CN101892159B - 一株衣藻藻株及其应用 - Google Patents
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Abstract
本发明提供一株衣藻藻株,经过鉴定所述藻株为一株新的藻株,其保藏号为CGMCC No.3577。所述衣藻藻株油脂含量较高,可应用于制备生物柴油,饲料或食用油。
Description
技术领域
本发明属于生物能源领域,具体地涉及一种衣藻及其制备生物柴油的应用,更具体地,本发明涉及一株新的衣藻藻株,其油脂含量较好,可用于制备生物柴油、饲料或食用油的应用。
背景技术
随着日益严重的环境恶化,控制汽车尾气排放和温室效应,保护人类赖以生存的自然环境成为人类急需解决的问题。同时全球能源需求不断扩大,寻求可以替代石油在能源结构中占主导地位的可再生清洁能源是目前普遍关注的热点。
微藻是一类在水中生长的种类繁多且分布极其广泛的低等植物,它是由阳光驱动的细胞工厂,通过微藻细胞高效的光合作用,吸收CO2,将光能转化为脂肪或淀粉等化合物的化学能,并放出O2。微藻是光合效率最高的原始植物,也是自然界中生长最为迅速的一种低等植物,而且某些微藻可以生长在高盐、高碱环境的水体中,可充分利用滩涂、盐碱地、沙漠进行大规模培养,也可利用海水、盐碱水、工业废水等非农用水进行培养,还可以利用工业废气中的CO2。利用微藻生产生物柴油能够解决目前使用植物原料发展生物柴油面临的耕地不足、气候变化对产量影响大和引起农作物价格上涨等突出问题。因此,微藻生物柴油作为可再生清洁能源成为了潜在的能源研究热点。
衣藻(Chlamydomonas)亦称“单衣藻”。绿藻门、团藻目、衣藻科中的衣藻属。藻体为单细胞,球形或卵形,前端有两条等长的鞭毛,能游动。鞭毛基部有伸缩泡两个;另在细胞的近前端,有红色眼点一个。载色体大型杯状,具淀粉核一枚。无性繁殖产生游动孢子;有性生殖为同配、异配和卵式生殖。在不利的生活条件下,细胞停止游动,并进行多次分裂,外围厚胶质鞘,形成临时群体称“不定群体”。环境好转时,群体中的细胞产生鞭毛,破鞘逸出。其中的莱茵衣藻是一种单细胞真核鞭毛藻类,是研究多种生命活动(如光合作用、生理节律和趋光性等)的模式生物,与酵母细胞有许多共同的特征,素有“光合酵母”之称。关于衣藻基因方面的研究有大量报道,如专利CN200610026203.3衣藻红色荧光标记蛋白基因CrmRFP、其合成方法及其真核表达载体的构建方法,CN200810066705.8表达人组织激肽释放酶的转基因莱茵衣藻的构建方法,CN 200610018306.5一种莱茵衣藻外源基因表达系统及其构建生产PHB转基因藻的方法,却未见衣藻生产生物柴油的报道,本发明提供了一种含油量较高的衣藻,其可以进一步进行基因改造。
发明内容
本发明的目的是提供一种产油衣藻及其在生物能源领域的应用。
在一个方面中,本发明提供一株衣藻藻株DQA-01(Chlamydomonas sp.DQA-01),保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏号为CGMCC No.3577。此衣藻藻粉含油量占干重量的1%----50%,其油脂可以做成生物柴油,饲料或食用油;所述的产油衣藻是属于衣藻属的一个新种,命名为DQA-01,与作为基因工程中微藻模式生物的莱茵衣藻(Chlamydomonas reinhardtii)在进化树上非常相近,可为能源微藻基因工程改造提供材料。在一个实施方案中,所述衣藻藻株DQA-01在15-35℃的温度范围内培养,温度优选为23℃。在一个实施方案中,所述衣藻藻株DQA-01在培养过程光照强度控制在50---200umol/m2.s。在一个实施方案中,所述衣藻藻株在光照强度150-300umol/m2.s诱导下积累油脂。在一个实施方案中,所述衣藻藻株首先在60umol/m2.s培养,在第3天时将光强加大到120umol/m2.s,在第7天时将光强加大到200umol/m2.s。在一个实施方案中,所述衣藻藻株DQA-01在培养期间将培养基的pH值调节在7-9之间,优选pH为7.2。
