CN114058515A - 一种利用海水微拟球藻生产蜂王浆主效成分24-亚甲基胆固醇的方法 - Google Patents

一种利用海水微拟球藻生产蜂王浆主效成分24-亚甲基胆固醇的方法 Download PDF

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CN114058515A
CN114058515A CN202111192566.5A CN202111192566A CN114058515A CN 114058515 A CN114058515 A CN 114058515A CN 202111192566 A CN202111192566 A CN 202111192566A CN 114058515 A CN114058515 A CN 114058515A
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nannochloropsis
cholesterol
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nannochloropsis oculata
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路延笃
邓湘子
周文序
甘琴华
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Hainan University
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Abstract

本发明涉及基因工程技术领域，特别涉及一种微拟球藻工程株及其制备方法和应用。该工程株为敲除DWARF1(DWF1)基因的微拟球藻。本发明利用CRISPR CAS9技术，在不引入任何外源分子标签的前体下，构建出DWF1基因的敲除株，得到胆固醇含量显著降低、24‑亚甲基胆固醇含量显著提高的工程株，其遗传性状稳定。4‑亚甲基胆固醇是蜂王浆主成分，具降血压、抗心律失常、血管解痉等活性。若将此工程株产业化生产，有助于填补高24‑亚甲基胆固醇微藻这一领域的空白，实现利用海洋微拟球藻开发高24‑亚甲基胆固醇、高二十碳五烯酸、低胆固醇的健康食品，并为利用海水和二氧化碳工业化生产人造蜂王浆提供理论依据。

Description

一种利用海水微拟球藻生产蜂王浆主效成分24-亚甲基胆固 醇的方法
技术领域
本发明涉及基因工程技术领域，特别涉及一种微拟球藻工程株及其制备方法和应用。
背景技术
微拟球藻(Nannocloropsis oceanica)是一种光合自养的圆球状的单细胞生物，在缺氮胁迫条件下能产生大量的脂质积累，累积量高达其生物量干重的60％，故常用作生物柴油的潜在替代品。除此之外，它也能用作一系列高附加值产品的开发，如食品、饲料添加剂、色素、化妆品、药品等。而其代谢产物如叶绿素、甾醇、类胡萝卜素和一些植物激素，决定了对生长或产生有价值的化学物质至关重要的细胞特性。微拟球藻富含油脂，且有特别高含量的PUFA(polyunsaturated fatty acid，多不饱和脂肪酸)，如EPA(eicosapentaenoic acid，二十碳五烯酸)，它在功能食品、功能饵料等方面具有巨大的经济价值及广阔的应用前景。
胆固醇是构成人体组织细胞所不可或缺的重要组成物质，它不仅参与细胞膜的形成，而且是合成维生素D、胆汁酸及甾体激素的重要原料。但当人体血清胆固醇含量过高时，易导致高胆固醇血症的发生，对人体产生不利的影响。现代研究发现，动脉粥样硬化性、心脑血管疾病及冠心病等的发生与高胆固醇血症有密切的联系。因此，降低血清中过多的胆固醇含量对人身体健康是有利的。微拟球藻因其富含EPA，被广泛用于功能食品和功能饵料开发，但却因含有高的胆固醇而饱受诟病。
微拟球藻中胆固醇的合成前体是24-亚甲基胆固醇(Lu et al.,2014 Regulationof the cholesterol biosynthetic pathway and its integration with fattyacidbiosynthesis inthe oleaginous microalgaNannochloropsis oceanica)。24-亚甲基胆固醇是蜂王浆主要的甾体化合物，具有降血压、降低血清胆固醇、减慢心率、抗心律失常、血管解痉等多种生物活性(Chakrabarti et al.,2020 Evaluating effects of acritical micronutrient(24-methylenecholesterol)on honey bee physiology)。
目前，还未见针对胆固醇和24-亚甲基胆固醇含量进行改造的微拟球藻工程株。
发明内容
