CN114672421A - 一种高生育酚含量微藻的培育方法和筛选方法 - Google Patents
一种高生育酚含量微藻的培育方法和筛选方法 Download PDFInfo
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Abstract
本发明公开了一种高生育酚含量微藻的培育方法和筛选方法,涉及藻类培育领域。培育方法包括:将微藻置于培养基中避光培养,微藻为浐灞湿地公园的人工湖生长的栅藻或新疆博斯腾湖生长的小球藻。筛选方法包括:在不同水域进行水样采集、分离纯化;检测生育酚合成途径中的关键基因表达量;测定初筛后样品的生物量和油脂含量,随后再对油脂含量高的微藻进行生育酚含量测定;进行18S rRNA基因扩增及序列分析鉴定。本申请筛选获得的高生育酚含量藻株经过培育后,可以获得高产量的生育酚,提高维生素E的产量。
Description
技术领域
本发明涉及领域,尤其涉及一种高生育酚含量微藻的培育方法和筛选方法。
背景技术
维生素E(生育酚)是光合作用细胞合成的脂溶性抗氧化剂,鉴于维生素E的多种生理作用,已被广泛应用于食品添加剂、防腐剂、化妆品(如防晒剂)的生产中,也被用作饲料添加剂。目前维生素E制备方法主要有化学合成、生物提取和生物合成。其中化学合成和生物提取法收率低,易污染环境,生物合成法操作复杂。
目前,利用微生物产维生素E以其绿色环保的特点成为新的研究方向,其中微藻是天然生育酚很好的来源,迄今为止发现能合成生育酚的微藻主要有螺旋藻(Spirulinaplatensis)、杜氏藻(Dunaliellatertiolecta)、集胞藻(Synechocystisaquatilis)、微绿球藻(Nannochloropsisoculata)、心形扁藻(Tetraselmissuecica)、衣藻(Chlamydomonas)、棕鞭藻(Ochromonas)、裸藻(Euglena gracilis)和等鞭金藻(Isochrysisgalbana)。不同种类的微藻其细胞内生育酚含量与比例依微藻种类、组织和培养条件的不同而不同。目前对于微藻中的维生素E相关研究较缺乏,导致利用微藻来生产生育酚的产业发展受限,急需寻求含油量高且生育酚含量高的藻种,并探索其合适的生长条件。
发明内容
本发明提供了一种高生育酚含量微藻的培育方法和筛选方法,以获得一种高生育酚含量的微藻,有利于促进生育酚生产产业的发展。
为了解决上述技术问题,本发明目的之一提供了一种高生育酚含量微藻的培育方法,包括以下步骤:将微藻置于培养基中,避光培养,其中微藻为浐灞湿地公园的人工湖生长的栅藻或新疆博斯腾湖生长的小球藻。
作为优选方案,所述培养基为BG-11培养基,所述BG-11培养基中Na2CO3的添加量为0,所述BG-11培养基还包括有碳源,所述碳源为葡萄糖、工业甘油和工业苹果酸中的一种或多种。
作为优选方案,所述碳源由葡萄糖和工业苹果酸按质量比为1:1混合而成。
作为优选方案,所述碳源的浓度为20g/L。
作为优选方案,避光培养条件为在温度27℃、转速120r/min的摇床中培养。
为了解决上述技术问题,本发明目的之二提供了一种高生育酚含量微藻的筛选方法,包括以下步骤:
S1、在不同水域进行水样采集,随后进行藻株的分离纯化;
S2、检测水样中的生育酚含量,进行高生育酚含量藻株的初筛;
S3、测定初筛后样品的生物量和油脂含量,随后再对油脂含量高的微藻进行生育酚含量测定,进行高生育酚含量藻株的复筛;
S4、对复筛后获得的藻株进行18S rRNA基因扩增及序列分析鉴定。
作为优选方案,在步骤S2和S3中,生育酚含量采用实时荧光定量PCR检测生育酚合成途径中的关键基因表达量。
作为优选方案,生育酚合成途径中的关键基因包括HPPD、GGH、MPBQMT、TC和γ-TMT中的一种或多种。
作为优选方案,在S1中,将藻株采用平板划线法进行分离纯化,采用BG-11培养基培养,所述BG-11培养基中Na2CO3的添加量为0,所述BG-11培养基还包括20g/L的葡萄糖。