在另一个方面中,本发明提供筛选所述含油衣藻的方法,所述方法包括下述步骤:
1)藻样采集,选择含有多种藻类的污染河流、湖泊或沼泽地,使用藻样采集器进行藻样采集;
2)藻种分离纯化,利用稀释铺平板法或毛细管法将混合在一起的多种藻种进行分离,得到单个藻种的纯藻株;
3)藻种培养及油脂诱导,将纯藻株在BG11培养基,25℃下进行培养,当藻的浓度达到1-8g/l的时候,加强光进行油脂诱导;
4)油脂含量测定,取油脂诱导后的藻进行尼罗红染色,在荧光显微镜下进行观察,选择荧光比较多的藻种,从而快速得到含油较高的藻种。在一个实施方案中,所述方法还包括对纯藻株进行藻种鉴定,通过形态和分子相结合的方法确定藻种在进化树中的位置的步骤。
在另一个方面中,本发明提供一种生产生物柴油的方法,所述方法特征在于使用本发明提供的所述衣藻藻株DQA-01进行。
在另一个方面中,本发明提供一种生产饲料的方法,所述方法特征在于使用本发明提供的所述衣藻藻株DQA-01进行。
在另一个方面中,本发明提供一种生产食用油的方法,所述方法特征在于使用本发明提供的所述衣藻藻株DQA-01进行。
在另一个方面中,本发明提供一种对衣藻藻株进行基因改造的方法,所述方法特征在于使用本发明提供的所述衣藻藻株DQA-01进行。
在又一个方面中,本发明提供所述衣藻藻株DQA-01用于制备生物柴油、饲料或食用油的应用。
在又一个方面中,本发明提供所述衣藻藻株DQA-01用于制备基因改造藻株的应用。
在又一个方面中,本发明提供一种饲料,其由本发明提供的所述衣藻藻株DQA-01制备而来。
在又一个方面中,本发明提供一种食用油,其由本发明提供的所述衣藻藻株DQA-01制备而来。
在又一个方面中,本发明提供一种生物柴油,其特征在于,其是通过培养本发明提供的衣藻藻株DQA-01,并从中分离油脂制备而来。
本发明中的衣藻藻体为单细胞,球形或卵形,前端有两条等长的鞭毛,能游动。鞭毛基部有伸缩泡两个;另在细胞的近前端,有红色眼点一个。载色体大型杯状,具淀粉核一枚。无性繁殖产生游动孢子;有性生殖为同配、异配和卵式生殖。在不利的生活条件下,细胞停止游动,并进行多次分裂,外围厚胶质鞘,形成临时群体称“不定群体”。环境好转时,群体中的细胞产生鞭毛,破鞘逸出。其用于藻种鉴定的ITS序列是(GGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTGAATCTATCACAATCCACAACCCGCGAACCATACTGTTGGCCTTCCTTGGTTTCGGCCAAGGAGCCAGGCTCTGTCGGTGCTCACGCGCCGTACGGCAGCCTGGGTCGCCTGCCCAATTAATTATTAATTAATTAGCTGGGCCGGCGTCGGTCTCTTAACCAACCACACACCAAACATAACAATAATAAAAAACGAGCGCTTGGCTTAGAGCCGACGCTCACCAACCAAAGACAACTCTCAACAACGGATATCTTGGCTCTCATAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGCGAATTGCAGAAATACGTGAATCATCGAATCTTTGAACGCAAATTGCGCTCGAGGCTTCGGCCAAGAGCATGTCTGCCTCAGCGTCGGGTTAATACTCGCCTGCTGCACTGCCTGTGCATGCAAGCGGAGCTGGCTGTCTCGGCCCCTCGTAAAACAGGTGCCGGGTCTGCTGAAGTACAGAGGTTGATGCATGGACCCGCTCATGGGCCTCAACTGGGTAGGCAACTCGTTGCTAATGCTTTAGTTGATGGCTTGGATCCGCGCTTGTCGACCCGAACCAGGAACTCGGCTTCTGCCGAGCAAACCCCTCATTTTCTCGACCTGAGCTCAGGCAAGAACACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA)(SEQ ID NO:1),与最相近的Chlamydomonas callosa的相似度为89%,是衣藻属的一个新种。
