含量(mg/g)=蝦青素質(zhì)量濃度/細(xì)胞生物量
1.3.4 藻細(xì)胞油脂含量和脂肪酸組分測定 按照Yu等(2012)的方法測定藻細(xì)胞中的油脂含量和脂肪酸組成。離心收集各組藻細(xì)胞,冷凍干燥,稱取重ω1的藻體充分研磨,加入3 ml氯仿–甲醇混合液(氯仿∶甲醇=2∶1,/)提取油脂,置于150 r/min的搖床中20 min,2000×離心10 min,收集上清液,重復(fù)提取2~3次至藻體發(fā)白,將上清液置于管重ω2的50 ml離心管中,39℃烘箱中干燥,稱重ω3,油脂含量計(jì)算公式如下:
油脂含量(%)=(ω3–ω2)/ω1×100
向上述含有油脂的離心管中加入2 ml硫酸甲醇溶液(硫酸∶甲醇=3∶97,/)混勻,70℃水浴4 h,加入2 ml正己烷,轉(zhuǎn)移至進(jìn)樣瓶中,利用Agilent 7890進(jìn)行脂肪酸GC-MS分析,色譜條件:色譜柱為HP-5MS (5% Phenyl Methyl Silox,30 mm×250 μm× 0.25 μm),二階升溫程序:170℃維持0 min,以10℃/min的速度升至190℃,維持1 min;再以0.8℃/min的速度升溫至207℃,維持1 min;進(jìn)樣分流比40∶1,分流進(jìn)樣1 μl;進(jìn)樣口溫度維持在250℃;載體為高純氦氣,以1 ml/min流速流入;質(zhì)譜條件:四級桿溫度為150℃,EI離子源溫度為230℃,溶劑延遲2 min;質(zhì)譜掃描范圍為50~550 amu。以NIST08.L作為數(shù)據(jù)庫,利用峰面積歸一化法進(jìn)一步計(jì)算脂肪酸各組分之間的相對百分含量。
1.3.5 藻細(xì)胞中碳水化合物和蛋白含量的測定 采用Jia等(2015)的方法測定藻細(xì)胞中的碳水化合物,取10 mg凍干藻粉加入0.5 ml乙酸,80℃水浴20 min,加入10 ml丙酮,3500 r/min離心10 min,棄上清液。將沉淀重懸于2.5 ml的4 mol/L三氟乙酸中,煮沸4 h,10000 r/min離心3 min。取20 μl上清液,加入硫酸–苯酚–水溶液900 μl(硫酸∶水∶苯酚= 15 ml∶7.5 ml∶0.15 g),煮沸20 min,在490 nm測吸光度,用葡萄糖制作標(biāo)準(zhǔn)曲線,以測定碳水化合物的總含量。
取10 mg凍干藻粉,加入100 μl的1 mol/L NaOH溶液,80℃水浴10 min,加入900 μl蒸餾水,12000×離心30 min,將上清液轉(zhuǎn)移至新的離心管中。重復(fù)上述操作2次,合并上清液,以牛血清蛋白制作標(biāo)準(zhǔn)曲線,利用Berges等(1993)的方法測定總蛋白含量。
1.3.6 測定雨生紅球藻蝦青素和脂肪酸合成相關(guān)酶基因的表達(dá)量 本實(shí)驗(yàn)使用Primer 5.0設(shè)計(jì)、、和酶基因的上下游擴(kuò)增引物(表1),擴(kuò)增后產(chǎn)物經(jīng)生工生物工程(上海)股份有限公司測序后BLAST比對,以此為模板設(shè)計(jì)熒光定量引物(表2)。收集藻細(xì)胞,無菌水洗2次,保存于–80℃?zhèn)溆?。在液氮中充分研磨藻?xì)胞,使用Trizol法提取總RNA,利用逆轉(zhuǎn)錄試劑盒TaKaRa逆轉(zhuǎn)錄合成cDNA,以此為模板進(jìn)行RT-PCR擴(kuò)增,通過ABI 7500熒光定量儀對、、和基因表達(dá)量進(jìn)行測定,以18S (引物:5′-CGGTCTGCCTCTGGTATG-3′與5′-GC TTGCTTTGAACACGCT-3′)基因作為內(nèi)標(biāo)來調(diào)節(jié)RNA的用量和循環(huán)數(shù),使內(nèi)標(biāo)基因在不同濃度誘導(dǎo)下的表達(dá)豐度一致。
表1 酶基因克隆引物
Tab.1 Primers for gene cloning
表2 酶基因熒光定量PCR引物
Tab.2 Primers for enzyme genes RT-PCR
實(shí)驗(yàn)各組均設(shè)置3個平行樣,數(shù)據(jù)處理采用ANOVA(SPSS 19.0)一步法分析和Duncan氏多重范圍比較進(jìn)行分析。最小顯著性差異進(jìn)行多重比較來檢驗(yàn)調(diào)查不同實(shí)驗(yàn)的組間差異,<0.05為具有顯著性。
圖1 不同濃度BHT處理對雨生紅球藻生物量和蝦青素積累的影響
*表示組間有顯著性差異(<0.05),**表示組間有極顯著性差異(<0.01)。下同
* represents significant difference between groups (<0.05), ** represents highly significant difference between groups. The same as below