有鉴于此，本发明提供了一种微拟球藻工程株及其制备方法和应用。本发明获得一株低胆固醇、高24-亚甲基胆固醇工程微拟球藻细胞，其胆固醇含量下降98.7％，24-亚甲基胆固醇含量提高了222倍，且性状可稳定遗传，可用于产业化生产高多不饱和脂肪酸、高24-亚甲基胆固醇、低胆固醇的功能食品和水产饵料，为绿色健康产业服务。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种微拟球藻工程株，其为敲除DWF1基因的微拟球藻。
本发明还提供了该微拟球藻工程株的制备方法，采用CRISPR/Cas9技术将微拟球藻中的DWF1基因敲除。
本发明还提供了该微拟球藻工程株的培养方法，将微拟球藻工程株接种于平板培养基中进行培养；平板培养基配方为：
Figure BDA0003301757630000021
优选地，平板培养基配方为：
Figure BDA0003301757630000022
Figure BDA0003301757630000031
作为优选，营养液母液的配方为：
硝酸钠 30～50g
一水合磷酸二氢钠 1～5g
水 补足至200mL。
优选地，营养液母液的配方为：
硝酸钠 40g
一水合磷酸二氢钠 2.66g
水 补足至200mL。
作为优选，维生素母液的配方为：
Figure BDA0003301757630000032
优选地，维生素母液的配方为：
Figure BDA0003301757630000033
作为优选，微量元素溶液的配方为：
Figure BDA0003301757630000034
优选地，微量元素溶液的配方为：
Figure BDA0003301757630000041
作为优选，抗生素溶液的配方为：
氨苄青霉素钠 0.5～2mg
头孢氨噻肟 0.5～2mg
水 补足至10mL。
优选地，抗生素溶液的配方为：
氨苄青霉素钠 1mg
头孢氨噻肟 1mg
水 补足至10mL。
本发明还提供了一种诱导微拟球藻工程株生产甾醇的方法，对上述微拟球藻工程株进行高光胁迫诱导培养。
作为优选，高光胁迫诱导培养的条件为：将经过验证后的转化子与野生型均接种至PBR中培养至对数生长期，即OD750＝2.0-3.5，然后转接至含120mLf/2培养基的250mL三角瓶中，每组设置三个平行，过夜暗适应后，置于光强为200μmol·photons·m-2·s-1，25℃，24h光照的光照培养箱中，于第10天离心收集藻液，将样品冷冻真空干燥后，用于后续GC-MS的分析。
本发明还提供了该微拟球藻工程株在制备低胆固醇、高24-亚甲基胆固醇功能食品或水产饵料中的应用。
本发明具有的技术效果如下：
目前还未见针对DWF1基因在微拟球藻中的研究，传统遗传工程策略往往引入外源抗性标签，会引发公众对转基因食品的顾虑。本发明利用CRISPR CAS9技术，在不引入任何外源分子标签的前体下，构建出微拟球藻DWF1基因的敲除株，得到胆固醇含量显著降低(从近70％下降至1.55％以下)、24-亚甲基胆固醇含量显著提高(从0.29％提高至超过60％，以提高最明显的dwf1-6为例)的工程株，这一工程株遗传性状稳定，若将此工程株进行产业化生产，有助于填补低胆固醇、高24-亚甲基胆固醇微藻这一领域上的空白，为将微拟球藻用于开发低胆固醇含量的健康食品的生产应用提供理论依据。将此工程株应用于开发成食品、保健品或特殊医学配方食品等，可以满足人民对于健康的需求。
附图说明
图1转化子验证；
图2野生型微拟球藻的甾醇峰图；
图3 dwarf1(即dwf1)敲除株的甾醇峰图；
图4野生型与突变体的甾醇峰图叠加图。
具体实施方式
本发明公开了一种微拟球藻工程株及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中所用试剂或仪器均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
1、平板培养基配制如下：
将10g琼脂粉溶入1L配制的人工海水中，配制成1％的琼脂液固体培养基中，放入灭菌锅中121℃，灭菌20min。灭菌完成后趁热在超净工作台上用移液器吸取5mL营养液母液、500μL微量元素溶液、1mL抗生素溶液以及3μL100mg/mL的潮霉素B加入琼脂液中，摇匀，倒平板。(注：营养液母液及微量元素溶液均需要提前灭菌，121℃，20min；维生素母液和抗生素溶液均需要预先0.22μm膜过滤除菌)
表1营养液母液配方(200mL)
物料名称 分子式 级别 称量量(g)
硝酸钠 NaNO<sub>3</sub> AR 40
一水合磷酸二氢钠 NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O AR 2.66
表2维生素母液配方(100mL)
物料名称 分子式 级别 称量量(mg)
维生素B<sub>12</sub> VitaminB12 AR 1
生物素 Biotin AR 1
维生素B<sub>1</sub> VitaminB1 AR 20
表3微量元素溶液母液配方(200mL)
物料名称 分子式 级别 称量量(mg)
乙二胺四乙酸二钠 Na<sub>2</sub>EDTA AR 874
六水合氯化铁 FeCl<sub>3</sub>.6H<sub>2</sub>O AR 730
五水硫酸铜 CuSO<sub>4</sub>·5H<sub>2</sub>O AR 3.92