作为优选方案,在S2中还包括预先对不同取样地的混合水样进行生育酚含量测定,以筛选取样地;在S3中,还包括采用预先采用尼罗红染色法检测荧光强度以判断油脂含量高低。
相比于现有技术,本发明实施例具有如下有益效果:
1、本申请针对浐灞湿地公园的人工湖生长的栅藻或新疆博斯腾湖生长的小球藻进行培养,通过筛查结果可知,这两种微藻的生育酚含量和油脂含量呈正相关,且含量相对于其他微藻较高,可以获得高生育酚含量的微藻。
2、首先收集不同水域的水样,通过实时荧光定量PCR比较生育酚合成途径中6个关键基因的表达量,得到高表达6个关键基因的藻株,同时测定其生物量、油脂含量和α-生育酚含量,更准确的筛选出高生育酚含量的藻株,最后对其中生育酚含量和油脂含量最高的藻株进行生物学鉴定,筛选出自身能够生产较高生育酚的微藻,有助于高生育酚微藻的培育。
附图说明
图1:为本发明实施例一S201中不同水域水样的q-PCR检测结果;
图2:为本发明实施例一S202中10个藻株q-PCR检测结果;
图3:为本发明实施例一S301中10个藻株荧光强度检测结果;
图4:为本发明实施例一S303中5个藻株的α-生育酚和油脂产量对比结果;
图5:为本发明实施例一S4中藻株CB-2基于18S rDNA基因部分序列构建的***发育树;
图6:为本发明实施例一S4中藻株BST-2基于18S rDNA基因部分序列构建的***发育树。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例一
一种高生育酚含量微藻的筛选方法,包括以下步骤:
S1、水样的采集和藻株的分离纯化:
S101、水样分别采自新疆天山天池、新疆内陆湖博斯腾湖、内陆湖巴里坤湖、无伦古湖、陕西境内咸阳湖、浐灞湿地公园的人工湖、丰庆公园人工湖以及陕西秦岭的天然水池,浮游微藻采用1L采水器采集水样,底栖微藻采用刷子从水体中的石块、枝条等物体上刮取,采集的水样用透明塑料瓶保存,然后带入实验室进行分离纯化。
S102、采集的水样采用平板划线法进行分离纯化,培养采用改良后BG-11培养基(改良方式为将Na2CO3换成15g/L葡萄糖,使其作为唯一碳源,培养基组成见表1);取100μL水样涂布于培养基中进行分离,分别在(27±1)℃条件下培养20d后挑取单藻在固体平板上划线分离,直至水浸片法镜检得到单一藻细胞,纯化后转移至50mL异养BG-11培养基中进行异养培养,以10%接种量接种对数生长期的藻液于BG-11异养培养基中,于120r/min、温度(27±1)℃下培养,摇床中避光培养。
表1改良后BG-11培养基的组成
S103、根据培养终期细胞液的颜色和性质观察,除去接团、粘壁、颜色浑浊的藻株,重点考察了20种性质优良的藻株,如表2所示。
表2-20株优良藻株的来源和形态分类
S2、高生育酚含量藻株的初筛:
对8个取样地收集的水样进行实时荧光定量PCR,比较8个水域水样中6个生育酚合成途径中关键基因的表达量,初步判断该水域是否含有可产生生育酚的藻种。
S201、将同一取样地的水样混合,对8个取样地收集的混合水样分别提取RNA,反转录获得cDNA,之后依据qPCRGreen Master Mix(Without ROX)试剂盒说明书,使用Primer 5.0软件设计引物,使扩增产物长度在100bp-150bp之间,选取+GAPDH作为内参基因进行qRT-PCR分析(6个基因引物的序列如表3);引物设计完成后采用NCBI数据库中的Blast工具检索引物的特异性,避免发生引物的非特异性扩增,在上海生工生物公司进行引物合成;实时荧光定量PCR(qRT-PCR)在LineGene 9600Plus荧光定量PCR仪(博日,杭州)上进行,定量反转录步骤按照PrimeScriptTM RT regent kit with gDNA Eraser说明书进行;荧光定量PCR操作步骤按照qPCRGreen Master Mix(Without ROX)试剂盒的说明进行。每个样品进行三次生物学重复和三次技术重复,采用2-ΔΔCt的方法计算基因相对定量的qRT-PCR表达量,比较8个水域的6个基因的qRT-PCR相对表达量。
实验方法与步骤具体如下:
(1)RNA提取:
①取5ml收集的水样离心,弃上清,加入1ml RNAiso,匀浆,并转移到RNase-free的离心管中,室温放置5min,使其充***解;
②12000rpm离心5min,转移上清到新的1.5ml离心管中;
③加入200ul(1/5RNAiso Plus体积)氯仿,振荡混匀,室温静置5min;
④4℃,12000g离心15min,将上清转移到新的1.5ml离心管中;
⑤加入0.5-1倍RNAiso Plus体积的异丙醇,混匀,室温放置10min;
⑥4℃,12000g离心10min,弃上清,RNA为沉在管底的白色沉淀;
⑦加入RNAiso Plus等体积的75%乙醇清洗沉淀,温和振荡离心管,悬浮沉淀;
⑧4℃,7500g离心5min,弃上清保留沉淀;
⑨室温晾干5-10min。注:RNA样品不能过于干燥,否则难以溶解;
⑩加入50ul DEPC处理水溶解,取3ul跑核酸胶验证。
(2)反转录反应(为了配制的准确性,应按反应数+2的量配制成混合液,然后再分别按照试剂量分装到每个小反应管中,冰上操作):
反应体系:
步骤1的反应混合液10.0uL;
PrimeScript RT Enzyme Mix I 1.0uL;
RT Primer Mix 1.0uL;
5×PrimeScript Buffer 2(for Real Time)4.0uL;
RNase Free dH2O 4.0uL;
总20.0uL;
PCR反应程序:37℃15min;85℃5sec;4℃10min。得到qRT-PCR的cDNA模版。
(3)qRT-PCR反应(冰上操作):
反应体系:
AceQqPCR SYBR Green Master Mix 10.0μL;
上游引物(10uM) 0.4uL;
下游引物(10uM) 0.4uL;
qRT-PCR cDNA模版2.0μL;
dH2O 7.2uL;
总20.0uL;
qRT-PCR的反应程序采用两步法进行:预变性:95℃ 5min;循环反应:95℃ 15s,60℃ 1min,重复40个循环;溶解曲线:95℃ 1min,65℃ 1min,95℃ 20s,步进0.5℃/s,30℃1min。
表3-α-生育酚合成途径中关键基因和内参基因来源及引物信息
结果如图1所示,8个水域中6个基因的表达量高低不一,其中陕西秦岭天然水池和乌伦古湖中的6个基因表达量均比较低,我们初步断定陕西秦岭天然水池和乌伦古湖中产生育酚的微藻较少或没有。6个基因在新疆天山天池、浐灞湿地森林公园的人工湖、咸阳湖、丰庆公园人工湖、新疆博斯腾湖和新疆巴里坤湖中均表现出较高的表达量,初步判断这几个取样地中可能含有较多的高生育酚含量的藻种。
S202、通过对分离纯化出来的16个藻株中生育酚合成途径中6个关键基因进行实时荧光定量PCR,实时荧光定量PCR步骤同步骤1中方法,测定其表达量进行比较,初步筛选出生育酚合成途径中关键基因高表达量的藻株,得到10株6个关键基因均高表达的藻株,结果如图2所示,其中藻株BST-2、BST-3、CB-2和CB-3的6个基因表达量均相对较高,进一步确定这四个藻株可能会产生较多的生育酚。
S3、高生育酚含量藻株的复筛:
S301、采用尼罗红染色法对微藻染色,尼罗红(NR)是一种亲脂性的荧光染料,微藻细胞被尼罗红染色后,在480nm的荧光激发下细胞内的油脂会发出黄色荧光,测定其荧光强度来初步筛选产油脂微藻,本研究使用的尼罗红染色条件为:尼罗红染料质量浓度为1.0ug/mL,DMSO溶剂体积分数为20%,染色时间为15min,藻液密度OD600为0.4-0.5。
如图3结果所示,尼罗红染色后10株微藻荧光强度比较发现藻株BST-2、BST-3、CB-2、CB-3和BLK-2的荧光值相对较高,这5株微藻的荧光强度达到7000a.u.以上,表明这5株微藻的油脂含量相对较高。
S302、采用BG-11培养基对初筛的10株产油脂微藻在温度27℃条件下培养,培养10天至稳定期收获,测定其生物量和油脂含量,并计算油脂产量,以油脂产量的高低评价其产油脂能力,从中筛选产油脂能力高的微藻进行后续实验,具体包括以下步骤:
(1)生物量测定:待微藻培养至稳定期后,通过离心收获藻体,然后置于冷冻干燥机中冻干,获得干藻粉,称其质量,生物量=干藻粉质量/藻液体积。
(2)油脂含量测定:用氯仿-甲醇法提取干藻粉体内油脂,称取培养收获的藻粉0.1g,加入V(氯仿)∶V(甲醇)∶V(水)为1∶2∶1的混合液20mL,50℃超声提取15min,然后加入V(氯仿)∶V(水)为1∶1的混合液10mL,50℃超声提取15min,取氯仿层于旋转蒸发器上蒸干,得到微藻油在50℃下干燥至恒质量,计算出油率,3个平行试验,取平均值,按照下面公式计算油脂质量分数和总脂质量浓度:
油脂质量分数(%)=(m1/0.1)×100
总脂质量浓度(g·L-1)=微藻藻体质量浓度×油脂质量分数
式中m1表示0.1g藻粉提取所得到的油脂质量(g);微藻藻体质量浓度(g·L-1)为单位体积培养液所收获的微藻量。
表4-10株微藻生物量、油脂含量及油脂产率的统计结果
测试结果如表4所示,通过测定初筛的10株微藻生物量和油脂含量,计算出油脂产量,藻株BST-2的生物量最大(0.93g/L),油脂含量较高(33.85%),油脂产量最高(0.315g/L);其次是CB-2,其生物量为0.87g/L,油脂含量为32.26%,油脂产量为0.281g/L;再次是BST-3,其生物量为0.86g/L,油脂含量为30.45%,油脂产量为0.262g/L,然后依次是CB-3和BLK-2,将油脂含量相对较高的这5个藻株进行下一步研究。
S303、对上述筛选到的5株油脂含量较高的微藻进行培养收获后测定其生育酚含量,步骤如下:
(1)检测样品的制备:取1ml藻株培养液于1.5ml离心管中,12000rpm离心5min,取上清于新的离心管,并加入40ul冰醋酸,再用0.22μm水系针孔式滤头过滤;
(2)HPLC检测条件:流动相:0.01M KH2PO4溶液(A)和甲醇(B);比例:90%A/10%B;流速:0.8ml/min;检测波长:290nm;色谱柱:YMC-Pack ODS-AQ(4.6×250mm);
(3)标准曲线制备方法如下:用万分之一天平称取10mgα-生育酚,溶于10ml纯甲醇溶液中,浓度定为1g/L的母液,依次用纯甲醇溶液梯度稀释,稀释得到浓度分别为250mg/L,100mg/L,50mg/L,20mg/L,5mg/L的α-生育酚溶液,并用相同的方法再稀释出两批同浓度的α-生育酚溶液,对所有样品经0.22um有机系针孔式滤头过滤,再进行HPLC,制作α-生育酚标准曲线。
结果如图4所示,比较5种藻株的α-生育酚含量,发现藻株BST-2的α-生育酚含量最高,为235.5ug/g DCW,其次是藻株CB-2,其α-生育酚含量为187.4ug/g DCW,含量最低的是BLK-2,其α-生育酚含量为102.9ug/g DCW,比较5种微藻的α-生育酚含量,发现其α-生育酚含量高低与油脂含量密切相关,含量高低趋势一致,其表现为油脂含量越高其α-生育酚也越高。
S4、高生育酚含量藻株的鉴定:
将上一步筛选到的5株生育酚含量较高的藻株中选择含量最高的藻株BST-2和CB-2进行18S rRNA基因扩增及序列分析,将已分离纯化的单藻落平板,送到生工生物工程(上海)有限公司进行18s rDNA测序,藻株CB-2的18S r DNA序列长度为1627bp,藻株BST-2的18S r DNA序列长度为1513bp,将得到的结果提交Genbank中进行Blast序列比对,基于MEGA软件进行多重序列分析,并构建***发育树,如图5-6所示。
结果显示,藻株CB-2的18S rDNA序列与多个栅藻序列的相似度达到99%,其与Scenedesmus abundans(X73995.1)的相似度最高。从GenBank数据库中选6株同源性相近的栅藻与藻株CB-2构建***进化树,结果见图5,图中藻株CB-2与Scenedesmus abundans聚为一簇,同源性支持率高达100%,确定藻种Y06为栅藻(Scenedesmus abundans)。结果图6表明,藻株BST-2与多个Chlorella sp.的亲缘关系最近,初步确定该藻属于Chlorella sp,即小球藻属。
实施例二