如圖1A所示,0、1和2 mg/L BHT處理組生物量最大值分別為0.62、0.58和0.56 g/L,3 mg/L BHT處理組藻細(xì)胞生長明顯受到限制。圖1B顯示,0、1和2 mg/L BHT處理組蝦青素含量逐漸升高,2 mg/L BHT處理組增加最為顯著,在13 d時(shí)達(dá)到最高31.66 mg/g,0和1 mg/L BHT處理組分別為16.94和22.30 mg/g;3 mg/L BHT處理組蝦青素含量則較低。
高光照、缺氮條件下,0和2 mg/L BHT處理組藻細(xì)胞中油脂的變化趨勢如圖2所示。0和2 mg/L BHT處理組油脂含量均逐步上升,最后趨于穩(wěn)定;2 mg/L BHT處理組在11 d時(shí)達(dá)最高,占細(xì)胞干重的45.56%,較對照組(39.06%)提高了16.6%。
表3為第11天時(shí)0和2 mg/L BHT處理組脂肪酸組成。培養(yǎng)至11 d,C16∶0、C18∶1n9t、C18∶2n6c和C18∶3n6為脂肪酸的主要組成成分,占總脂肪酸的88%以上。2 mg/L BHT處理組和對照組相比,脂肪酸各組分含量變化不大,僅C18∶1n9t含量略微下降。
圖2 不同濃度BHT處理對雨生紅球藻油脂積累的影響
碳水化合物和蛋白質(zhì)是藻細(xì)胞內(nèi)重要的生理參數(shù),本研究檢測了在添加BHT條件下二者的變化情況。2 mg/L BHT處理組和對照組的碳水化合物含量均呈現(xiàn)下降趨勢,2 mg/L BHT處理組從第9天開始顯著低于對照組,13 d時(shí)至最低,為細(xì)胞干重的13.45%,此時(shí)對照組為16.12%(圖3A);微藻細(xì)胞內(nèi)蛋白含量總體呈降低趨勢,2 mg/L BHT在5 d、7 d時(shí)顯著低于對照組(圖3B)。
圖3 不同濃度BHT處理對雨生紅球藻碳水化合物和蛋白含量的影響
表3 不同濃度BHT對雨生紅球藻脂肪酸組成的影響(%)
Tab.3 Effects of BHT on fatty acid profile of H.pluvialis during induction process (%)
圖4 BHT對dxs和bkt基因表達(dá)量的影響
和是蝦青素合成途徑中的關(guān)鍵酶基因,圖4為培養(yǎng)過程中和基因的相對表達(dá)量變化。2 mg/L BHT處理組的基因相對表達(dá)量在3、5、9、11、13和15 d均顯著高于對照組,第9天時(shí)為對照組的5.19倍(圖4A);與對照組相比,2 mg/L BHT處理組表達(dá)量從第3天開始顯著增加,第9天時(shí)達(dá)到最高,為對照組的2.04倍,7和11 d也顯著高于對照組,分別為1.97倍和1.96倍(圖4B)。
用qRT-PCR方法檢測不同誘導(dǎo)時(shí)間內(nèi),2 mg/L BHT誘導(dǎo)組和對照組雨生紅球藻脂肪酸合成基因和的表達(dá)量(圖5A、圖5B),2 mg/L BHT處理組的相對表達(dá)量從第1天起顯著高于對照組,為對照組的3.36倍,第5天達(dá)到最高,為對照組的4.57倍;整個誘導(dǎo)培養(yǎng)期間,2 mg/L BHT處理組基因的相對表達(dá)量也顯著高于對照組。
本研究中,外源添加不同濃度BHT的結(jié)果顯示,低濃度(1和2 mg/L)對雨生紅球藻的生物量影響并不顯著,而高濃度(3 mg/L)對生物量影響較大,對藻細(xì)胞有明顯的毒害作用。2 mg/L BHT誘導(dǎo)組的蝦青素積累量顯著高于其他組;1 mg/L的添加劑量組高于對照組,但效果不如2 mg/L的處理組;3 mg/L添加量的毒害作用導(dǎo)致蝦青素的積累無法進(jìn)行。BHT是一種抗氧化劑,高濃度可能對機(jī)體產(chǎn)生促氧化作用(Prooxidant),從而導(dǎo)致藻細(xì)胞死亡。Nanou等(2010)利用高濃度的BHT處理真菌時(shí)發(fā)現(xiàn),BHT與分子氧相互作用,產(chǎn)生苯氧自由基和超氧陰離子,表現(xiàn)出氧化性,致使細(xì)胞死亡;于威等(2018)研究發(fā)現(xiàn),利用高濃度抗氧化劑谷胱甘肽處理黃瓜幼苗呈現(xiàn)明顯的致毒作用,這均與本研究結(jié)果一致。Wen等(2015)研究發(fā)現(xiàn),適宜濃度的乙醇誘導(dǎo)雨生紅球藻能促進(jìn)其蝦青素的積累,達(dá)到25.10 mg/g;丁巍等(2017)利用胺鮮酯誘導(dǎo)雨生紅球藻提高了蝦青素的含量,最高可達(dá)24.94 mg/g,這均低于本研究中蝦青素積累量(31.66 mg/g)。