七水硫酸锌 ZnSO<sub>4</sub>·7H<sub>2</sub>O AR 8.8
六水氯化钴 CoCl<sub>2</sub>·6H<sub>2</sub>O AR 2.184
四水氯化锰 MnCl<sub>2</sub>·4H<sub>2</sub>O AR 72
二水钼酸钠 Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O AR 2.52
表4抗生素溶液配方(10mL)
物料名称 分子式 级别 称量量(mg)
氨苄青霉素钠 C<sub>16</sub>H<sub>18</sub>N<sub>3</sub>NaO<sub>4</sub>S AR 1
头孢氨噻肟 C<sub>16</sub>H<sub>16</sub>N<sub>5</sub>O<sub>7</sub>S<sub>2</sub>Na AR 1
2、利用CRISPR CAS9技术，构建出微拟球藻DWARF1基因的敲除株。
需敲除的微拟球藻DWARF1基因序列为：
ATGCCGCAACCAAGTCTGAAGTCCTTCGCCTCCAAAGGCAAAGGGAAGCCCCAGCGAGAGGAGAACTTTTTTGAGTATATGATTACTCACCACCGATGGGTCTTTTGCGTCTTCTTGCTAATGCCACTGTCGCTTGCGTTTGATATTGTCCTCTATGTCCGCAATAGCGTCCAGTTTTATCTCCGAAATTGGGCACCCAAGCAACACGAGGCCAAAGTGGAGGAGATCAAGAAGCAGATTTTAGAGTGGGCTAAGCATGACGGCAAGAACAAGCTGTGCACCGCTCGCCCGGGTTGGCAGACCATGTCCCTGCGTGTCGGAAAATACAAGAAGACATTCCGTAAGGTCAAGCTGGATCTACATGACATCCTCTCCATCACCACCACCGGTCAATCTCCCTCCGTTTGCGTCGAGCCCCTCGTCACCATGGGGCAGCTGACTGCCGCCCTCCTCCCCGTTGGTTGGACCGTGCCTGTCTTACCTGAACTAGACGACTTGACTATCGGGGGTTTGATTGCTGGAGTGGGTGTGGAAAGCTCTAGTCATATTTACGGGCTCTTCCAACACATTTGCATGGAGTTTGAGGTGGCATTGGCGGATGGCTCGGTCGTCTTTTGCAGCCCTGAGAAGAATGCCGAGTTGTTTTACAACTTGCCTTGGTCGCATGGCACGCTTGGCTTTTTGTTATCCGCTACAATTAGGATCATCCCTGCGAAACAATACGTGAAATTAGAGTACTTTGCGTTTACCAATGGCGGGAAAGCCCTGGAGATGTTTGAGAGGGAGAGCAGGAAAGGCGTGAACCGTCCTCCGCTCGCTGGGACGCAAGTCGCGTATGCGGCCGCTGCGGACTTGAAAAAAAGGATGGAGGGAGGGGTGACTGGGAAGGGGGAGGGCGATGAGGGGTCCTCGCCTGTAGGGGGAGGGAGGGAGGACGCGGCGGACTATGTGGAGGCGCTTGCGTATAGCCGAGAGCATGTAGTGGTGATGTTGGGGACGATGGTGGATACGCCTGAGGGGGGTAAGGAGGCAGGGAAGGTAAACGCGTTGGGCAAGTTTTGGAAGCCGTGGTTTTTCAAGCACGTGGAGGGGTTTCTGGTCAAGGGAGGGAAGGAGGGAGAGAGGGTGGCCGTGGAGTATGTGCCATTGCGGCATTACTACCACCGGCACAGCAAGAGTTTGTTCTGGGAGATTCAGGACATCATTCCCTTTGGGAATAACGTGGTGTTCCGGTACCTGTTTGGGTGGATGATGCCTCCGCGCATTTCGCTTCTGAAACTGACTCAAACGGAAGCTTTGCGGAAGCTGTACGAGGAGCATCACGTGGTACAGGACATGCTGGTACCCGTCAAGGACCTGGGAGAGGGCTTGGATGTGTTCGAGGAGGTGTTCGGCGTGTACCCGCTCTGGCTGTGCCCCATGCGCATCCCCAAGAATCCGGATTATGCCAAGTTTGGCGGGTTTGTCAAGCCGTTGGAAGGGGGGAAAGACGAGATGTTTGTGGACGTGGGCGCGTACGGCAACCCAAGCATGGAGGGATTTAATGCCCGAGAGGCGTGTCGAAAGGCAGAGGACTGGGTGCGGGCCAAGAAAGGTTACCAAATGATGTACGCCGACTCCTTCATGACGCGAGAAGAGTTCCGGGAGATGTTTGATCACTCCAAGTACGACGAGCTCAGGCGGCGGATGGATCTATGCCTGCAGGCGTTTCCTGAGGTGTACGATAAGGTCAGCAAGGCTGCACGGGTCTAG