发明人已将所述衣藻DQA-01藻株(Chlamydomonas sp.DQA-01)于2010年1月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,简称CGMCC),其保藏号为CGMCCNo.3577。
发明效果
本发明分离的衣藻藻株在高光的情况下可以积累占干重20%以上的油脂,其油具有很好的流动性,是品质比较好的油脂。此株衣藻本身就含有比较高油脂的特征,将对微藻生物柴油利用基因工程改造突破现有能源微藻产量低、成本高等难题具有十分重要的意义。
附图说明
图1为根据DQA-01藻株的ITS及5.8S部分序列构建的进化树。
图2为DQA-01藻株的显微照片
图3为DQA-01藻株的油脂尼罗红染色荧光显微照片
图4为DQA-01藻ITS序列与genebank中Chlamydomonas callosa比对结果照片
本发明的创新性:
目前用于基因工程改造的衣藻是不产油或产油非常少的藻种,而其它含油量高的藻株在基因改造方面难度比衣藻大。
本发明中提供的微藻不仅产油,而且是衣藻属的一个物种,为产生物柴油的微藻基因改造提供了操作性简便的材料。
下面将通过具体实施方式来对本发明的优选实施方案进行说明。但是本发明的保护范围并不受所述实施例的限制。
具体实施方式
实施例1 筛选含油衣藻藻株
取无菌水稀释后的从达旗地区取回的小河水水样,在400倍显微镜观察后,大约细胞浓度为800-1200个/ml,用毛细管取大约1ul的藻液,接种于48孔装有BG11培养基的培养板中,在25℃、光照强度为50umol/m2.s情况下进行培养,当达到一定细胞浓度时,显微镜观察,选择只有单藻株的孔,进行铺平板,得到单藻株。
将纯藻株在BG11培养基中,25℃、光照强度为50umol/m2.s下进行培养,当藻的浓度达到3g/l的时候,加开灯管,使光强达到250umol/m2.s,进行油脂诱导;
将单藻株进行尼罗红染色,藻内油脂被染上了色,在荧光显微镜下进行观察(见图3),选择荧光多的藻株,即为油脂含量高的藻株。将分离出来的含油量较高的藻株命名为DQA-01。
实施例2 确定藻种在进化树上的分类。
藻株鉴定采取形状鉴定和分子鉴定相结合。
形状鉴定(见图2):对上述分离出来的藻株DQA-01在1000倍显微镜进行观察,发现藻体为单细胞,球形或卵形,前端有两条等长的鞭毛,能游动。鞭毛基部有伸缩泡两个;另在细胞的近前端,有红色眼点一个。载色体大型杯状,具淀粉核一枚。无性繁殖产生游动孢子;有性生殖为同配、异配和卵式生殖。在不利的生活条件下,细胞停止游动,并进行多次分裂,外围厚胶质鞘,形成临时群体称“不定群体”。检索《中国淡水藻类--系统、分类及生态》,发现此藻在形态上分类相同的为绿藻门、绿藻纲、团藻目、衣藻科、衣藻属。
分子鉴定:
A,从分离的衣藻扩增其ITS及5.8S部分核苷酸序列
用CTAB法提取培养4天的DQA-01藻基因组DNA。取一定量的细胞,研磨处理后加入适量CTAB缓冲液,匀浆,加入等体积的酚:氯仿抽提,等体积异丙醇沉淀,75%乙醇洗涤后溶于一定体积灭菌双蒸水中,紫外分光光度计检测其浓度和纯度。
ITS序列扩增采用真核生物ITS扩增通用引物(引物合成由上海生工生物工程公司合成)。
引物1 5’GGAAGTAAAAGTCGTAACAAGG 3’
引物2 5’GCATATCAATAAGCGGAGGA 3’
取1μl总DNA为模板。扩增条件如下:94℃变性5min,然后94℃40s,56℃ 40s,72℃ 2min 30个循环,最后72℃ 10min。1.0%的琼脂糖凝胶电泳检测其扩增产物,PCR扩增获得约750bp片断。序列如SEQ IDNO:1所显示。
B,所克隆的核苷酸序列同源性搜索
将获得的DQA的ITS及5.8S部分序列登录GenBank数据库进行BLAST比对,结果显示与ACCESSION号为U66945的Chlamydomonascallosa的内转录间隔区1(ITS1),5.8S核糖体RNA及内转录间隔区2(ITS2)序列具有较高的相似性达89%(见图4),该Chlamydomonas callosa序列,1-.217bp为ITS1序列,218-378为5.8S核糖体RNA基因序列,377-625为ITS2序列。