和是蝦青素合成途徑中的關(guān)鍵酶基因,這2個基因表達(dá)量與蝦青素的積累量呈正相關(guān),Gao等(2012)研究發(fā)現(xiàn),外源添加水楊酸上調(diào)了基因的相對表達(dá)量,促進(jìn)了蝦青素的合成。Ding等(2018)發(fā)現(xiàn),添加褪黑素提高了雨生紅球藻細(xì)胞和基因表達(dá)量,同時(shí)提高了蝦青素的積累量。本研究中,圖4和圖1對比可知,基因表達(dá)量和蝦青素積累之間不具有同步性,蝦青素的大量積累滯后于和的基因表達(dá)量增加。Gao等(2012)使用水楊酸誘導(dǎo)蝦青素積累時(shí)發(fā)現(xiàn),蝦青素合成關(guān)鍵酶基因分為轉(zhuǎn)錄后水平和轉(zhuǎn)錄水平基因,即有的基因在轉(zhuǎn)錄后上調(diào)蝦青素生物合成,有的基因在轉(zhuǎn)錄時(shí)上調(diào)蝦青素的合成。Zhao等(2015)也證明了這一結(jié)論。
利用抗氧化劑作為誘導(dǎo)子來提高生物體內(nèi)次生代謝產(chǎn)物的含量,逐步成為研究熱點(diǎn)。Franz等(2013)研究發(fā)現(xiàn),添加適宜濃度的沒食子酸誘導(dǎo)微藻能增加細(xì)胞內(nèi)油脂含量,比對照組提高了2.17倍;Li等(2017)外源添加褪黑素提高了單針藻sp. QLY-1中的油脂含量。本研究使用BHT作為誘導(dǎo)子,提高了雨生紅球藻中油脂的含量,達(dá)到細(xì)胞干重的45.56%,高于其他文獻(xiàn)報(bào)道的微藻油脂含量(Bogen, 2013; Holbrook, 2014; Shrivastav, 2015)。
此外,Che等(2016)研究發(fā)現(xiàn),外源添加黃腐酸能提高油脂含量,脂肪酸各組分變化不大,這與本研究的結(jié)果一致。和是脂肪酸合成通路中的關(guān)鍵酶基因,本研究中外源添加BHT提高了和的相對表達(dá)量,最高為對照組的3.36倍和3.02倍,促進(jìn)了脂肪酸的合成。Lei等(2012)也發(fā)現(xiàn)脂肪酸合成的增加往往伴隨著和表達(dá)量的提高。Shang等(2016)添加茴香醚誘導(dǎo)雨生紅球藻合成蝦青素,和的相對表達(dá)量均升高,提高了脂肪酸含量,與本實(shí)驗(yàn)呈現(xiàn)一致結(jié)果;油脂積累延遲于基因表達(dá)水平的上調(diào),說明和起到了轉(zhuǎn)錄后水平的調(diào)節(jié)作用。
碳水化合物和蛋白質(zhì)是藻細(xì)胞重要的生理指標(biāo),Ho等(2017)發(fā)現(xiàn)高鹽誘導(dǎo)下,sp. JSC4通過降低細(xì)胞內(nèi)淀粉含量增加了脂質(zhì)積累;Chokshi等(2017)研究發(fā)現(xiàn),微藻在缺氮條件下,細(xì)胞內(nèi)的蛋白含量隨著培養(yǎng)時(shí)間的進(jìn)行而逐步降低。本研究中,BHT處理組和對照組的碳水化合物和蛋白均呈下降趨勢,BHT處理組下降更為顯著。因此,在脅迫條件下,雨生紅球藻可能通過分解自身的碳水化合物和蛋白來合成油脂和蝦青素等代謝產(chǎn)物來提高抗性。
雨生紅球藻蝦青素的積累與油脂合成緊密相關(guān),脂肪酸在蝦青素合成中有兩個作用,一是與蝦青素酯化,形成蝦青素酯,有利于蝦青素的合成;另一個是形成脂質(zhì)體(Lipid bodies, LBs)以貯存蝦青素(Zhang, 2016)。雨生紅球藻中95%的蝦青素與脂肪酸發(fā)生酯化反應(yīng),形成蝦青素酯,儲存于富含甘油三酯的脂質(zhì)體中,這一反應(yīng)降低了底物的量,減少末端產(chǎn)物抑制作用,促進(jìn)了蝦青素的積累(Chen, 2015)。增加脂肪酸的合成,合成的蝦青素進(jìn)入LBs后得以分隔,可作為減少末端產(chǎn)物抑制、增加蝦青素積累的策略(Zhekisheva, 2005)。
在高光照、缺氮條件下,外源添加適宜濃度抗氧化劑褪黑素能夠抑制細(xì)胞內(nèi)的活性氧(ROS),增加細(xì)胞內(nèi)抗氧化酶活性和抗氧化物含量,維持細(xì)胞內(nèi)的氧化還原處于平衡狀態(tài),促進(jìn)蝦青素和油脂的積累(Zhao, 2018; Ding, 2018)。同樣,BHT作為抗氧化劑,可提高真菌中抗氧化酶活性,從而清除胞內(nèi)過量ROS,進(jìn)而促進(jìn)β-胡蘿卜素的合成(Nanou, 2010)。另一方面,Ding等(2018)通過外源添加抗氧化劑褪黑素誘導(dǎo)雨生紅球藻發(fā)現(xiàn),上調(diào)了細(xì)胞內(nèi)一氧化氮(NO)和水楊酸(SA)水平,NO和SA通過一系列級聯(lián)反應(yīng)促進(jìn)了蝦青素和脂肪酸的積累;SA作為植物激素可促進(jìn)雨生紅球藻中蝦青素的積累(Gao, 2012);添加NO供體,SNP可通過激活胞內(nèi)抗氧化酶活性,猝滅過量ROS,進(jìn)而調(diào)控微藻在鉻脅迫下的抗性(Ková?ik, 2015)。