一种高生育酚含量微藻的培育方法,包括以下步骤:
主要对碳源进行筛选,选择的碳源分别为:①葡萄糖,②工业甘油,③工业苹果酸,④葡萄糖加工业甘油(1:1)(葡甘),⑤葡萄糖加工业苹果酸(1:1)(葡果),⑥葡萄糖加工业甘油加工业苹果酸(1:1:1)(葡甘果),碳源浓度为20g/L,其他成分同实施例1中改良后BG-11培养基,对筛选出的BST-2和CB-2微藻在温度27℃,摇床转速120r/min的条件下避光培养,培养10天至稳定期收获,然后测定其油脂含量和α-生育酚含量。
表5-不同碳源培养微藻的油脂产量和α-生育酚含量的比较
结果如表5所示,其中葡萄糖加工业苹果酸的效果是最好的,其中藻株BST-2的油脂产量达到0.462g/L,α-生育酚含量达到336.2ug/g DCM,藻株CB-2的油脂产量达到0.395g/L,α-生育酚含量达到253.3ug/g DCM,起到了提高微藻的油脂含量和α-生育酚含量的作用。因此将葡萄糖和工业苹果酸视作藻株BST-2和藻株CB-2的最佳碳源进行培养,可达到提高其α-生育酚含量的作用。
进一步优选地,葡萄糖和工业苹果酸的浓度为20g/L,可提高微藻α-生育酚含量。
进一步优选地,葡萄糖和工业苹果酸的质量比例为1:1时,可提高微藻α-生育酚含量。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步的详细说明,应当理解,以上所述仅为本发明的具体实施例而已,并不用于限定本发明的保护范围。特别指出,对于本领域技术人员来说,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种高生育酚含量微藻的培育方法,其特征在于,包括以下步骤:将微藻置于培养基中,避光培养,其中微藻为浐灞湿地公园的人工湖生长的栅藻或新疆博斯腾湖生长的小球藻。
2.如权利要求1所述的一种高生育酚含量微藻的培育方法,其特征在于,所述培养基为BG-11培养基,所述BG-11培养基中Na2CO3的添加量为0,所述BG-11培养基还包括有碳源,所述碳源为葡萄糖、工业甘油和工业苹果酸中的一种或多种。
3.如权利要求2所述的一种高生育酚含量微藻的培育方法,其特征在于,所述碳源由葡萄糖和工业苹果酸按质量比为1:1混合而成。
4.如权利要求2或3所述的一种高生育酚含量微藻的培育方法,其特征在于,所述碳源的浓度为15-25g/L。
5.如权利要求1所述的一种高生育酚含量微藻的培育方法,其特征在于,避光培养条件为在温度27℃±2℃、转速100-150r/min的摇床中培养。
6.一种高生育酚含量微藻的筛选方法,其特征在于,用于筛选如权利要求1-5任一所述的一种高生育酚含量微藻的培育方法中的微藻,其特征在于,包括以下步骤:
S1、在不同水域进行水样采集,随后进行藻株的分离纯化;
S2、检测水样中的生育酚含量,进行高生育酚含量藻株的初筛;
S3、测定初筛后样品的生物量和油脂含量,随后再对油脂含量高的微藻进行生育酚含量测定,进行高生育酚含量藻株的复筛;
S4、对复筛后获得的藻株进行18SrRNA基因扩增及序列分析鉴定。
7.如权利要求6所述的一种高生育酚含量微藻的筛选方法,其特征在于,在步骤S2和S3中,生育酚含量采用实时荧光定量PCR检测生育酚合成途径中的关键基因表达量。
8.如权利要求7所述的一种高生育酚含量微藻的筛选方法,其特征在于,生育酚合成途径中的关键基因包括HPPD、GGH、MPBQMT、TC和γ-TMT中的一种或多种。
9.如权利要求6所述的一种高生育酚含量微藻的筛选方法,其特征在于,在S1中,将藻株采用平板划线法进行分离纯化,采用BG-11培养基培养,所述BG-11培养基中Na2CO3的添加量为0,所述BG-11培养基还包括20g/L的葡萄糖。
10.如权利要求6所述的一种高生育酚含量微藻的筛选方法,其特征在于,在S2中还包括预先对不同取样地的混合水样进行生育酚含量测定,以筛选取样地;在S3中,还包括采用预先采用尼罗红染色法检测荧光强度以判断油脂含量高低。
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