电穿孔转化参照Wang等(Wang et al.,Genome editing ofmodel oleaginousmicroalgaeNannochloropsis spp.by CRISPR/Cas9)。基因敲除参照Lu等(Lu et al.,Roleof an ancient light-harvesting protein of PSI in light absorption andphotoprotection)。转化后的藻液涂于含300μg/L潮霉素的f/2平板中，挑选至液体培养基(步骤1中配制的平板培养基不加琼脂即为液体培养基)中进行培养，平板培养温度为25℃，光照强度为50μmol·photons·m-2·s-1。培养21天。
3、单克隆培养
待平板上长出肉眼可见的较大的单克隆时，挑取若干单克隆至含f/2液体培养基的50mL三角瓶中。置于培养箱中25℃，50μmol·photons·m-2·s-1下培养10-15天。
4、PCR验证
待生长至肉眼可见绿色时取藻液进行藻液PCR验证，用XD-001F与XD-001R引物对进行扩增，PCR产物大小约为660bp。
XD-001F：TGGATCAGCAAGACGAAGACCT
XD-001R：CGGAGATAAAACTGGACGCTATTG
并将PCR产物送测序，用基因组序列进行blastn比对，确认是否在PAM区发生移码突变，使得该基因失效。验证的PCR电泳图见图1。
实验得到了三株转化子，分别命名为dwf1-1、dwf1-6、dwf1-7。
5、高光胁迫处理
将经过验证后的转化子与野生型均接种至PBR中培养至对数生长期，即OD750＝2.0-3.5，然后转接至含120mLf/2培养基的250mL三角瓶中，每组设置三个平行，过夜暗适应后，置于光强为200μmol·photons·m-2·s-1，25℃，24h光照的光照培养箱中，于第10天离心收集藻液，将样品冷冻真空干燥后，用于后续GC-MS的分析。峰图见图2、图3、图4。(注：无论野生型还是突变体，均选用了三个生物学重复中差异最大的一个用作展示说明。)
6、GC-MS分析
GC-MS分析技术参数：流量1mL/min，起始温度170℃，持续1min，然后以20℃/min上升到280℃，停留至少15min，离子源温度为150℃，进样量1μL，柱箱温度为170℃。
表5微拟球藻在高光胁迫处理下的GC/MS甾醇数据分析
Figure BDA0003301757630000091
为了评价不同处理的效果，每个样品在上述条件下建立三个生物重复。采用单因素方差分析评价各处理的差异，然后进行p值检验。数据以平均值±标准差(n≥3)表示。p值＜0.05时，差异被认为是显著的。
由表可见，突变体的胆固醇相对含量与野生型相比，显著降低。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 海南大学
<120> 一种利用海水微拟球藻生产蜂王浆主效成分24-亚甲基胆固醇的方法
<130> MP21025541
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgccgcaac caagtctgaa gtccttcgcc tccaaaggca aagggaagcc ccagcgagag 60
gagaactttt ttgagtatat gattactcac caccgatggg tcttttgcgt cttcttgcta 120
atgccactgt cgcttgcgtt tgatattgtc ctctatgtcc gcaatagcgt ccagttttat 180
ctccgaaatt gggcacccaa gcaacacgag gccaaagtgg aggagatcaa gaagcagatt 240
ttagagtggg ctaagcatga cggcaagaac aagctgtgca ccgctcgccc gggttggcag 300
accatgtccc tgcgtgtcgg aaaatacaag aagacattcc gtaaggtcaa gctggatcta 360
catgacatcc tctccatcac caccaccggt caatctccct ccgtttgcgt cgagcccctc 420
gtcaccatgg ggcagctgac tgccgccctc ctccccgttg gttggaccgt gcctgtctta 480
cctgaactag acgacttgac tatcgggggt ttgattgctg gagtgggtgt ggaaagctct 540
agtcatattt acgggctctt ccaacacatt tgcatggagt ttgaggtggc attggcggat 600
ggctcggtcg tcttttgcag ccctgagaag aatgccgagt tgttttacaa cttgccttgg 660