而適量ROS可以促進(jìn)微藻中次級代謝產(chǎn)物的積累(Shi, 2017)。因此,外源添加BHT可能是通過調(diào)控ROS和胞內(nèi)相關(guān)信號分子水平,進(jìn)而促進(jìn)蝦青素和油脂的積累(Zhao, 2018; Ding, 2018; Nanou, 2010)。此外,BHT上調(diào)了蝦青素和脂肪酸合成途徑中關(guān)鍵酶基因的相對表達(dá)量,促進(jìn)了蝦青素和脂肪酸的積累。
外源添加適宜濃度的BHT能促進(jìn)誘導(dǎo)條件下雨生紅球藻中蝦青素和油脂的積累。2 mg/L BHT處理雨生紅球藻的效果最佳,蝦青素積累量為31.66 mg/g,是對照組的1.87倍,促進(jìn)了蝦青素合成關(guān)鍵酶基因和的表達(dá);同時(shí),提高了脂肪酸合成酶基因和表達(dá)量,油脂含量增加到干重的45.56%,比對照組提高了16.6%;油脂的積累,有利于蝦青素酯的形成,從而增加了微藻細(xì)胞中蝦青素的生物合成。
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Effects of Butylated Hydroxytoluene on Accumulation of Astaxanthin and Lipids inLUGU
YUE Chenchen1, YU Xuya1, ZHAO Yongteng1, WANG Huiping2①
(1. Faculty of Life Sciences and Technology, Kunming University of Science and Technology, Kunming 650500; 2. Kunming Children's Hospital, Kunming 650228)
is a microalga, and this species is economically important since it is a rich source of natural astaxanthin, which is considered a “super anti-oxidant.” The study was conducted usingLUGU filtrated from Lugu Lake in Yunnan Province, China. Studies have shown that under stress, the exogenous addition of appropriate concentration of butylated hydroxytoluene (BHT) can effectively promote the accumulation of astaxanthin and increase the content of lipids in the algal cells. The effects of BHT on several traits were investigated under stress conditions (high illumination and nitrogen deficiency), including the impact on growth of algae; the accumulation of astaxanthin; the synthesis of lipids; the composition of fatty acids, carbohydrates and protein; the expression level of the key enzyme gene of astaxanthin; and fatty acid biosynthesis. In this study, the different concentrations ofBHT (0, 1, 2, and 3 mg/L) were achieved through single factor experiments in algal cell culture medium. Results from these experiments showed that BHT treatment does not effectively promote the algae growth, but it does affect the accumulation of astaxanthin in algae cells. There was a significant