C,根据上述序列作出进化树,见图1。
利用NCBI数据库中的BLAST工具对藻株DQA的ITS部分序列进行序列相似性分析。选取部分同源序列和该藻株的ITS部分序列利用clustaLx软件包进行序列同源进化比对,形成一个多重序列匹配排列矩阵。然后,运用Mega4.0软件,采用邻位相连(Neighbor-joining)算法,自展数据集bootstraps值为1000构建系统发育进化树。
根据形态和ITS blast的结果,限定DQA-01为衣藻属-ChlamydomonasSp的一个新种。
发明人已将所述藻株于2010年1月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,简称CGMCC),其保藏号为CGMCC No.3577。
实施例3 衣藻油脂积累
将处在对数生长期的藻种接种在配制好的培养基中,通过光强的调节进行生长培养和油脂诱导培养。
进行衣藻油脂积累的培养基和一般方法如下:
A,培养基配制:
根据下表BG11配方进行培养基配制
NaNO3 0.8--1.5g/l
K2HPO4.3H2O 0.02-0.08g/l
MgSO4.7H2O 0.075g/l
CaCl2.2H2O 0.036g/l
citric acid 0.006g/l
Ferric ammonium citrate 0.006g/l
EDTA(dinatrium-salt) 0.001g/l
Na2CO3 0.02g/l
A5+Co solution* 1ml
去离子水 919ml
*A5+Co solution的组成成分
加到1000ml去离子水中
H3BO3 2.86g
MnCl2.H2O 1.81g
ZnSO4.7H2O 0.222g
CuSO4.5H2O 0.079g
Na2MoO4.2H2O 0.390g
Co(NO3)2.6H2O 0.049g
B,接种
将所述衣藻藻株DQA-01绿色游动细胞接种在配置好的培养基中,使细胞密度达到OD750为0.2---0.8之间。
C,培养
培养过程光照强度控制在50-200umol/m2.s,利用通气使藻株在培养基中保持均匀,温度调控在15-35℃范围内,在培养期内,通过向培养液中通人二氧化碳,将培养基的pH值调节在7-9之间。
D,油脂积累诱导
从培养的第5天起,加大光照强度,使光照强度在150-300umol/m2.s,在这一阶段内,衣藻开始油脂积累。
E,采收藻
培养进行到第12天,藻液颜色变黄或乳白时,将藻液收集,通过离心或自然沉降的方法获得藻泥,将藻泥在100℃下进行干燥。
藻粉油脂含量积累及测定
按上述步骤进行培养,其初始接种细胞密度OD750为0.5,使用BG11培养基,其中NaNO3浓度为0.9g/l、K2HPO4 3H2O浓度为0.04g/l,光照强度为60umol/m2.s,pH为7.2,温度维持在23℃左右,在第3天时将光强加大到120umol/m2.s,在第7天时将光强加大到200umol/m2.s,培养到第12天,藻液颜色变黄或乳白时,将藻液收集,通过离心或自然沉降的方法获得藻泥,将藻泥在100℃下进行干燥。
测定干燥后的藻粉油脂含量,其测定方法:取50mg或100mg冻干藻粉放置在具Telfnon螺口瓶盖的体积为15-20ml的小玻璃瓶中,再放置一小磁力棒,加入2-4ml 10%DMSO-Methanol溶液,40℃砂浴(盛砂的烧杯放置恒温加热磁力搅拌器上)5分钟;然后在4℃下磁力搅拌抽提30分钟,3500转离心,转移上清液到另一小瓶中。剩下藻渣再加入1∶1的乙醚、正己烷4-8ml 4℃下磁力搅拌抽提1小时,3500转离心,转移上清液到上述一小瓶中。可重复上述过程直到藻渣变白。在上述合并抽提液中加入纯水使四者(水、DMSO-Methanol、乙醚、正己烷)比例为1∶1∶1∶1,震荡分相,移取有机相转移到另一小玻璃瓶中,在通风橱中用氮气吹至成浓缩液,然后转移到事先称重过的1.5ml塑料离心管中,再用氮气吹干至恒重,称量恒重后的管重,用此管重减去事先称重过的离心管重得到油脂重量,然后用油脂重量除以藻粉重,计算出其油脂总含量为藻粉干重31%。