tcgcatggca cgcttggctt tttgttatcc gctacaatta ggatcatccc tgcgaaacaa 720
tacgtgaaat tagagtactt tgcgtttacc aatggcggga aagccctgga gatgtttgag 780
agggagagca ggaaaggcgt gaaccgtcct ccgctcgctg ggacgcaagt cgcgtatgcg 840
gccgctgcgg acttgaaaaa aaggatggag ggaggggtga ctgggaaggg ggagggcgat 900
gaggggtcct cgcctgtagg gggagggagg gaggacgcgg cggactatgt ggaggcgctt 960
gcgtatagcc gagagcatgt agtggtgatg ttggggacga tggtggatac gcctgagggg 1020
ggtaaggagg cagggaaggt aaacgcgttg ggcaagtttt ggaagccgtg gtttttcaag 1080
cacgtggagg ggtttctggt caagggaggg aaggagggag agagggtggc cgtggagtat 1140
gtgccattgc ggcattacta ccaccggcac agcaagagtt tgttctggga gattcaggac 1200
atcattccct ttgggaataa cgtggtgttc cggtacctgt ttgggtggat gatgcctccg 1260
cgcatttcgc ttctgaaact gactcaaacg gaagctttgc ggaagctgta cgaggagcat 1320
cacgtggtac aggacatgct ggtacccgtc aaggacctgg gagagggctt ggatgtgttc 1380
gaggaggtgt tcggcgtgta cccgctctgg ctgtgcccca tgcgcatccc caagaatccg 1440
gattatgcca agtttggcgg gtttgtcaag ccgttggaag gggggaaaga cgagatgttt 1500
gtggacgtgg gcgcgtacgg caacccaagc atggagggat ttaatgcccg agaggcgtgt 1560
cgaaaggcag aggactgggt gcgggccaag aaaggttacc aaatgatgta cgccgactcc 1620
ttcatgacgc gagaagagtt ccgggagatg tttgatcact ccaagtacga cgagctcagg 1680
cggcggatgg atctatgcct gcaggcgttt cctgaggtgt acgataaggt cagcaaggct 1740
gcacgggtct ag 1752
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tggatcagca agacgaagac ct 22
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cggagataaa actggacgct attg 24

Claims (10)

1.一种微拟球藻工程株，其特征在于，其为敲除DWF1基因的微拟球藻。
2.权利要求1所述微拟球藻工程株的制备方法，其特征在于，采用CRISPR/Cas9技术将微拟球藻中的DWF1基因敲除。
3.权利要求1所述微拟球藻工程株的培养方法，其特征在于，将所述微拟球藻工程株接种于平板培养基中进行培养；所述平板培养基配方为：
Figure FDA0003301757620000011
4.根据权利要求3所述的培养方法，其特征在于，所述营养液母液的配方为：
硝酸钠 30～50g
一水合磷酸二氢钠 1～5g
水 补足至200mL。
5.根据权利要求3所述的培养方法，其特征在于，所述维生素母液的配方为：
Figure FDA0003301757620000012
6.根据权利要求3所述的培养方法，其特征在于，所述微量元素溶液的配方为：
Figure FDA0003301757620000013
Figure FDA0003301757620000021
7.根据权利要求3至6中任一项所述的培养方法，其特征在于，所述抗生素溶液的配方为：
氨苄青霉素钠 0.5～2mg
头孢氨噻肟 0.5～2mg
水 补足至10mL。
8.一种诱导微拟球藻工程株生产甾醇的方法，其特征在于，对权利要求1所述微拟球藻工程株进行高光胁迫诱导培养。
9.根据权利要求8所述的方法，其特征在于，所述高光胁迫诱导培养的条件为：200μmol·photons·m-2·s-1，25℃，24h光照。
10.权利要求1所述微拟球藻工程株在制备低胆固醇、高24-亚甲基胆固醇、高二十碳五烯酸功能食品、保健品或水产饵料中的应用。
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