dose effect depending on the BHT treatment applied to. After examining BHT additions of different concentrations, astaxanthin accumulation was determined to be the highest after the 2 mg/L BHT treatment, and it was significantly higher than that resulting from other treatments or the control group (<0.05). Moreover, astaxanthin accumulation was 1.87 times higher than that in the control group, reaching 31.66 mg/g. The lipid content of the 2 mg/L BHT treatment was 45.56%, which was also higher than that in the control (39.06%). Under these conditions, the expression levels of the key enzyme genes of astaxanthin synthesis,andwere 5.19 folds and 2.04 folds, respectively, as those of the control, and the expression levels of the key enzyme genes of fatty acid synthesis,andwere significantly higher than those of the control (<0.05), being 4.56 folds and 3.02 folds, respectively, as those of the control. Contrastingly, the carbohydrate and protein contents decreased compared to those of the control group. Overall, our results also show that BHT can increase accumulation of astaxanthin and increase the content of lipids in the algal cells when administered at an appropriate dose.
; Butylated hydroxytoluene; Astaxanthin; Lipids; Astaxanthin synthesis genes; Fatty acid synthesis genes
S968.41
A
2095-9869(2019)06-0145-09
10.19663/j.issn2095-9869.20180831001
http://www.yykxjz.cn/
岳陳陳, 余旭亞, 趙永騰, 王惠萍. 二丁基羥基甲苯對雨生紅球藻蝦青素和油脂積累的影響. 漁業(yè)科學(xué)進(jìn)展, 2019, 40(6): 145–153
Yue CC, Yu XY, Zhao YT, Wang HP. Effects of butylated hydroxytoluene on accumulation of astaxanthin and lipids inLUGU. Progress in Fishery Sciences, 2019, 40(6): 145–153
* 國家自然科學(xué)基金地區(qū)科學(xué)基金項(xiàng)目(21766012;21666012)和云南省重大科技專項(xiàng)計(jì)劃(2018ZG003)共同資助[This work was supported by Regional Science Foundation Project of National Nature Science Foundation of China (21766012; 21666012), Yunnan Province Major Science and Technology Special Program (2018ZG003)]. 岳陳陳,E-mail: 18468235975@163.com
王惠萍,主任醫(yī)師,E-mail: 2500016390@qq.com
2018-08-31,
2018-10-05
WANG Huiping, E-mail: 2500016390@qq.com
(編輯 馮小花)