取25mg-100mg藻粉照上面方法进行提取后,用正己烷溶解,使用Agilent 6820气相色谱仪进行气相色谱分析(色谱条件为载气:氮气流量1ml/min、氢气流量30ml/min、空气流量300ml/min,进样口温度:280℃,检测器温度:280℃,检测器类型:FID,分析方法:内标法,进样口类型:分流/不分流进样口),其油脂组分含量如下表1。
表1 气相色谱测定的样品总脂组分表(组分含量为重量百分比)
本发明中的衣藻具有一定的油含量,可用于提炼生物柴油;此藻种为衣藻属下的一个种,其与做了大量基因方面研究的莱茵衣藻是同一属,在进化上具有许多相似处,故可以借鉴莱茵衣藻基因方面的研究经验对此产油衣藻进行基因改造,有助于能源微藻基因改造方面的研究。
序列表
<110>新奥科技发展有限公司
<120>一株衣藻藻株及其应用
<130>IB096695
<160>1
<170>Patent In version 3.1
<210>1
<211>728
<212>DNA
<213>衣藻藻株(Chlamydomonas sp.DQA-01)
<400>1
ggaagtaaaa gtcgtaacaa ggtttccgta ggtgaacctg cggaaggatc attgaatcta 60
tcacaatcca caacccgcga accatactgt tggccttcct tggtttcggc caaggagcca 120
ggctctgtcg gtgctcacgc gccgtacggc agcctgggtc gcctgcccaa ttaattatta 180
attaattagc tgggccggcg tcggtctctt aaccaaccac acaccaaaca taacaataat 240
aaaaaacgag cgcttggctt agagccgacg ctcaccaacc aaagacaact ctcaacaacg 300
gatatcttgg ctctcataac gatgaagaac gcagcgaaat gcgatacgta gtgcgaattg 360
cagaaatacg tgaatcatcg aatctttgaa cgcaaattgc gctcgaggct tcggccaaga 420
gcatgtctgc ctcagcgtcg ggttaatact cgcctgctgc actgcctgtg catgcaagcg 480
gagctggctg tctcggcccc tcgtaaaaca ggtgccgggt ctgctgaagt acagaggttg 540
atgcatggac ccgctcatgg gcctcaactg ggtaggcaac tcgttgctaa tgctttagtt 600
gatggcttgg atccgcgctt gtcgacccga accaggaact cggcttctgc cgagcaaacc 660
cctcattttc tcgacctgag ctcaggcaag aacacccgct gaacttaagc atatcaataa 720
gcggagga 728
Claims (7)
1.一株衣藻Chlamydomonas sp.藻株DQA-01,保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏号为CGMCC No.3577。
2.权利要求1的衣藻藻株,其特征在于在15-35℃的温度范围内培养。
3.权利要求1的衣藻藻株,其特征在于在培养过程中光照强度控制在50---200umol/m2.s。
4.权利要求1的衣藻藻株,其特征在于,其在光照强度150-300umol/m2.s下被诱导积累油脂。
5.权利要求1的衣藻藻株,其特征在于,在培养期间将培养基的pH值调节在7-9之间。
6.权利要求1-5中任一项的衣藻藻株用于制备生物柴油、饲料或食用油的应用。
7.一种生产生物柴油的方法,所述方法特征在于使用权利要求1-5任一项的衣藻藻株进行生产。
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